luv. A... .K ”9% fii LEE“: . .. i..l..|4 . ‘Oh'az 1d- . .mrz-mfla‘djw. . . n V r”? if...) '13—. 4a....“ x, ‘ I... as»; 5 a. .. ’v—fi: I .. , .19. t1. $011!. ‘1“! ’W LIBRARIES STATE UNIVERSITY EngHLANIGASqNG, MICH 48824-1048 This is to certify that the dissertation entitled Dynamin-related Proteins are Involved in Chloroplast Division and Morphogenesis presented by Hongbo Gao has been accepted towards fulfillment of the requirements for the Ph. D degree in Genetics Major Professor’s (Sjbnature Mad Hiya/100$ Date MSU is an Affirmative Action/Equal Opportunity Institution PLACE IN RETURN BOX to remove this checkout from your record. To AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 2/05 czzlnmooueindd-pts Dynamin-related Proteins are Involved in Chloroplast Division and Morphogenesis By Hongbo Gao A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHOLOSOPHY Genetics Program 2005 Abstract Dynamin-related Proteins are Involved in Chloroplast Division and Morphogenesis By Hongbo Gao Chloroplasts are specialized organelles evolved fiom cyanobacteria as endosymbionts in plant cells. Homologs of the cyanobacterial cell division genes F tsZ, MinD and MinE have been identified previously in plants and shown to be important for chloroplast division. In this work, a new chloroplast division gene, ARC5, was identified by positional cloning in Arabidopsis. The arc5 mutant has 1-15 enlarged dumbbell- shaped chloroplasts per cell, suggesting that ARCS may be involved in the constriction of chloroplasts during division. A GFP-ARCS fusion protein rescues the mutant phenotype of arc5 and localizes to a ring at the chloroplast division site. Chloroplast import and protease protection assays indicate that the ARCS ring is positioned on the outer surface of the chloroplast. Thus, ARCS is the first cytosolic component of the chloroplast division complex to be identified. Phylogenetic analysis suggests that ARCS is in the dynamin family of mechanochemical enzymes in eukaryotes, which are large GTPases involved in membrane fission and fusion in a variety of cellular processes. These results indicate that the chloroplast division machinery is of mixed evolutionary origin and that it shares structural and mechanistic similarities with both the cell division machinery of bacteria and the dynamin-mediated organellar fission machineries of eukaryotes. Additional experimental results demonstrate that a homolog of ARCS, ARCSH, is not involved in chloroplast division. Therefore, ARCS is important but not essential for chloroplast division and it probably evolved to facilitate the division of chloroplasts during endosymbiosis. FZO is a more diverged dynamin-related protein involved in mitochondrial fusion in metazoa and fungi. However, an FZO—like protein in Arabidopsis, FZL, is targeted to chloroplasts.le knockout mutants show a chloroplast division defect with heterogeneity in chloroplast size and thinner thylakoid sacs with disorganized stacking. An F ZL-GFP gene rescued the 1%] mutant phenotype. Overexpression of FZL-GF P causes very long thylakoid sacs with considerably less stacking. F ZL-GFP is a membrane protein present in both the thylakoids and chloroplast inner membrane. FZL-GFP is mainly localized to punctate or vesicle-like structures associated with chloroplasts and infrequently in the cytosol, but is not associated with mitochondria. The level of FZL- GFP expression correlates with the number of punctate and vesicle-like structures on the surface of the chloroplast, suggesting that these structures are coincident with FZL and that F ZL may be involved in their formation. Mutation of a conserved lysine residue in the GTPase domain abolishes the punctate localization pattern and the ability of FZL- GFP to complement le mutant. FZL seems to be involved in the formation and trafficking of vesicles from the thylakoid to the chloroplast envelope and cytosol, and blocking of this process affects both thylakoid morphology and chloroplast division. The finding that two dynamin-related proteins of eukaryotic origin, ARCS and F ZL, are required for chloroplast division and morphogenesis indicates that the chloroplast division machinery has been modified from the cyanobacterial cell division machinery during evolution of the organelle. T 0 my wife, F ulz', for her love and support. iv ACKNOWLEDGEMENTS First I would like to thank Genetics Program for giving me the chance to study in Michigan State University. I will be benefited from this study experience in all my life. I want to thank my advisor Dr. Katherine Osteryoung for allowing me to study in her lab and work on the wonderful area of chloroplast division, and for her outstanding guidance throughout my doctoral program. I would also like to thank my committee members Dr. Kenneth Keegstra, Dr. Steve Heidemann and Dr. Helmut Bertrand for their excellent guidance, helpfiil suggestions during my PhD studies. I would like to thank many of the excellent professors who gave classes to me. They not only taught me knowledge but also help me to open my mind and fly in the sky of science. I also want to thank all the past and present members of Osteryoung lab which I was fortunate to interact with: Dr. Deena Kadiryan-Kalbach, Dr. Stan Vitha, Dr. Rosemary McAndrew, Dr. Kevin Stokes, Dr. David Yoder, Dr. Cecilia Chi-Ham, Dr. Shinya Miyagishima, Brad Olson, Mary Fantacone, Jonathan Glynn, Magali Tshiamala and Qian Li. I want to specially thank Dr. Stan Vitha for his help on microscopy when I started in the lab and Dr. Deena Kadirjan-Kalbach, our lab manager, for keeping everything running smoothly. Thanks to all of the members of the lab for helpful comments and Kevin Stokes for help with phylogenetic analysis on chapter two. I would also like to thank Mary F antacone for reading chapter four. Magali Tshiamala and Qian Li are two undergraduate students worked with me and I want to thank them for their technical assistance in my work. I would like to thank my friends Dr. Changchen Xu, Dr. J ianping Yu, Dr. Hui Chen, Dr. Hua Zhang, and Dr. Bin Yu for their helps in my experiments. Especially, I would like to give my deep thanks to all the people who helped us after my son, Andrew, was born in the year 2002. Finally, I would like to thank all the people who helped me but their names were not listed here. I would also like to acknowledge Michigan State University Intramural Research Grants Program and National Science Foundation for the support. vi fl TABLE OF CONTENTS LIST OF FIGURES ............................................................................... xi LIST OF TABLES ............................................................................... xiii KEY TO ABBREVIATIONS ................................................................. xiv Chapter 1 Evolution of the Chloroplast Division Machinery ............................. 1 1. Conservation between cyanobacterial cell division proteins and chloroplast division proteins .............................................................................................. 6 FtsZ ........................................................................................... 7 Min system ................................................................................... 9 FTNZ and ARC6 ........................................................................... 15 SulA, Lon and HslVU ..................................................................... 16 II. Innovations in the chloroplast division machinery during evolution 19 Ftle ........................................................................................ 19 ARC3 ....................................................................................... 21 ARCS ....................................................................................... 22 III. Components that were probably lost or replaced by others during evolution ..... 27 FtsA and MreB 28 F tn6 ......................................................................................... 30 F tsI .......................................................................................... 31 FtsW ........................................................................................... 32 IV. Perspective .................................................................................. 33 Literature cited .................................................................................. 36 Chapter 2 ARC5, a Cytosolic Dynamin-like Protein from Plants, Is Part of the Chloroplast Division Machinery ................................................................ 52 Abstract .......................................................................................... 53 Introduction ....................................................................................... 54 Materials and Methods ........................................................................ 56 vii Plant Material .............................................................................. S6 Microscopy ................................................................................ 56 F inc-mapping of ARCS .................................................................. 56 Identification of a Candidate ARCS Gene ............................................. S7 Amplification of ARCS cDNA .......................................................... 57 Sequence Alignment and Phylogenetic Analysis ..................................... 58 Complementation Analysis .............................................................. 58 GFP-ARCS Localization ................................................................. 59 In Vitro Chloroplast hnport and Protease Protection Assays ....................... 60 Results .......................................................................................... 60 Phenotype of arc5 ......................................................................... 60 Fine Mapping of ARCS and a Novel Antisense Strategy for Identification of an ARCS Candidate Gene ................................................................ 62 Sequencing of arc5 and Mutant Complementation ................................... 66 Analysis of ARCS cDNAs and Gene Products ........................................ 67 Localization of ARCS ................................................................... 72 Discussion ...................................................................................... 77 Acknowledgement ............................................................................. 80 Online supporting Information ................................................................ 81 Literature cited .................................................................................. 83 Chapter 3 ARCSH, a Homolog of ARC5, is not Involved in Chloroplast Division..86 Abstract ......................................................................................... 87 Introduction ..................................................................................... 88 Results ........................................................................................... 94 ARCSH is a homolog of ARCS in Arabidopsis ....................................... 94 Knock-out of ARCSH has no effect on chloroplast division ........................ 94 arc5/arc5h double mutant has a similar phenotype to arc5 ......................... 9S 3SS-ARC5H has no effect on chloroplast division in arc5 or wild—type plants . 100 ARCS-GFP is localized to speckles in the nucleus .................................. 100 Discussion ..................................................................................... 103 Material and methods ....................................................................... 106 Plant material ............................................................................ 106 Sequence Alignment and Phylogenetic Analysis ................................... 106 viii Microscopy ............................................................................... 107 PCR identification of the Salk lines and verification of the arc5 mutation 107 RT-PCR .................................................................................. 108 Overexpression of ARCSH ............................................................ 108 ARCSH-GFP Localization ............................................................. 109 Literature cited ................................................................................ 110 Chapter 4 FZL, an FZO-like Protein in Plants, Links Thylakoid Morphogenesis to Chloroplast Division ............................................................................ 114 Abstract ........................................................................................ 1 15 Introduction ................................................................................... 1 16 Result .......................................................................................... 1 18 FZL is an F ZO-like protein in plants ................................................. 118 F ZL has a chloroplast transit peptide .................................................. 124 Loss of FZL fimction alters chloroplast size and morphology .................... 127 F ZL level affects the structure and the morphology of the thylakoid ............ 128 Topology of F ZL ........................................................................ 131 Localization of F ZL ..................................................................... 135 Increased levels of FZL result in more punctate and vesicle-like structures on the surface of chloroplasts .................................................................. 140 The GTPase domain is important for F ZL function ................................ 143 Discussion .................................................................................... 146 Materials and Methods ..................................................................... 153 Plant material ............................................................................ 153 Microscopy ............................................................................... 153 Sequence analysis of FZL .............................................................. 155 Test of the transit peptide of FZL ..................................................... 156 Complementation analysis and FZL-GFP localization ............................. 157 Protease protection assay, fractionation of chloroplasts and immunoblot. . 157 Mutagenesis of FZL ..................................................................... 158 Literature cited ................................................................................. 159 Chapter 5 Conclusion and Future Directions ............................................ 166 Literature cited ................................................................................. 176 ix LIST OF FIGURES Chapter 1 Figure 1 A model for Min oscillation in E. coli ....................................... 11 Figure 2 Phylogenetic analysis of dynamin-related proteins with an unrooted neighbor-joining tree ............................................. 24 Chapter 2 Figure 1 Comparison of chloroplasts in Arabidopsis leaf mesophyll cells 61 Figure 2 Cloning of ARC5 ................................................................ 63 Figure 3 ARC5 is a dynamin-like protein ............................................. 68 Figure 4 GFP-ARCS is localized to the constriction site of dividing chloroplasts ...................................................................... 73 Figure 5 ARC5 is on the outside surface of chloroplast .......................... 74 Chapter 3 Figure 1 ARC5H is a homolog of ARC5 and the closest relative of ARC5 in plants .......................................................................... 90 Figure 2 ARC5H is not involved in chloroplast division ........................... 96 Figure 3 35S—ARC5H has no effect on chloroplast division ...................... 98 Figure 4 ARCSH-GFP is localized to a cluster of punctate structures in the nucleus ..................................................................... 101 I)‘ v e Chapter 4 Figure 1 FZL is an FZO-like protein involved in chloroplast division ........ 119 Figure 2 FZL has a chloroplast targeted transit peptide ........................ 125 Figure 3 FZL level is related to the structure of thylakoid. ..................... 129 Figure 4 FZL is a chloroplast-targeted membrane protein ..................... 132 Figure 5 FZL-GFP is localized to punctate structures in FZL-GFP complemented fzI mutants .................................................. 136 Figure 6 FZL-GFP is localized to vesicle-like stmcture in FZL-GFP complemented le mutant with immuno gold labeling ............... 138 Figure 7 Correlation between number of puntate and vesicle-like structures and the level of FZL-GFP ............................................................... 141 Figure 8 Mutations in the GTPsae domain affect the localization and function of mutant FZL-GFP ....................................................................... 144 xi (5.) ”-K LIST OF TABLES Chapter 2 Table 1 PCR markers used for fine mapping ARC5 ............................... 82 xii KEY TO ABBREVIATIONS arc ATPase BAC bar BLAST bp C-terminus CaMV CAPS cDNA co-IP DAPI EST EMS fts FZL FZO GFP GTPase INDEL IPTG kb accumulation and replication of chloroplasts adenosine triphosphatase bacterial artificial chromosome BASTA resistant basic local alignment search tool base pair carboxy terminus cauliflower mosaic virus cleaved-amplified polymorphic sequence complementary DNA co-immunoprecipitation 4',6-diamidino-Z-phenylindole, dihydrochloride expressed sequence tag ethyl methanesulfonate filarnentation temperature-sensitive FZO-like fuzzy onions green fluorescence protein guanosine triphosphatase insertion-deletion isopropylthio-B-galactoside kilobase xiii LHC min MS N-terminus OCS PAGE PCR PD Pfu PS RT SDS T-DNA UTR kilodalton light-harvesting complex minicell Murashige and Skoog amino terminus octopine synthase polyacrylamide gel eletrophoresis polymerase chain reaction plastid-dividing Pyrococcus furiosus photosystem reverse transcription sodium dodecyl sulfate transfer DNA untranslated region xiv Chapter 1 Evolution of the Chloroplast Division Machinery Chloroplasts are specialized organelles that carry out photosynthesis and many other important processes such as fatty acid and amino acid biosynthesis in plant cells. Many of the biological pathways in chloroplasts are similar to those in cyanobacteria. Chloroplasts have a small circular genome of about 60-200 genes without associated histones (Gray, 1999; Howe et al., 2003; Raven and Allen, 2003). They have their own protein-synthesizing machinery, which more closely resembles that of prokaryotes than that found in the cytoplasm of eukaryotes (Harris et al., 1994). Molecular phylogenetic studies of the genomic sequences of chloroplasts in many plants and several species of cyanobacteria strongly support the idea that chloroplasts are derived from cyanobacteria (Chu et al., 2004). After the Arabidopsis and rice genomes were sequenced (Arabidopsis_Genome__Initiative, 2000; Yu et al., 2002), it was revealed that most of the cyanobacterial genes were transferred into the nuclear genome in the past, and that most of them have acquired sequences encoding chloroplast transit peptides. Many of the functional proteins of chloroplasts were also found to be homologous to cyanobacterial proteins (Douglas, 1998; Durnford et al., 1999; McFadden, 1999; Raven and Allen, 2003). Thus, it is now widely accepted that chloroplasts originated from cyanobacteria through endosymbiosis. Higher plants have many chloroplasts per cell in green tissue. For example, the mature mesophyll cells of wild-type Arabidopsis thaliana contain about 80-110 chloroplasts per cell (Marrison et al., 1999). New chloroplasts are generated by binary division of existing chloroplasts (Leech et a1. 1981). Chloroplasts divide during cell division and expansion (Possingh, 1969; Saurer, 1970; Dean, 1982; Ellis, 1985). Dumbbell-shaped chloroplasts, which represent the division phase, can frequently be found in young leaf tissue. Chloroplast division has physiological importance for plant cells. Suppression of chloroplast division results in fewer and larger chloroplasts per cell (Osteryoung et al., 1998; Pyke, 1999; Stokes et al., 2000). A chloroplast division mutant was reported to have a defect in gravitropism (Yamamoto et al., 2002). Smaller and more numerous chloroplasts per cell provide an advantage compared to larger and fewer chloroplasts: smaller chloroplasts have more total surface area and may exchange materials with other parts of the cell more efficiently. They can also migrate faster in response to changes in light intensity and direction (J eong et al., 2002). Multiple chloroplasts in one cell seems also very important in the history of chloroplast genome evolution: cells with multiple chloroplasts can withstand the breaking of some chloroplasts, leading to the gene transfer fiom the chloroplast genome to the nuclear genome (Timmis et al., 2004). This transfer is important for the establishment of the modern chloroplast and nuclear genomes (Martin, 2003). Early studies of chloroplast division were limited to observation by either light or electron microscopy. More details of chloroplast division were obtained following improvement of electron microscopy techniques (Leech RM, 1981; Gross and Possingham, 1989; Robertson et al., 1996). Observation of proplastids, which are the progenitor plastid type, reveals a central constriction that has been interpreted to represent the division phase of proplastids. Similar to proplastid division, chloroplast division includes elongation of the chloroplasts to form a peanut shape, constriction of the division firrrow, firrther narrowing of the division furrow to a thin isthmus that joins (9‘2 the two daughter chloroplasts, and finally pinching off of the narrow isthmus. The envelope membranes of daughter chloroplasts are then resealed (Leech RM, 1981; Cross and Possingham, 1989). Electron-dense deposits are observed at the division site of plastids (Kuroiwa et al., 1998). They form ring-like structures on the stromal face of the inner plastid membrane and the cytosolic surface of the outer plastid membrane. They were named the inner plastid-dividing (PD) ring and outer PD ring. A ring-like structure called the middle PD ring is also found between the two envelope membranes of the plastid in some red algae, but not in higher plants (Kuroiwa et al., 1998). The inner PD ring forms first and is belt-like, while the outer PD ring is rope-like and narrower than the inner ring. In the process of constriction of the plastid division furrow, the width and thickness of the outer PD ring increase linearly (Miyagishima et al., 2001a; Miyagishima et al., 2001b). The width of the inner PD ring remains the same throughout the plastid division. It has been suggested that the inner PD ring decomposes during constriction, and the outer PD ring wraps around the chloroplast with a constant number of molecules during the constriction (Miyagishima et al., 2001a; Miyagishima et al., 2001b). To document the division and development of chloroplasts, Leech’s group isolated 11 chloroplast division mutants in Arabidopsis, and named them arc, for accumulation and replication of chloroplasts (Pyke and Leech, 1994; Marrison et al., 1999). In each of the mutants, the mutation is recessive and represent single nuclear locus. Among these mutants, arc] and arc7 have smaller and more numerous chloroplasts while the other nine mutants show larger and fewer chloroplasts. arc6 shows the most severe phenotype, with only 1-2 chloroplasts per cell (Robertson et al., 1995). arc5 has about 10 large chloroplasts per cell, which are frequently dumbbell-shaped (Robertson et al., 1996; Gao et al., 2003). arc3 and arc] 1 have about 20 chloroplasts of various sizes per cell (Marrison et al., 1999). Double-mutants were made to elucidate the role of some arc genes in chloroplast development and division. Based on the phenotypes of single and double mutants, several hypotheses were developed (Pyke and Leech, 1994; Marrison etal., 1999). It was suggested that ARC6 controlled the initiation of both proplastid and chloroplast division; ARC3 had an important role in the initiation of chloroplast division and controlled the rate of chloroplast expansion; ARC] 1 could function just before chloroplast division is initiated or at any time earlier than the chloroplast expansion phase, and controlled the central positioning of the final division plane; ARC5 worked in the final stage of chloroplast division and facilitated the separation of the two daughter chloroplasts; ARC 1 was in a different pathway and down- regulated proplastid division. However, later cloning of several chloroplast division genes and their further characterization indicated that most of the hypotheses above were inaccurate (Itoh et al., 2001; Gao et al., 2003; Vitha et al., 2003; Fujiwara et al., 2004; Shimada et al., 2004). With the rapid developments molecular biology and genomics, many bacterial cell division genes were cloned and the functions of their gene products have been extensively studied (Bramhill, 1997; Rothfield et al., 1999; Errington et al., 2003). Some of the chloroplast division genes have also been cloned recently (Osteryoung and Pyke, 1998; Strepp et al., 1998; Colletti et al., 2000; Itoh et al., 2001; Gao et al., 2003; Vitha et al., 2003; Fujiwara et al., 2004; Maple et al., 2004; Raynaud et al., 2004; Shimada et al., 2004). The results indicated that the chloroplast division machinery has inherited some of the important features of the cyanobacterial cell division machinery and it also has acquired some eukaryotic features from the host cell (Osteryoung and Nunnari, 2003). This review illustrates the conservation and innovation of the chloroplast division machinery during evolution by comparing some of the key components of the division machineries between bacteria and chloroplasts. 1. Conservation between cyanobacterial cell division proteins and chloroplast division proteins Since chloroplasts are derived fi'om endosymbiotic cyanobacteria and many chloroplast—targeted proteins also originated from cyanobacteria, it is likely that most of the components of the chloroplast division machinery are derived from the cell division machinery of cyanobacteria (Osteryoung and Pyke, 1998; Osteryoung, 2000). Indeed, a search for homologs of bacterial cell division genes in the sequenced Arabidopsis genome led to the discovery of several chloroplast division genes: the F tsZ gene family, MinD, MinE, ARC6 and SulA (Osteryoung and Vierling, 1995; Osteryoung et al., 1998; Colletti et al., 2000; Stokes et al., 2000; Itoh et al., 2001; Vitha et al., 2003; Maple et al., 2004; Raynaud et al., 2004). FtsZ FtsZ is a GTPase that was first found in E. coli and is conserved in almost all bacteria and archaea (Bramhill, 1997; Rothfield et al., 1999). FtsZ is related to tubulin, and is suggested to be the ancestor of tubulin (de Boer et al., 1992a; RayChaudhuri and Park, 1992; Lowe and Amos, 1998; Nogales et al., 1998b). The structures of FtsZ (Erickson, 1998) and the aB-tubulin heterodimer (Nogales et al., 1998a) also show a high degree of similarity. F tsZ self-polymerizes, forms a ring at the equator of the bacterial cell, and determines the division site of the cell (Bi and Lutkenhaus, 1991). Blocking the function of F tsZ or changing the level of FtsZ blocks bacterial cell division and causes long filamentous cells (Dai and Lutkenhaus, 1992; Dewar et al., 1992). During different stages of the cell cycle FtsZ exhibits a series of different behaviors: assembly to form the Z ring at midcell, maintenance of the ring through continual subunit turnover, and constriction and disassembly of the ring (Romberg and Levin, 2003). The polymerization mechanism of FtsZ is also similar to that of tubulin (Scheffers and Driessen, 2001; Scheffers et al., 2002; Scheffers and Driessen, 2002). In vivo, Z rings can be assembled within 1 to 3 min and disassembled within 1 min (Sun and Margolin, 1998; Sun et al., 1998). Fluorescence recovery after photobleaching showed that the Z ring is highly dynamic throughout its existence and constitutes about 30% of cellular FtsZ protein (Stricker et al., 2002). In addition, the FtsZ protein in the Z ring can exchange with FtsZ protein in the cytosol very quickly (Stricker et al., 2002). These characteristics are based on the GTP-dependent, reversible polymerization of FtsZ. GTP is hydrolyzed immediately after F tsZ polymerization, and the F tsZ polymer contains both GDP and inorganic phosphate (Scheffers and Driessen, 2002). Preformed F tsZ polymers can be stabilized by addition of nonhydrolyzable GTP-y -S with more than 95% of the nucleotide associated with the FtsZ polymer in the GDP form, and be rapidly destabilized by addition of GDP (Scheffers et al., 2000; Mukherjee et al., 2001; Scheffers and Driessen, 2001; Scheffers and Driessen, 2002). These data indicated that, similar to tubulin, phosphate release may also be important for FtsZ polymer dynamics. In plants, there are at least two classes of F tsZ: F tle and FtsZ2 (Osteryoung and Vierling, 1995; Osteryoung et al., 1998; Strepp et al., 1998; Stokes et al., 2000; Stokes and Osteryoung, 2003). They are all targeted into chloroplasts by a cleavable transit peptide (McAndrew et al., 2001). The C-terminal core domain is conserved in FtsZ2 but not Ftle. Phylogenetic analysis indicates that they are all closely related to cyanobacterial F tsZ (Rensing et al., 2004). Both F tle and F tsZZ were shown to be important for chloroplast division and they may have distinct roles in chloroplast division. Antisense suppression, knock-out and over-expression of F tle or F tsZ2 can cause enlarged and fewer chloroplasts in the cell (Osteryoung et al., 1998; Strepp et al., 1998; Stokes etal., 2000) (Deena Kadirj an-Kalbach, personal communication). In extreme cases, there is only one chloroplast per cell. Ftle and FtsZ2 were shown to form a ring and colocalize at the division site of chloroplasts by irnmunofluorescence microscopy and expression of GP P fiision proteins in Arabidopsis thaliana (Mori et al., 2001; Vitha et al., 2001). Even in the antisense and overexpressing FtsZ plants, Ftle and F tsZZ are still colocalized (McAndrew et al., 2001) (Stan Vitha, personal communication). It was shown that in wild-type plants the molecular ratio between F tle and FtsZZ is fixed at 1:2 (McAndrew, personal communication). Thus, it seems that the correct ratio between the two types of FtsZ is important for their proper function in chloroplast division. Mitochondria are also derived from bacteria as endosymbionts but at a time earlier than chloroplasts (Dyall et al., 2004). The division machinery of mitochondria seems to be simpler than that of chloroplasts (Osteryoung and Nunnari, 2003). In animals, fungi and higher plants, there is no mitochondrial division protein with a prokaryotic origin (Osteryoung and Nunnari, 2003). However, FtsZ was found in some red algae and protists and shown to be localized to the middle of mitochondria and required for the normal morphology of mitochondria (Beech et al., 2000; Takahara et al., 2000; Gilson et al., 2003; Kiefel et al., 2004). In Dictyostelium, two mitochondrial F tsZs, FszA and F 523, were found (Gilson et al., 2003). FszA is localized to the future or recent division sites of mitochondria (Gilson et al., 2003). However, FszB was found in an electron-dense, submitochondrial body usually located at one end of the organelle (Gilson et al., 2003). The evolution of the mitochondrial division machinery seems to be an ongoing process. The mitochondrial FtsZ probably is the last bacteria-derived division protein to be left and it has been lost many times in different species (Kiefel et al., 2004). Min system Proper cell division in bacteria requires accurate positioning of the division plane in the middle of the cell, which is determined by the placement of the F tsZ ring. The site of FtsZ ring assembly is in turn controlled by the min (mini-cell) operon, which suppresses F tsZ polymerization at sites other than the middle of the cells (de Boer et al., 1989). MinC, MinD, and MinE are the proteins encoded by the min operon (de Boer et al., 1989), which is conserved in many bacteria including cyanobacteria. The Min proteins in Escherichia coli, when expressed at a certain ratio, undergo a highly dynamic localization cycle, during which they oscillate between the membrane of both cell halves, to ensure the proper placement of the cell division site (Figure 1) (de Boer et al., 1991; Mulder et al., 1992; Margolin, 2001; Meinhardt and de Boer, 2001). The min system spatially regulates division through the topological regulation of MinCD, an inhibitor of cell division. MinCD inhibits division by preventing assembly of the Z ring (Bi and Lutkenhaus, 1993). A MalE-MinC fusion protein with full biological activity can interact with F tsZ and prevent polymerization without inhibiting F tsZ's GTPase activity (Hu et al., 1999). A functional GFP-MinC was shown to oscillate rapidly between the halves of the cell independently of F tsZ (Hu and Lutkenhaus, 1999; Raskin and de Boer, 1999b). However, GFP-MinC is a cytoplasmic protein with no oscillation in the absence of the other Min proteins (Hu and Lutkenhaus, 1999). The addition of MinD, which interacts with MinC, results in the localization of GFP-MinC on the membrane (Hu and Lutkenhaus, 1999). MinD is an ATPase located on the the inner membrane region of the cell envelope (de Boer et al., 1991). A functional GFP-MinD was also shown to have a rapid oscillation cycle which is imposed by MinE (Raskin and de Boer, 1999a). These results support a model in which MinD recruits MinC to inhibit F tsZ ring formation near the cell ends and force F tsZ assembly in the middle. 10 Figure 1 A model for Min oscillation in E. coli (adapted from Margolin, 2001 ). MinCD complexes first assemble on the membrane at one pole of the cell, while MinE is concentrated at the other pole (A). MinE diffuses towards MinCD (B), assembles into the MinE ring (C) and begins to disassemble the MinCD (D, E and F), causing MinCD to diffuse away from one pole (D, E and F). After the MinE ring has moved to one end of the cell, it begins to diffuse (F and G), lagging behind MinCD by a few seconds. In the meantime, MinCD is assembling in the other pole of the cell (F and G). The cycle then repeats. Images in this figure are presented in color. 11 w... MinE ' MinCD 12 general i MinE re a imam then to tl behavior formation To series of ii. vesicles in folms the v activating th djSSOClallon hl’drolysis (t MinD~bllayel MinE induces release “Min Ci'lopIaSmiC XI In the absence of MinE, the coordinated expression of MinC and MinD leads to a general inhibition of cell division (de Boer et al., 1992b). In the yeast two-hybrid system, MinE reduces the interaction between MinC and MinD (Huang et al., 1996). MinE forms a dynamic ring that undergoes a repetitive cycle of movement first to one cell pole and then to the opposite pole (Fu et al., 2001). Taken together with studies of the dynamic behavior of MinCD, the MinE ring represents a cell structure that allows F tsZ ring formation at midcell by suppressing MinCD activity at this site. To further investigate the molecular basis of the oscillation of the Min system, a series of in vitro experiments was done. MinD was shown to bind to phospholipid vesicles in the presence of ATP and to assemble into a well-ordered helical array that forms the vesicles into tubes in a cooperative fashion (Hu et al., 2002; Lackner et al., 2003). MinD can recruit either MinC or MinE in an ATP-dependent manner (Hu et al., 2003). MinE can promote bundling of MinD filaments as well as their disassembly by activating the ATPase activity of MinD (Suefuji et al., 2002). MinE stimulates dissociation of MinC from MinDzATP-membrane complexes even without ATP hydrolysis (Hu et al., 2003). In contrast, MinC is unable to displace MinE bound to the MinD-bilayer complex (Hu et al., 2003; Lackner et al., 2003). These results suggest that MinE induces conformational changes in membrane-bound MinD, which result in the release of MinC and then the conversion of membrane-bound MinD (MinD:ATP) to cytoplasmic MinD (MinD:ADP). l3 in the 1997) prodw 2000; . Fujiwa exhibit chlorop al., 200i; Arabido, FtsZ in p the giam along the 2003). Gt,- exPressior Specificall rm'Splaced, Chloromwf P 1amS rESerr SuggeSUn Q [1 Based Chlamydomon In some green algae, such as Chlorella vulgaris, minD and minE genes are present in the chloroplast genome and arranged in the same order as in E. coli (Wakasugi et al., 1997). In higher plants, these two genes are present in the nuclear genome and the gene products are targeted to chloroplasts by an N-terminal transit peptides (Colletti et al., 2000; Dinkins et al., 2001; Itoh et al., 2001; Maple et al., 2002; Reddy etal., 2002; Fujiwara et al., 2004). Transgenic Arabidopsis plants with reduced MinD expression exhibit variability in chloroplast size and number and asymmetrically constricted chloroplasts, suggesting that the chloroplast division machinery is misplaced (Colletti et al., 2000). Overexpression of AtMinD also inhibits chloroplast division in both Arabidopsis and tobacco (Colletti et al., 2000; Dinkins et al., 2001). Immuno-staining of FtsZ in plants over-expressing MinD shows random and short FtsZ filaments throughout the giant chloroplast, while parallel FtsZ rings are distributed ectopically at multiple sites along the enlarged chloroplast in the anti-sense MinD transgenic plants (Vitha et al., 2003). Giant chloroplasts were also observed in transgenic plants with higher or lower expression levels of MinE (Itoh et al., 2001; Maple et al., 2002; Reddy et al., 2002). Specifically, in the plants overexpressing AtMinE, the chloroplast division sites were misplaced, giving rise to either asymmetric or multiple constrictions along the length of chloroplasts (Maple et al., 2002). The phenotypes observed in minD and minE mutant plants resemble those of bacterial mutants with altered minD and minE expression levels, suggesting that their working mechanism may be similar. Based on BLAST searches of the genome sequences of Arabidopsis, rice and Chlamydomonas and of the EST data from many plant species, MinC is missing in plants. 14 Accor prever with a chloro; Bad/[u Stewart But m0: that its 5 cyanoba are mucl Still, the approach be more 1 baCIEfia (1 fold 10m (D (l' lKoksharO [minus a; ’ ‘ < According to the model of the Min system in bacteria, MinC is the protein that directly prevents FtsZ ring formation at non-mid- cell sites. Therefore, plants must have a protein with a role similar to that of MinC in order to allow the Min system to function in chloroplasts. One possibility is that the role of MinC is taken by another protein. In Bacillus subtilis, MinE is missing and its role probably is taken by DivIVA (Cha and Stewart, 1997; Edwards and Errington, 1997; Howard, 2004), supporting this hypothesis. But more likely, MinC is present in plants and the reason it has not been discovered is that its sequence is not well conserved. This is supported by the fact that all known cyanobacterial species have MinC, MinD and MinE but that protein sequences of MinC are much less conserved than those of MinD, MinE and many other cell division proteins. Still, the function of MinC may be well conserved. To find the MinC homolog in plants, approaches other than BLAST searches, such as searches based on protein structure, may be more useful. FTN2 and ARC6 F tn2 is a cell division gene conserved only in cyanobacteria but not in other bacteria (Koksharova and Wolk, 2002). A transposon insertion in the F tn2 gene in Synechococcus sp. strain PC C 7942 blocks cell division. The mutant cells are up to 100- fold longer than wild-type cells and their colonies show an irregular spreading phenotype (Koksharova and Wolk, 2002). FTN2 has a domain of DnaJ cochaperones at its N- terminus and interacts with FtsZ in a bacterial two-hybrid system (Koksharova and Wolk, 2002; Mazouni et al., 2004). In vitro experiments with the purified proteins showed that 15 FTN2 decor chloro; mappe: unsger nnnant‘ plants (‘ disorgan kvelofi proteins a GP P was ; 2003). FTN2 can also decorate the F tsZ filaments and that the DnaJ domain is critical for the decoration (Mazouni et al., 2004). arc6 is a chloroplast division mutant in Arabidopsis with only one or two chloroplasts per cell (Pyke et al., 1994; Marrison et al., 1999). The arc6 locus was mapped to a region containing the F m2 homolog (Marrison et al., 1999). Sequencing of this gene revealed a nonsense mutation (V itha et al., 2003). Complementation of the mutant phenotype by the wild-type gene confirmed that ARC6 is the F m2 homolog in plants (V itha et al., 2003). Chloroplasts in the arc6 mutant contain numerous short, disorganized FtsZ filament fragments (Vitha et al., 2003). arc6 plants have a reduced level of FtsZ proteins but a normal level of F tsZ mRN A, indicating that their FtsZ proteins are less stable than that in wild type (Vitha et al., 2003). A functional ARC6- GFP was shown to be localized to a ring at the center of the chloroplasts (V itha et al., 2003). The data from Fth in cyanobacteria and ARC6 in plants suggest that they may function similarly in stabilizing the constricting FtsZ ring. SulA, Lon and HslVU SulA, which is induced as part of the DNA-damage response (Huisman et al., 1980), inhibits bacterial cell division by interacting with F tsZ and preventing its polymerization (Mukherj ee et al., 1998; Trusca et al., 1998). In sulA and 31413 mutants, cell division is not blocked after DNA-damaging treatments as in wild type (Jones and 16 Holland, 1985). Induction of the expression of the wild-type sulA gene with the lac promoter by IPTG is sufficient to cause inhibition of cell division (Huisman et al., 1984). This inhibition can be suppressed by mutations in the suIB gene if sulA is not highly expressed (Huisman et al., 1984). Cloning of the 31418 gene indicated that it encodes FtsZ (Jones and Holland, 1985). In the absence of F tsZ or in the 314181 14 mutant, SulA is extremely unstable with a half-life of only 3 min, in contrast to its normal half life of 12 min in the presence of F tsZ (Jones and Holland, 1985). These data suggest that SulA interacts directly with FtsZ in vivo to block cell division. In the presence of GTP and Mg2+, SulA protein was shown to interact with F tsZ and form a stable complex in a molar ratio of approximately one to one (Higashitani et al., 1995). The role of GTP cannot be replaced by GDP or GTP-y-S, suggesting that hydrolysis of GTP is required for the interaction between SulA and FtsZ (Higashitani et al., 1995). SulA inhibits the polymerization of F tsZ but not the polymerization of purified SulB mutant protein (Mukherjee et al., 1998; Trusca et al., 1998). By alanine scanning mutagenesis, the central region of SulA was shown to be essential for the FtsZ polymerization inhibition (Higashitani et al., 1997). By using deletion mutants, residues 3-27 and the 21 residues at the C-terminal end were shown not to be required for the inhibition, while the mutant protein lacking N-terminal residues 3-47 or 34 residues at the C-terminal end was inactive (Higashitani et al., 1997). SulA forms into a dimer either alone or in complex with FtsZ (Cordell et al., 2003). SulA blocks the polymerization of F tsZ and inhibits cell division by binding to the T7 loop surface of F tsZ (opposite to the nucleotide-binding site) without inducing conformational change (Cordell et al., 2003). 17 (Mi; ATP termir of cell or a C - and An 1 (Maple e targeted t Cl’llOIOpla; GF P prote ClllOl’OplaS Chloroplast many FtsZ filTlCllonS Sl Hom. prcdlCIed Wll filflCllOnS in C dlspenSab] egc SulA is induced by DNA damage and other stresses and has a very short half-life (Mizusawa and Gottesman, 1983). SulA is degraded by Lon protease and HslVU, an ATP-dependent protease (Mizusawa and Gottesman, 1983; Schoemaker et al., 1984; Kanemori et al., 1999; Seong et al., 1999). The cleavage is preferred at the central and C- terminal regions of SulA, which are important for the interaction with FtsZ and inhibition of cell division (N ishii and Takahashi, 2003). Either the deletion of the C-terminal region or a C-terminal addition of a protein tag can greatly increase the stability of SulA (Ishii and Amano, 2001). Homologs of SulA are also found in plants and play a role in chloroplast division (Maple et al., 2004; Raynaud et al., 2004). The gene product of AtSulA is predicted to be targeted to the chloroplast. The expression pattern of AtSulA is similar to that of other chloroplast division genes, such as F tsZs, MinD and MinE. In transgenic plants, AtSulA- GFP protein is imported into chloroplasts and the overexpression of AtSulA inhibits chloroplast division in various cell types. Overexpression of AtSulA can overcome the chloroplast division defect caused by overexpresion of F tsZ. Since the formation of too many FtsZ polymers can block chloroplast division, these data suggest that SulA in plants functions similarly to SulA in bacteria by preventing FtsZ polymerization. Homologs of Lon and HslVU are also found in plants by BLAST search and are predicted with high scores to be chloroplast-targeted (Gao, unpublished). But their functions in chloroplast division have not been studied. Since chloroplasts have a non- dispensable genome, it is also important for chloroplasts to repair DNA damage before 18 they . f in plc 11.an ago. Si evoluti plants (" machint ('Reumai beht'een outer em- ellkar}-’oti. division 0, Componen. FtsZ] In Cy,- FtsZ in Plants closely TClat e d Mc‘lndfeut', 2O they divide. It is likely that the functions of the Lon and HslVU proteases are conserved in plants. 11. Innovations in the chloroplast division machinery during evolution The endosymbiotic origin of chloroplasts occurred more than one billon years ago. Since then, chloroplasts have experienced great changes in the long history of evolution and acquired new protein components to function as an organelle in modern plants (Dyall et al., 2004). For example, chloroplasts have evolved protein import machinery complexes to import thousands of proteins encoded by the nuclear genome (Reumann et al., 1999; Reumann and Keegstra, 1999). Cyanobacteria have a cell wall between the inner and outer membrane, but there is no cell wall between the inner and outer envelope of chloroplasts and the outer envelope of chloroplasts has acquired many eukaryotic properties (Douce and J oyard, 1990; Dyall et al., 2004). Moreover, the division of chloroplasts must be under control of the host cells. Therefore, new components of the chloroplast division machinery must have evolved to adapt to the environment in host cells. Fle In cyanobacteria, there is only one type of FtsZ. However, there are two types of FtsZ in plants: Ftle and FtsZZ (Osteryoung and McAndrew, 2001). Ftle and FtsZ2 are closely related and very similar to the FtsZs in cyanobacteria (Osteryoung and McAndrew, 2001). FtsZs in most bacteria have a conserved N-terminal GTPase domain, 19 C -te sequ FtsZ Yan t (Oste in Fts. Ftle indicatc' evolutic division FlrSL the form a m “In and th KOksharO‘ may be 10p chJomplast funCthns f0 CterTm.néll e C-terminal domain and the C-terminal core domain (Lowe and Amos, 1998; Mosyak et al., 2000). The C-terminal core domain at the extreme end contains a highly conserved sequence motif, D/E-I/V-P-X-F/Y-L, which is required for direct interactions between F tsZ and two other essential cell division proteins, ZipA and F tsA (Mosyak et al., 2000; Yan et al., 2000; Lowe and van den Ent, 2001). All three domains are conserved in FtsZZ (Osteryoung and McAndrew, 2001). However, the C-terrninal core domain is not present in Ftle of plants (Osteryoung and McAndrew, 2001). Similar to FtsZ2, suppression of F tle expression or overexpression of Ftle causes severe division phenotypes, suggesting that Ftle and FtsZ2 are both essential for chloroplast division, but they may have different roles (Osteryoung etal., 1998; Stokes et al., 2000). Phylogenetic analysis indicates that F tle and F tsZZ may have diverged at the very early stages of plant evolution, further supporting the distinct role of Ftle in the evolution of the chloroplast division machinery (Stokes and Osteryoung, 2003; Reusing et al., 2004). There are three possible explanations for the role of F tle in chloroplast division. First, the appearance of Ftle during evolution may provide spacers for F tsZ proteins to form a ring suitable for chloroplasts. Since the diameter of bacterial cells is typically ~l um and the diameter of chlorOplasts is typically 4-8 pm (Osteryoung et al., 1998; Koksharova and Wolk, 2002), one can envision that if there is only F tsZZ in plants, there may be topological hindrances for FtsZZ proteins to form a ring as large as required for chloroplast division. Second, the C-terminal end of F tle may have acquired new functions for the regulation of its activity. This is supported by the fact that the lack of the C-terminal end of Ftle causes an intermediate division phenotype (Deena Kadirj an- 20 Kolbz provit This s FtsZ] h}poti ARC3 cell (P) encodes 2004). 1 and FtsZ (MORX‘; (PH’SK) . in F152 ar 2004). this from that c alleles ofth 2004), ARC Preliminary, chloropjast ir. ‘31" 20041 in work on lhe Cl". Kolbach, personal communication). The third possibility is that the role of Ftle is to provide enough proteins to be assembled into a ring together with FtsZZ in chloroplasts. This seems to be less likely and can be tested by replacing F tle with F tsZZ. If in the F tsZI knockout plants a transgene of F tsZZ can rescue the mutant phenotype, the third hypothesis will be supported and the first and the second hypothesis will be invalidated. ARC3 arc3 is a chloroplast division mutant in Arabidopsis with 10-20 chloroplasts per cell (Pyke and Leech, 1994; Marrison et al., 1999). Cloning of ARC3 indicated that it encodes an F tsZ-like protein and that the arc3 mutant is a null allele (Shimada et al., 2004). The N-terminus of ARC3 shares low similarity with the GTPase domains of Ftle and FtsZZ. The C-terminus of ARC3 has a membrane-occupation-and-recognition—nexus (MORN) repeat motif similar to that of phosphatidylinositol-4-phosphate S-kinases (PIPSK) (Shimada et al., 2004). Because many residues essential for the GTPase activity in FtsZ are not conserved in ARC3 (Osteryoung and McAndrew, 2001; Shimada et al., 2004), this protein may not have GTPase activity and its function might be quite different fi'om that of Ftle and FtsZZ. Since chloroplast division is still occuring in the null alleles of the arc3 mutant (Pyke and Leech, 1994; Marrison et al., 1999; Shimada et al., 2004), ARC3 may have evolved to modify the chloroplast division machinery. Preliminary data show that ARC3 is a soluble protein located on the outer surface of the chloroplast in a ring-like structure at the early stage of chloroplast division (Shimada et al., 2004). These results are very surprising and it cannot yet be explained how ARC3 can work on the cytosolic side of the chloroplast envelope. However, the data on ARC3 21 top exp role ARC shape al., 19 may be 1994; P gene by mUlant ; Site (Gac ARC5 m results ha al., 2003 j COmplex It be unjqu e t (Figure 2). . Billet organellar flSS topology and localization were very preliminary (Shimada et al., 2004). Further experiments are needed to confirm these data before we can gain more insights about the role of ARC3 in chloroplast division. ARC5 arcS is a chloroplast division mutant in Arabidopsis with 1-15 enlarged dumbbell- shaped chloroplasts per cell (Pyke and Leech, 1994; Robertson et al., 1996; Marrison et al., 1999; Gao et al., 2003). The phenotype of arc5 indicated that the ARC5 gene product may be involved in the constriction of chloroplasts during division (Pyke and Leech, 1994; Robertson et al., 1996; Marrison et al., 1999; Gao et al., 2003). I cloned the ARC5 gene by positional cloning (Gao et al., 2003). A GFP-ARC5 fusion gene rescues the arc5 mutant phenotype and the gene product is localized to a ring at the chloroplast division site (Gao et al., 2003). Chloroplast import and protease protection assays indicate that the ARC5 ring is positioned on the outer surface of the chloroplast (Gao et al., 2003). Similar results have also been observed for the homolog of ARC5 in a red alga (Miyagishima et al., 2003). Thus, ARC5 is the first cytosolic component of the chloroplast division complex to be identified. ARC5 is related to a group of dynamin-like proteins shown to be unique to plants by phylogenetic analysis (Gao et al., 2003; Miyagishima et al., 2003) (Figure 2). It has no obvious counterparts in prokaryotes, suggesting that it evolved from a dynamin-related protein present in the eukaryotic ancestor of plants. Dynarnin and its relatives are large GTPases that participate in a variety of organellar fission and fusion events in eukaryotes, including budding of endocytic and 22 Golgi- forum 1993; Jensen dynam interco: Purifier tubes th Takei et djvnamin membrar Pll-lil\'e Sl reminisce mbular in‘ dense ling YESUIIS, in E Slippon [he Hlnshaw, ll In Cor r elated prOIej; Golgi-derived vesicles, mitochondrial fission, mitochondrial fusion, and plant cell plate formation (Chen et al., 1991; van der Bliek and Meyerowitz, 1991; Wilsbach and Payne, 1993; Gu and Verma, 1996; Pelloquin et al., 1998; Bleazard etal., 1999; Sesaki and Jensen, 1999; Hinshaw, 2000; Danino and Hinshaw, 2001). Structural analysis of dynamin indicates that it spontaneously self-assembles into rings and stacks of interconnected rings (Hinshaw and Schmid, 1995 ; Kelly, 1995; Carr and Hinshaw, 1997). Purified recombinant dynamin binds to a lipid bilayer in a regular pattern to form helical tubes that constrict and vesiculate upon GTP addition (Sweitzer and Hinshaw, 1998; Takei et al., 1998). In the shibire mutant of Drosophila, which has a mutation in the dynamin-I gene, many pit-like structures were observed to accumulate on the plasma membrane near presynaptic sites (Kosaka and Ikeda, 1983a, b). The neck portion of the pit-like structures was surrounded by cytoplasmic dense material, about 10 nm thick, reminiscent of a "collar" (Kosaka and Ikeda, 1983a). In GTP-y-S-treated nerve terminals, tubular invaginations of the plasma membrane were surrounded by transverse electron- dense rings that were positive for dynamin immunoreactivity (Takei et al., 1995). These results, in addition to the finding that dynamin is capable of generating force, strongly support the hypothesis that dynamin is active in the fission reaction (Sweitzer and Hinshaw, 1998). Thus, it was suggested that dynamin is a mechanoenzyme directly involved in membrane remodeling when the vesicles are pinched off. In contrast to most of the other members of the dynamin family, two dynamin- related proteins, F20 and Mgml, are involved in membrane fusion in fungi and animals 23 Figure 2 Phylogenetic analysis of dynamin-related proteins with an unrooted neighbor-joining tree. Bootstrap values are shown at selected nodes with <50% bootstrap support. yjdA and engA in E. coli are GTPases involved in ribosome function and are used as the outgroup here. ARC5 is involved in chloroplast division. The function of ARC5H in rice and Arabidopsis is not clear. At1g59610 and At1g10290 are involved in the trafficking of Golgi—derived vesicles. At1g14830, At3960190, At2944590 and At3g61760 are also phragmoplastins and involved in cell plate formation in Arabidopsis. DRP-1, Dnm1, Dnm1 p, At4g33650, AT2914120 and rice ADL2 are involved in mitochondrial division. Dynamin-2 and dynamin-1 are involved in the budding of clathrin-coated endocytotic vesicles. Mx1 and Mx2 are Myxovirus resistance protein. The function of At1g60540 and At1g60500 is unknown. MSP1and Mgm1 p mediates the fusion of the inner mitochondrial membrane during mitochondrial fusion. AtFZL is an FZO-like protein in Arabidopsis and is involved in the morphogenesis and division of chloroplasts. FZO, human mitofusin2, rat FZO1A, rat mitofusin1 and on1 p are involved in the fusion of the outer mitochondrial membrane during mitochondria fusion. 24 E. coli yjdA E. coli engA ARC5 1000 Rica ARC5H Arabidopsis ARC5H 1000 r— At1 959610 ._ At1g10290 1000l , 997 806 1000‘ 90__e_ 772‘ 914 . 1000‘ At1g14830 1000 At3960190 5 At2944590 115942000 soybean Phragmoplastin tobacco Phragmoplastln At3g61760 C. elegans DRP-1 Human Dnm1 Yeast Dnm1 p - 974 1000i 1 0—— At4933650 At29141 20 Rice ADL2 1 000 Human Dynamin-Z Human Dynamln-1 Human Mx1 1 000 H777 Dog Mx2 1 000 ———— At1960540 At1 960500 Fission yeast MSP1 Budding yeast Mgm1p AtFZL Drosophila FZO ' 1000 rfl 1ooor— Human mitofusinZ 1 000 l— Rat FZO1A Rat mitofusin1 Budding yeast on1p 25 199 7; outer r Mgm1. ’ the mlll inner m. coiled-ct between between 1 2004). H _\ generating the other 5: Mos domain, a m dOmain (GEl GTPase 3Cliv SWEitzer and, interactions be (Smlmol’a et al dim“ { Salim (Hales and Fuller, 1997; Sesaki and Jensen, 1999; Shaw and Nunnari, 2002; Sesaki et al., 2003). Mutations in F 20 and Mgrnl block mitochondrial fusion and result in a fragmented mitochondrial morphology and loss of mitochondrial DNA (Hales and Fuller, 1997; Sesaki and Jensen, 1999; Sesaki et al., 2003). F20 is directly inserted into the outer membrane of mitochondria by two transmembrane domains (Westermann, 2003). Mgm1, which is closely related to the mitochondrial fission protein Dnm1, is located in the mitochondrial intermembrane space and involved in the fusion of the mitochondrial inner membrane (Wong et al., 2000; Wong et al., 2003). The GTPase domain and two coiled-coil domains of FZO are exposed to the cytosol (Westermann, 2003). Interactions between the coiled-coil domains of F 20 molecules was proposed to mediate the tethering between membranes from different mitochondria (Westermann, 2003; Koshiba et al., 2004). Hydrolysis of GTP may mediate the fusion of mitochondrial outer membranes by generating force for the movement of proteins and opening the attached membranes on the other side (Westermann, 2003; Meeusen et al., 2004). Most of the dynamin-related proteins have four conserved domains: a GTPase domain, a middle-domain, a Pleckstrin homology (PH) domain and a GTPase effector domain (GED) (Hinshaw, 2000). Dynamin is proposed to be a mechano-enzyrne and its GTPase activity may be involved in the generation of force (Warnock and Schmid, 1996; Sweitzer and Hinshaw, 1998). The middle-domain is involved in protein-protein interactions between dynamin molecules and is also essential for the function of dynamin (Smirnova et al., 1999). The PH domain is involved in the specific membrane binding of dynamin (Salim et al., 1996; Zheng et al., 1996). The GED domain interacts with the 26 GTP: more GTPa relatet thereft ditTere ring is . protein: evolutic cell divi: machine; Ill. Com l Sir bauen'ai a, assOCiated é Cl'anobaCter genes fol. Hit ChlomplaSI d GTPase domain and modifies its activity (F ukushima et al., 2001). ARCS is relatively more divergent from dynamin than other dynamin-related proteins (Gao et al., 2003). The GTPase domain and the middle domain of ARC5 can be aligned with other dynamin- related proteins better than can the PH and GED domains (Gao et al., 2003). ARC5 may therefore generate force to facilitate chloroplast division, but it functions at a site quite different fiom those of other dynamin-related proteins, and the diameter of the ARC5 ring is much larger than the diameter of the rings formed by other dynamin-related proteins. These results indicate that the chloroplast division machinery is of mixed evolutionary origin and that it shares structural and mechanistic similarities with both the cell division machinery of bacteria and the dynamin-mediated organellar fission machineries of eukaryotes. III. Components that were probably lost or replaced by others during evolution. Since chloroplasts lost their autonomy during their evolution, some of the bacterial functions are apparently no longer important for chloroplast function and the associated genes were lost. For example, nitrogen fixation is important for the survival of cyanobacteria but not important for the function of chloroplasts, so that many of the genes for nitrogen fixation were lost in evolution. This may also be partly true for chloroplast division genes. The morphology of plant chloroplasts is somewhat different from that of cyanobacteria. It is not surprising that some of the cell division proteins found in cyanobacteria are not conserved in plants. FtsA, F tn6, F tsI and F tsW may be examples. 27 F tsA Park-i simi la simila: also re partiallj have the This kint but also I Here I my and shape Plants. FtsA and MreB F tsA belongs to a superfamily of ATPases that includes FtsA, DnaK, Hsp70, ParM, MreB, actin and hexokinase (Bork et al., 1992; Lowe et al., 2004). They all have similar structures and ParM and MreB have been shown to form filaments structurally similar to actin filments (van den Ent et al., 2002). FtsA, DnaK, Hsp70 and MreB are also very similar in sequence and may be homologs in different species with at least partially similar fimctions (Amos et al., 2004; Lowe et al., 2004). Sometimes, they may have the same function but have been annotated with different names in different species. This kind of confiision may be due to conservation and variations among these proteins but also to the fact that they were studied in different species from different perspectives. Here I will focus on FtsA and MreB, which function respectively in bacterial cell division and shape determination, and HSP7OS, the homologs of cyanobacterial FtsA or MreB in plants. FtsA was initially found by an fts screen (Wijsman and Koopman, 1976). FtsA is a bacterial cell division protein localized to a ring at the division site (Ma et al., 1996); Its localization depends on FtsZ, whereas FtsZ localization does not depend on FtsA (Addinall and Lutkenhaus, 1996). FtsA has no membrane-binding site and interacts with the C-terminal core domain of FtsZ (Ma and Margolin, 1999; Yan et al., 2000). In some ftsZ mutants with mutations in the coding region of the C-terminal core domain, FtsZ can form a ring but cannot recruit F stA (Ma and Margolin, 1999). In the absence of FtsA, several other cell division proteins cannot be localized to the division site (Errington et 28 al.. 30( be not t and Ft: ofthis r species a al.. 200 l; pefipheo' Knocking al., 2001). rePeat Sim ("an den E Shows th at in am“ (Va iImportant n Illler‘acn'OnS Hom, OereB or F al., 2003; Rico et al., 2004). F tsA can be phosphorylated and bind ATP, but this seems to be not essential for its function (Sanchez et al., 1994). The molecular ratio between F tsA and FtsZ in E. coli is 1:100 (Dai and Lutkenhaus, 1992; Dewar et al., 1992). Alteration of this ratio will affect cell division. MreB regulates the rod shape of bacterial cells in Bacillus subtilis and many other species and is believed to be the prokaryotic form actin (Jones et al., 2001; van den Ent et al., 2001a; Esue etal., 2005). MreB forms helical filamentous structures that surround the periphery of the cell, presumably just under the cytoplasmic membrane (Egelman, 2003). Knocking out of MreB in rod-shaped bacteria results in a spherical morphology (Jones et al., 2001). In vitro, MreB can assemble into two-stranded protofilarnents with a subunit repeat similar to that of F -actin, except that the strands do not twist around each other (van den Ent et al., 2001a; van den Ent et al., 2001b). The crystal structure of MreB shows that its folding is also very similar to that of actin, except that there are insertions in actin (van den Ent et al., 2001a; van den Ent et al., 2001b). These insertions are important for the allosteric interactions within the actin subunit, subunit—subunit interactions in the filament, and interactions with other proteins. Homologs of MreB or F tsA are also present in cyanobacteria and plants. The role of MreB or FtsA homologs in cyanobacteria is unknown. However, their homologs in plants are called HSP70. There are multiple copies of HSP70 genes related to chloroplast function. In plant cells, when proteins are imported into chloroplasts, the HSP7OS on the cytosolic side of the chloroplast import machinery may help to recognize the chloroplast 29 transit requir stromz 1995: . mRNA centres mostly of F tsA chloropl F tn 6 [TanSposo SP- Strain j The fimcti not all the ‘Z‘Tinobame PTOIeins in ‘ f 3126 mUtam: the PFOIein S There is alSo Important for transit peptide and unfold the proteins, HSP70s in the intermembrane space may be required for the translocation of chloroplast-targeted proteins, and the HSP7OS on the stromal side may help the translocation and refolding of the proteins (Gray and Row, 1995; J ackson-Constan et al., 2001; Jarvis and S011, 2002). Based on EST data, the mRNAs of these HSP70 genes are much more abundant than those of FtsZ genes, in great contrast to the molecular ratio between F tsA and F tsZ in bacteria. Also, chloroplasts mostly have a spherical shape, similar to the bacteria that lack MreB. Thus, the homologs of F tsA or MreB in plants, chloroplast-targeted HSP7OS, may not have a role in chloroplast division or morphology. Ftn6 F m6 is a cyanobacteria-specific cell division protein. Knockout of F tn6 either by transposon insertion or homologous recombination affects cell division in Synechococcus sp. strain PCC 7942 and Anabaena sp. strain PCC 7120 (Koksharova and Wolk, 2002). The function of F m6 is unknown and there is no homolog of Ftn6 in other bacteria. Since not all the known cell division proteins in E. coli are found to have homologs in cyanobacteria and vice versa, F tn6 may either have a role similar to some division proteins in E. coli or have a role unique to cyanobacterial cell division. The phenotype of ftn6 mutants is not as severe as that of fth mutants (Koksharova and Wolk, 2002) and the protein sequences of F m6 are not well conserved in different cynaobacteria species. There is also no homolog of Ftn6 in plants, suggesting that the function of F m6 is not important for chloroplast division and that Ftn6 was lost during chloroplast evolution. 30 F tsl F1 for cross- (Nakamm cyanobact domain, a 2004). The staining ant division sitc- Ftsl blocks l al., 1997; W: membrane ar. Weiss, 2002 ). interaction(_s ) t0 the division dingo“ (Weis: of the cell fer s, dam Suggest tha CthI‘oPIE cl'élnobacteria H c Obsm'ed in seed pl Ftsl F tsl, also called penicillin-binding protein 3 (PBP3), is a transpeptidase required for cross-linking of the peptidoglycan cell wall at the bacteria cell division site (Nakamura et al., 1983; Weiss et al., 1997). Ftsl is conserved in many bacteria including cyanobacteria (Margolin, 2000). It has a small cytoplasmic domain, a transmembrane domain, a domain of unknown function, and a transpeptidase domain (Wissel and Weiss, 2004). The last two domains reside in the periplasm (Wissel and Weiss, 2004). Immuno- staining and a functional GFP fusion protein indicated that F tsI is localized to the cell division site (Weiss et al., 1997; Weiss et al., 1999). Inhibition of the catalytic activity of Ftsl blocks bacteria cell division but doesn’t affect the localization of Ftsl (Pogliano et al., 1997; Weiss et al., 1999). Localization of Ftsl to the division site requires its membrane anchor, FtsZ, FtsW, FtsA, FtsQ, and FtsL (Weiss et al., 1999; Mercer and Weiss, 2002). It was suggested that the catalytic activity of PBP3 is stimulated by interaction(s) with other division proteins (Eberhardt et al., 2003). GFP-Ftsl is localized to the division site during the later stages of cell growth and throughout the process of division (Weiss et al., 1999). Unconstricted FtsZ rings are stably trapped at the midpoint of the cell for several generations after inactivation of Ftsl (Pogliano et al., 1997). These data suggest that Ftsl functions in the late stage of cell division. Chloroplasts do not have a peptidoglycan wall in the intermembrane space as do cyanobacteria. However, penicillin can inhibit chloroplast division in the moss Physcomitrella patens (Kasten, 1997; Katayama et al., 2003). This phenomenon is not observed in seed plants, such as tomato and Arabidopsis (Kasten, 1997). So far, it is 31 unclear WI Arabidops land plants moss requi can OVCI‘CO F tsW F ts ll domains and the N- and th FtsW is also ' FtsW is some mutations in r is localized to al., 2002). In If that F ts W may Mill-Zing F tsZ unclear whether F tsI is conserved in moss or not. But in the sequenced rice and Arabidopsis genomes, no F tsI homologs are found. This indicates that, at least in higher land plants, Ftsl probably was lost during evolution. If it is true that lower plants like moss require Ftsl for chloroplast division, it will be interesting to learn how higher plants can overcome the loss of Ftsl. FtsW FtsW is an essential bacterial cell division protein with 10 transmembrane domains and a large periplasmic loop (Ishino et al., 1989; Lara and Ayala, 2002). Both the N- and the C terminus of F tsW are located in the cytoplasm (Lara and Ayala, 2002). FtsW is also well conserved in cyanobacteria (Margolin, 2000). Although the sequence of FtsW is somewhat similar to that of the bacterial cell shape-deterrnining protein RodA, mutations in FtsW only affect cell division and not cell shape (Khattar et al., 1994). FtsW is localized to the division site and interacts with F tsZ through its C-terminal tail (Datta et al., 2002). In the absence of FtsW, the formation of the FtsZ ring is affected, indicating that F tsW may have a role similar to that of another membrane protein, ZipA, in stabilizing F tsZ filaments. F tsW is also required for the localization of FstI to the cell division site (Mercer and Weiss, 2002). Thus, FtsW may link FtsZ ring formation in the cytoplasm to peptidoglycan synthesis in the periplasm at the bacteria cell division site. Neither FtsW nor RodA was found to have a homolog in Arabidopsis or rice. Since there is no F tsI in higher plants, FtsW may not be required to be conserved for the localization Ftsl. Moreover, the homolog of Fth, ARC6, is conserved in plants (Vitha 32 et al.. 200 and its C-l a role in st possibly re IV. Perspec The l cell division proteins and ; chlorOplast d 1‘ F182 p ChlorOplasts ( F Ellington et al, ling series as a et al., 2003). ARC6 has a transmembrane domain, its N-terminus is located in the stroma, and its C-terminus is located in the intermembrane space. ARC6 is also proposed to have a role in stabilizing FtsZ filaments at the chloroplast division site (Vitha et al., 2003) possibly replacing the fimction of F tsW. IV. Perspective The last decade has seen rapid growth of the knowledge in the areas of bacterial cell division and chloroplast division. The identification of many bacterial cell division proteins and some chloroplast division proteins indicates that the evolution of the chloroplast division machinery has involved both conservation and innovation. FtsZ probably is the most important division protein for both bacteria and chloroplasts (Rothfield et al., 1999; Margolin, 2000; Osteryoung and McAndrew, 2001; Errington et al., 2003). F tsZ polymerizes and forms a ring at the division site. The FtsZ ring serves as a scaffold for the localization of many other division proteins. The Min system regulates the localization of the F tsZ ring to the mid-cell or mid-chloroplast (Colletti et al., 2000; Itoh et al., 2001; Lutkenhaus, 2002). SulA inhibits the assembly of FtsZ as part of the SOS system in bacteria (Lowe et al., 2004) and its homolog in plants also has a role in chloroplast division (Maple et al., 2004; Raynaud et al., 2004). F m2 is a cyanobacteria-specific cell division protein with a DnaJ domain (Koksharova and Wolk, 2002); it is also conserved in plants (Vitha et al., 2003). On the other hand, many bacteria cell division proteins may have been lost during evolution as chloroplasts 33 evoll"?d : kno\Vl'l- ‘\ machineri Wt were know comparall“ al., 2000; ltc mutageneSis protein of e“: ARC5H was Therefore, AF family-related 2001), ARC5 1 generating fOF C Bacteria known to be in\' these proteins art cyanobacterial ce l'Koksharora and SPECific cell divisi dll iston proteins It evolved from bacteria into organelles. So far, only a few chloroplast division proteins are known. Most of these proteins are derived fiom the cynaobacteria] cell division machinery. When I started to clone ARC5 in the fall of 2001, only FtsZ, MinD and MinE were known to be involved in chloroplast division and they had all been identified by a comparative genome approach (Osteryoung et al., 1998; Strepp et al., 1998; Colletti et al., 2000; Itoh et al., 2001). arc5 is a chloroplast division mutant induced by EMS mutagenesis. Map-based cloning of ARC5 indicated that it encodes a dynamin-related protein of eukaryotic origin (Gao et al., 2003) (see also chapter II). A homolog of ARC5, ARC5H, was shown not to have a function in chloroplast division (see chapter III). Therefore, ARCS is important but not essential for chloroplast division. Since dynamin family-related proteins are proposed to be mechanoenzymes (Danino and Hinshaw, 2001), ARC5 may have evolved from the host to facilitate the division of chloroplasts by generating force from the outside of the organelles. Bacterial cell division has been studied mostly in E.. coli. About 15 proteins are known to be involved in cell division in E. coli (Margolin, 2000). However, not all of these proteins are conserved in cyanobacteria (Margolin, 2000). Also, homologs of the cyanobacterial cell division proteins F m2 and Ftn6 are not found in other bacteria (Koksharova and Wolk, 2002). This indicates that different kinds of bacteria may have specific cell division proteins. Therefore, identification of more cynobacteria—specific cell division proteins may help to identify new chloroplast division proteins by comparative 34 genomics. Ti saturation (’Pj also help to ic (Miyagishima cause a phenot that interact wi yeast two-hybr proteins identit will be discove In chap: in [h3'"lak0id mc invoked in mit. “'CSIEI‘mann, 2‘ ofFZO, Sugges Protein localize genomics. The previous mutant screening of chloroplast division mutants was far from saturation (Pyke and Leech, 1994; Marrison et al., 1999). A further mutant screening may also help to identify chloroplast division proteins that are not derived from cyanobacteria (Miyagishima et al., 2005). However, mutations in the genes with redundancy may not cause a phenotype and mutations in essential genes may be lethal. Identifying proteins that interact with the known chloroplast division proteins by biochemical methods and yeast two-hybrid assays can overcome this problem. With more chloroplast division proteins identified in the future, we can expect that more proteins of eukaryotic origin will be discovered. In chapter IV, F ZL, an F ZO-like protein in Arabidopsis was shown to have a role in thylakoid morphogenesis and chloroplast division. FZO is a dynamin-like protein involved in mitochondrial fusion in fungi and animals (Sesaki and Jensen, 1999; Westerrnann, 2003; Meeusen etal., 2004). The domain structure of FZL is similar to that of FZO, suggesting that it may have similar biochemical function. FZL is a membrane protein localized to both the thylakoid and the chloroplast inner envelope, but not to mitochondria. Loss of function of F ZL results in disorganization of thylakoid stacks and defects in chloroplast division. Overexpression of FZL causes very long thylakoid sacs with less stacking. Thus, the morphogenesis of thylakoids may also affect the division of chloroplasts. 35 Lite Addh Amos. Literature cited Addinall, S.C., and Lutkenhaus, J. (1996). FtsA is localized to the septum in an F tsZ- dependent manner. J Bacteriol 178, 7167-7172. Amos, L.A., van den Ent, F., and Lowe, J. (2004). Structural/functional homology between the bacterial and eukaryotic cytoskeletons. Curr Opin Cell Biol 16, 24- 3 1 . Arabidopsis_Genome_Initiative. (2000). Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408, 796-815. Beech, P.L., Nheu, T., Schultz, T., Herbert, S., Lithgow, T., Gilson, RR, and McFadden, GJ. (2000). Mitochondrial FtsZ in a chromophyte alga. Science 287, 1276-1279. Bi, E., and Lutkenhaus, J. (1993). Cell division inhibitors SulA and MinCD prevent formation of the FtsZ ring. J Bacteriol 175, 1118-1125. Bi, ER, and Lutkenhaus, J. (1991). FtsZ ring structure associated with division in Escherichia coli. Nature 354, 161-164. Bleazard, W., McCaffery, J .M., King, E.J., Bale, S., Mozdy, A., Tieu, Q., Nunnari, J., and Shaw, J.M. (1999). The dynamin-related GTPase Dnm1 regulates mitochondrial fission in yeast. Nat Cell Biol 1, 298-304. Bork, P., Sander, C., and Valencia, A. (1992). An ATPase domain common to prokaryotic cell cycle proteins, sugar kinases, actin, and hsp70 heat shock proteins. Proc Natl Acad Sci U S A 89, 7290-7294. Bramhill, D. (1997). Bacterial cell division. Annu Rev Cell Dev Biol 13, 395-424. Carr, J.F., and Hinshaw, J.E. (1997). Dynamin assembles into spirals under physiological salt conditions upon the addition of GDP and garnma-phosphate analogues. J Biol Chem 272, 28030-28035. Cha, J.H., and Stewart, G.C. (1997). The divIV A minicell locus of Bacillus subtilis. J Bacteriol 179, 1671-1683. Chen, M.S., Obar, R.A., Schroeder, C.C., Austin, T.W., Poodry, C.A., Wadsworth, S.C., and Vallee, RB. (1991). Multiple forms of dynamin are encoded by shibire, a Drosophila gene involved in endocytosis. Nature 351, 583-586. Chu, K.H., Qi, J., Yu, LG, and Anh, V. (2004). Origin and phylogeny of chloroplasts revealed by a simple correlation analysis of complete genomes. Mol Biol Evol 21, 200-206. 36 C ollett Cordell ( / Dai, K, d; Danino, l Oi Datta, P,, Fts de Boer, P divi d9 Baer, P. tOpo Plate: de Boer, Put the si Coli. ,‘ de Boer, P.A PTOIeii Escher Dean, C5 and 1- Cellu ofdjm,‘ 904% n. .. war, SJ, BE mltlatiOn 6316. Dinkins, R., Red Arabidops Colletti, K.S., Tattersall, E.A., Pyke, K.A., Froelich, J.E., Stokes, K.D., and Osteryoung, K.W. (2000). A homologue of the bacterial cell division site- determining factor MinD mediates placement of the chloroplast division apparatus. Curr Biol 10, 507-516. Cordell, S.C., Robinson, E.J., and Lowe, J. (2003). Crystal structure of the SOS cell division inhibitor SulA and in complex with F tsZ. Proc Natl Acad Sci U S A 100, 7889-7894. Dai, K., and Lutkenhaus, J. (1992). The proper ratio of F tsZ to F tsA is required for cell division to occur in Escherichia coli. J Bacteriol 174, 6145-6151. Danino, D., and Hinshaw, J.E. (2001). Dynamin family of mechanoenzymes. Current Opinion in Cell Biology 13, 454-460. Datta, P., Dasgupta, A., Bhakta, S., and Basu, J. (2002). Interaction between FtsZ and FtsW of Mycobacterium tuberculosis. J Biol Chem 277, 24983-24987. de Boer, P., Crossley, R., and Rothfield, L. (1992a). The essential bacterial cell- division protein FtsZ is a GTPase. Nature 359, 254-256. de Boer, P.A., Crossley, R.E., and Rothfield, L]. (1989). A division inhibitor and a topological specificity factor coded for by the minicell locus determine proper placement of the division septum in E. coli. Cell 56, 641-649. de Boer, P.A., Crossley, R.E., and Rothfield, L.I. (1992b). Roles of MinC and MinD in the site-specific septation block mediated by the MinCDE system of Escherichia coli. J Bacteriol 174, 63-70. de Boer, P.A., Crossley, R.E., Hand, A.R., and Rothfield, LL (1991). The MinD protein is a membrane ATPase required for the correct placement of the Escherichia coli division site. Embo J 10, 4371-4380. Dean, C., and Leech, RM. (1982). Genome expression during normal leaf development. 1. Cellular and chloroplast numbers and DNA, RNA and protein levels in tissues of different ages within a seven-day old wheat leaf. PLANT PHYSIOLOGY 69, 904-910. Dewar, S.J., Begg, K.J., and Donachie, W.D. (1992). Inhibition of cell division initiation by an imbalance in the ratio of F tsA to F tsZ. J Bacteriol 174, 6314- 63 16. Dinkins, R., Reddy, M.S., Leng, M., and Collins, GB. (2001). Overexpression of the Arabidopsis thaliana MinDl gene alters chloroplast size and number in transgenic tobacco plants. Planta 214, 180-188. 37 DOUC99 J Douglas 1 Durnfo ( l Dyall, S t Eberha Douce, R., and Joyard, J. (1990). Biochemistry and fimction of the plastid envelope. Annu Rev Cell Biol 6, 173-216. Douglas, SE. (1998). Plastid evolution: origins, diversity, trends. Curr Opin Genet Dev 8, 655-661. Durnford, D.G., Deane, J.A., Tan, 8., McFadden, G.I., Gantt, E., and Green, B.R. (1999). A phylogenetic assessment of the eukaryotic light-harvesting antenna proteins, with implications for plastid evolution. J Mol Evol 48, 59-68. Dyall, S.D., Brown, M.T., and Johnson, P.J. (2004). Ancient invasions: from endosymbionts to organelles. Science 304, 253-257. Eberhardt, C., Kuerschner, L., and Weiss, BS. (2003). Probing the catalytic activity of a cell division-specific transpeptidase in vivo with beta-lactams. J Bacteriol 185, 3726-3734. Edwards, D.H., and Errington, J. (1997). The Bacillus subtilis DivIVA protein targets to the division septum and controls the site specificity of cell division. Mol Microbiol 24, 905-915. Egelman, EH. (2003). A tale of two polymers: new insights into helical filaments. Nat Rev Mol Cell Biol 4, 621-630. Ellis, J.L., RM. (1985). Cell-size and chloroplast size in relation to chloroplast replication in light-grown wheat leaves. Planta 165, 120-125. Erickson, H.P. (1998). Atomic structures of tubulin and FtsZ. Trends Cell Biol 8, 133- 137. Errington, J., Daniel, R.A., and Scheffers, DJ. (2003). Cytokinesis in bacteria. Microbiol Mol Biol Rev 67, 52-65, table of contents. Esue, 0., Cordero, M., Wirtz, D., and Tseng, Y. (2005). The assembly of MreB, a prokaryotic homolog of actin. J Biol Chem 280, 2628-2635. Fu, X., Shih, Y.L., Zhang, Y., and Rothfield, LL (2001). The MinE ring required for proper placement of the division site is a mobile structure that changes its cellular location during the Escherichia coli division cycle. Proc Natl Acad Sci U S A 98, 980-985. Fujiwara, M.T., N akamura, A., Itoh, R., Shimada, Y., Yoshida, S., and Moller, S.G. (2004). Chloroplast division site placement requires dimerization of the ARCl l/AtMinDl protein in Arabidopsis. J Cell Sci 117, 2399-2410. 38 In“ Fukushima. N The GT assemb. 12, 275 Can, 11., Kadit ARC 5 . . division Gilson, PR, 1' Fisher. onhol0g and are tJ Eultaryo Gray, J.C., and membrar' Gray, MW. (19 Cu, X, and Ver cell platel Hales, KG, ant mediated Harris’ E'H's BO protein 3);; HigaSllltani’ A“ l 3111A of El Biochem l cell-divisitl L0n. Mol < H, l mShaW,J.E. (2‘ BlOl 16, 4b lllllShaw, 1E an '9 a mechanisiI . l HOHaI-d’ M. (2004 Fukushima, N.H., Brisch, E., Keegan, B.R., Bleazard, W., and Shaw, J.M. (2001). The GTPase effector domain sequence of the Dnmlp GTPase regulates self- assembly and controls a rate-limiting step in mitochondrial fission. Mol Biol Cell 12, 2756-2766. Gao, H., Kadirjan-Kalbach, D., Froehlich, J.E., and Osteryoung, K.W. (2003). ARC5, a cytosolic dynamin-like protein from plants, is part of the chloroplast division machinery. Proc Natl Acad Sci U S A 100, 4328-4333. Gilson, P.R., Yu, X.C., Hereld, D., Barth, C., Savage, A., Kiefel, B.R., Lay, S., Fisher, P.R., Margolin, W., and Beech, P.L. (2003). Two Dictyostelium orthologs of the prokaryotic cell division protein FtsZ localize to mitochondria and are required for the maintenance of normal mitochondrial morphology. Eukaryot Cell 2, 1315-1326. Gray, J.C., and Row, RE. (1995). Protein translocation across chloroplast envelope membranes. Trends Cell Biol 5, 243-247. Gray, M.W. (1999). Evolution of organellar genomes. Curr Opin Genet Dev 9, 678-687. Gu, X., and Verma, D. (1996). Phragrnoplastin, a dynamin-like protein associated with cell plate formation in plants. EMBO J. 15, 695-704. Hales, K.G., and Fuller, M.T. (1997). Developmentally regulated mitochondrial fusion mediated by a conserved, novel, predicted GTPase. Cell 90, 121-129. Harris, E.H., Boynton, J.E., and Gillham, NW. (1994). Chloroplast ribosomes and protein synthesis. Microbiol Rev 58, 700-754. Higashitani, A., Higashitani, N., and Horiuchi, K. (1995). A cell division inhibitor SulA of Escherichia coli directly interacts with FtsZ through GTP hydrolysis. Biochem Biophys Res Commun 209, 198-204. Higashitani, A., Ishii, Y., Kato, Y., and Koriuchi, K. (1997). Emotional dissection of a cell-division inhibitor, SulA, of Escherichia coli and its negative regulation by Lon. Mol Gen Genet 254, 351-357. Hinshaw, J.E. (2000). Dynamin and its role in membrane fission. Annu Rev Cell Dev Biol 16, 483-519. Hinshaw, J.E., and Schmid, S.L. (1995). Dynamin self-assembles into rings suggesting a mechanism for coated vesicle budding. Nature 374, 190-192. Howard, M. (2004). A mechanism for polar protein localization in bacteria. J Mol Biol 335, 655-663. 39 Howe, Hu.Z« Huang“ 0 Huisma. Si Huisma. Cl 5: lShiL ‘7'! li CUM? ltoh‘ R., ht cl Jack$01]. in Howe, C.J., Barbrook, A.C., Koumandou, V.L., Nisbet, R.E., Symington, H.A., and Wightman, T.F. (2003). Evolution of the chloroplast genome. Philos Trans R Soc Lond B Biol Sci 358, 99-106; discussion 106-107. Hu, Z., and Lutkenhaus, J. (1999). Topological regulation of cell division in Escherichia coli involves rapid pole to pole oscillation of the division inhibitor MinC under the control of MinD and MinE. Mol Microbiol 34, 82-90. Hu, Z., Gogol, E.P., and Lutkenhaus, J. (2002). Dynamic assembly of MinD on phospholipid vesicles regulated by ATP and MinE. Proc Natl Acad Sci U S A 99, 6761-6766. Hu, Z., Saez, C., and Lutkenhaus, J. (2003). Recruitment of MinC, an inhibitor of Z- ring formation, to the membrane in Escherichia coli: role of MinD and MinE. J Bacteriol 185, 196-203. Hu, 2., Mukherjee, A., Pichoff, S., and Lutkenhaus, J. (1999). The MinC component of the division site selection system in Escherichia coli interacts with FtsZ to prevent polymerization. Proc Natl Acad Sci U S A 96, 14819-14824. Huang, J., Cao, C., and Lutkenhaus, J. (1996). Interaction between FtsZ and inhibitors of cell division. J Bacteriol 178, 5080-5085. Huisman, 0., D'Ari, R., and George, J. (1980). Further characterization of sfiA and sfiB mutations in Escherichia coli. J Bacteriol 144, 185-191. Huisman, 0., D'Ari, R., and Gottesman, S. (1984). Cell-division control in Escherichia coli: specific induction of the SOS function SfiA protein is sufficient to block septation. Proc Natl Acad Sci U S A 81, 4490-4494. Ishii, Y., and Amano, F. (2001). Regulation of SulA cleavage by Lon protease by the C- terminal amino acid of SulA, histidine. Biochem J 358, 473-480. Ishino, F., Jung, H.K., Ikeda, M., Doi, M., Wachi, M., and Matsuhashi, M. (1989). New mutations its-36, Its-33, and fisW clustered in the mra region of the Escherichia coli chromosome induce therrnosensitive cell growth and division. J Bacteriol 171, 5523-5530. Itoh, R., Fujiwara, M., Nagata, N., and Yoshida, S. (2001). A chloroplast protein homologous to the eubacterial topological specificity factor minE plays a role in chloroplast division. Plant Physiol 127, 1644-1655. J ackson-Constan, D., Akita, M., and Keegstra, K. (2001). Molecular chaperones involved in chloroplast protein import. Biochim Biophys Acta 1541, 102-113. 40 Jarvis Jeong Jones, Jarvis, P., and Soil, J. (2002). Toc, tic, and chloroplast protein import. Biochim Biophys Acta 1590, 177-189. Jeong, W.J., Park, Y.I., Suh, K., Raven, J.A., Yoo, O.J., and Liu, J.R. (2002). A large population of small chloroplasts in tobacco leaf cells allows more effective chloroplast movement than a few enlarged chloroplasts. Plant Physiol 129, 112- 121. Jones, C., and Holland, LB. (1985). Role of the SulB (FtsZ) protein in division inhibition during the SOS response in Escherichia coli: F tsZ stabilizes the inhibitor SulA in maxicells. Proc Natl Acad Sci U S A 82, 6045-6049. Jones, L.J., Carballido-Lopez, R., and Errington, J. (2001). Control of cell shape in bacteria: helical, actin-like filaments in Bacillus subtilis. Cell 104, 913-922. Kanemori, M., Yanagi, H., and Yura, T. (1999). The ATP-dependent HslVU/CpoY protease participates in turnover of cell division inhibitor SulA in Escherichia coli. J Bacteriol 181, 3674-3680. Kasten, B.a.R., R. (1997). B-lactam antibiotics inhibit chloroplast division in a moss (Physcomitrella patens) but not in tomato (Lycopersicon esculentum). J. Plant Physiol. 150, 137—140. Katayama, N., Takano, H., Sugiyama, M., Takio, S., Sakai, A., Tanaka, K., Kuroiwa, H., and Ono, K. (2003). Effects of antibiotics that inhibit the bacterial peptidoglycan synthesis pathway on moss chloroplast division. Plant Cell Physiol 44, 776-781. Kelly, RB. (1995). Endocytosis. Ringing necks with dynamin. Nature 374, 116-117. Khattar, M.M., Begg, K.J., and Donachie, W.D. (1994). Identification of FtsW and characterization of a new ftsW division mutant of Escherichia coli. J Bacteriol 176, 7140-7147. Kiefel, B.R., Gilson, RR, and Beech, P.L. (2004). Diverse eukaryotes have retained mitochondrial homologues of the bacterial division protein F tsZ. Protist 155, 105- 115. Koksharova, C.A., and Wolk, GP. (2002). A novel gene that bears a DnaJ motif influences cyanobacterial cell division. J Bacteriol 184, 5524-5528. Kosaka, T., and Ikeda, K. (1983a). Possible temperature-dependent blockage of synaptic vesicle recycling induced by a single gene mutation in Drosophila. J Neurobiol 14, 207-225. 41 Kosaka l l Koshib: ( C & Ku roiw. l Lackner Kosaka, T., and Ikeda, K. (1983b). Reversible blockage of membrane retrieval and endocytosis in the garland cell of the temperature-sensitive mutant of Drosophila melanogaster, shibiretsl. J Cell Biol 97, 499-507. Koshiba, T., Detmer, S.A., Kaiser, J.T., Chen, H., McCaffery, J.M., and Chan, DC. (2004). Structural basis of mitochondrial tethering by mitofirsin complexes. Science 305, 858-862. Kuroiwa, T., Kuroiwa, H., Sakai, A., Takahashi, H., Toda, K., and Itoh, R. (1998). The division apparatus of plastids and mitochondria. Int Rev Cytol 181, 1-41. Lackner, L.L., Raskin, D.M., and de Boer, RA. (2003). ATP-dependent interactions between Escherichia coli Min proteins and the phospholipid membrane in vitro. J Bacteriol 185, 735-749. Lara, B., and Ayala, J.A. (2002). Topological characterization of the essential Escherichia coli cell division protein FtsW. FEMS Microbiol Lett 216, 23-32. Leech RM, T.W., Plattaloia KA. (1981). Observations on the mechanism of chloroplast division in higher-plants. New Phytologist 87, 1-&. Lowe, J., and Amos, L.A. (1998). Crystal structure of the bacterial cell-division protein FtsZ. Nature 391, 203-206. Lowe, J., and van den Ent, F. (2001). Conserved sequence motif at the C-terminus of the bacterial cell-division protein FtsA. Biochimie 83, 117-120. Lowe, J., van den Ent, F., and Amos, L.A. (2004). Molecules of the bacterial cytoskeleton. Annu Rev Biophys Biomol Struct 33, 177-198. Lutkenhaus, J. (2002). Dynamic proteins in bacteria. Curr Opin Microbiol 5, 548-552. Ma, X., and Margolin, W. (1999). Genetic and functional analyses of the conserved C- terrninal core domain of Escherichia coli FtsZ. J Bacteriol 181, 7531-7544. Ma, X., Ehrhardt, D.W., and Margolin, W. (1996). Colocalization of cell division proteins FtsZ and FtsA to cytoskeletal structures in living Escherichia coli cells by using green fluorescent protein. Proc Natl Acad Sci U S A 93, 12998-13003. Maple, J., Chua, N.-H., and Moller, S.G. (2002). The topological specificity factor AtMinEl is essential for correct plastid division site placement in Arabidopsis. Plant J 31, 269-277. Maple, J., Fujiwara, M.T., Kitahata, N., Lawson, T., Baker, N .R., Yoshida, S., and Moller, S.G. (2004). GIANT CHLOROPLAST 1 is essential for correct plastid division in Arabidopsis. Curr Biol 14, 776-781. 42 Margt Margo .Vlarris Martin Mazout z I McAndi (. c it NlcF add E Meeusel It Meinha. ft (1 MEI'CEr, If E .\ ' . . . 1'} agls; Margolin, W. (2000). Themes and variations in prokaryotic cell division. FEMS Microbiol Rev 24, 531-548. Margolin, W. (2001). Bacterial cell division: a moving MinE sweeper boggles the MinD. Curr Biol 11, R395-398. Marrison, J.L., Rutherford, S.M., Robertson, E.J., Lister, C., Dean, C., and Leech, RM. (1999). The distinctive roles of five different ARC genes in the chloroplast division process in Arabidopsis. Plant J 18, 651-662. Martin, W. (2003). Gene transfer from organelles to the nucleus: Frequent and in big chunks. PNAS 100, 8612-8614. Mazouni, K., Domain, F., Cassier-Chauvat, C., and Chauvat, F. (2004). Molecular analysis of the key cytokinetic components of cyanobacteria: FtsZ, ZipN and MinCDE. Mol Microbiol 52, 1 145-1 158. McAndrew, R.S., Froehlich, J.E., Vitha, S., Stokes, K.D., and Osteryoung, K.W. (2001). Colocalization of plastid division proteins in the chloroplast stromal compartment establishes a new functional relationship between Ftle and F tsZZ in higher plants. Plant Physiol 127, 1656-1666. McFadden, GJ. (1999). Endosyrnbiosis and evolution of the plant cell. Curr Opin Plant Biol 2, 513-519. Meeusen, S., McCaffery, J.M., and Nunnari, J. (2004). Mitochondrial Fusion Intermediates Revealed in Vitro. Science 305, 1747-1752. Meinhardt, H., and de Boer, RA. (2001). Pattern formation in Escherichia coli: a model for the pole-to-pole oscillations of Min proteins and the localization of the division site. Proc Natl Acad Sci U S A 98, 14202-14207. Mercer, KL, and Weiss, BS. (2002). The Escherichia coli cell division protein F tsW is required to recruit its cognate transpeptidase, Ftsl (PBP3), to the division site. J Bacteriol 184, 904-912. Miyagishima, S., Takahara, M., and Kuroiwa, T. (2001a). Novel filaments 5 nm in diameter constitute the cytosolic ring of the plastid division apparatus. Plant Cell 13, 707-721. Miyagishima, 8., Takahara, M., Mori, T., Kuroiwa, H., Higashiyama, T., and Kuroiwa, T. (2001b). Plastid division is driven by a complex mechanism that involves differential transition of the bacterial and eukaryotic division rings. Plant Cell 13, 2257-2268. 43 Miyagish cy‘ M Miyagish. 11.. tin Mizusawa lon 362 Mori, T., I V15; Cell Mosrak. L. “IS FtsZ Mukherjee, by s 95, 2 Mukherjee, defic Z tin; “'"lder, E., mUtat MinC 174, 3 Nal"alllura, 5 (1983‘ SeqUer “Shit W., an Cell (111 COli. F] xogales’ E., “ dimer b Miyagishima, S.Y., Wolk, CR, and Osteryoung, K.W. (2005). Identification of cyanobacterial cell division genes by comparative and mutational analyses. Mol Microbiol 56, 126-143. Miyagishima, S.Y., Nishida, K., Mori, T., Matsuzaki, M., Higashiyama, T., Kuroiwa, H., and Kuroiwa, T. (2003). A plant-specific dynamin-related protein forms a ring at the chloroplast division site. Plant Cell 15, 655-665. Mizusawa, S., and Gottesman, S. (1983). Protein degradation in Escherichia coli: the lon gene controls the stability of sulA protein. Proc Natl Acad Sci U S A 80, 358- 362. Mori, T., Kuroiwa, H., Takahara, M., Miyagishima, S.Y., and Kuroiwa, T. (2001). Visualization of an F tsZ ring in chloroplasts of Lilium longiflorum leaves. Plant Cell Physiol 42, 555-559. Mosyak, L., Zhang, Y., Glasfeld, E., Haney, S., Stahl, M., Seehra, J., and Somers, W.S. (2000). The bacterial cell-division protein ZipA and its interaction with an F tsZ fragment revealed by X-ray crystallography. Embo J 19, 3179-3191. Mukherjee, A., Cao, C., and Lutkenhaus, J. (1998). Inhibition of FtsZ polymerization by SulA, an inhibitor of septation in Escherichia coli. Proc Natl Acad Sci U S A 95, 2885-2890. Mukherjee, A., Saez, C., and Lutkenhaus, J. (2001). Assembly of an FtsZ mutant deficient in GTPase activity has implications for FtsZ assembly and the role of the Z ring in cell division. J Bacteriol 183, 7190-7197. Mulder, E., Woldringh, C.L., Tetart, F., and Bouche, J.P. (1992). New minC mutations suggest different interactions of the same region of division inhibitor MinC with proteins specific for minD and dicB coinhibition pathways. J Bacteriol 174, 35-39. N akamura, M., Maruyama, I.N., Soma, M., Kato, J., Suzuki, H., and Horota, Y. (1983). On the process of cellular division in Escherichia coli: nucleotide sequence of the gene for penicillin-binding protein 3. Mol Gen Genet 191, 1-9. Nishii, W., and Takahashi, K. (2003). Determination of the cleavage sites in SulA, a cell division inhibitor, by the ATP-dependent HslVU protease from Escherichia coli. FEBS Lett 553, 351-354. Nogales, E., Wolf, S.G., and Downing, K.H. (1998a). Structure of the alpha beta tubulin dimer by electron crystallography. Nature 391, 199-203. Nogales, E., Downing, K.H., Amos, L.A., and Lowe, J. (1998b). Tubulin and FtsZ form a distinct family of GTPases. Nat Struct Biol 5, 451-458. Oross, J.W., and Possingham, J.V. (1989). Ultrastructural features of the constricted region of dividing plastids. Protoplasma 150, 131-138. Osteryoung, K.W. (2000). Organelle fission. Crossing the evolutionary divide. Plant Physiol 123, 1213-1216. Osteryoung, K.W., and Vierling, E. (1995). Conserved cell and organelle division. Nature 376, 473-474. Osteryoung, K.W., and Pyke, KA. (1998). Plastid division: evidence for a prokaryotically derived mechanism. Curr Opin Plant Biol 1, 475-479. Osteryoung, K.W., and McAndrew, RS. (2001). The Plastid Division Machine. Annu Rev Plant Physiol Plant Mol Biol 52, 315-333. Osteryoung, K.W., and Nunnari, J. (2003). The division of endosymbiotic organelles. Science 302, 1698-1704. Osteryoung, K.W., Stokes, K.D., Rutherford, S.M., Percival, A.L., and Lee, W.Y. (1998). Chloroplast division in higher plants requires members of two fimctionally divergent gene families with homology to bacterial fisZ. Plant Cell 10, 1991-2004. Pelloquin, L., Belenguer, P., Menon, Y., and Ducommun, B. (1998). Identification of a fission yeast dynamin-related protein involved in mitochondrial DNA maintenance. Biochem Biophys Res Commun 251, 720-726. Pogliano, J., Pogliano, K., Weiss, D.S., Losick, R., and Beckwith, J. (1997). Inactivation of Ftsl inhibits constriction of the FtsZ cytokinetic ring and delays the assembly of FtsZ rings at potential division sites. Proc Natl Acad Sci U S A 94, 559-564. Possingh, J.S., W. (1969). Changes in chloroplast number per cell during leaf development in spinach. Planta 86, 186-&. Pyke, KA. (1999). Plastid division and development. Plant Cell 11, 549-556. Pyke, K.A., and Leech, RM. (1994). A Genetic Analysis of Chloroplast Division and Expansion in Arabidopsis thaliana. Plant Physiol 104, 201-207. Pyke, K.A., Rutherford, S.M., Robertson, E.J., and Leech, RM. (1994). arc6, A Fertile Arabidopsis Mutant with Only Two Mesophyll Cell Chloroplasts. Plant Physiol 106, 1169-1177. 45 Raskin, D.M., and de Boer, P.A. (1999a). Rapid pole-to-pole oscillation of a protein required for directing division to the middle of Escherichia coli. Proc Natl Acad Sci U S A 96, 4971-4976. Raskin, D.M., and de Boer, P.A. (1999b). MinDE-dependent pole-to-pole oscillation of division inhibitor MinC in Escherichia coli. J Bacteriol 181, 6419-6424. Raven, J.A., and Allen, J.F. (2003). Genomics and chloroplast evolution: what did cyanobacteria do for plants? Genome Biol 4, 209. RayChaudhuri, D., and Park, J.T. (1992). Escherichia coli cell-division gene ftsZ encodes a novel GTP-binding protein. Nature 359, 251-254. Raynaud, C., Cassier-Chauvat, C., Perennes, C., and Bergounioux, C. (2004). An Arabidopsis homolog of the bacterial cell division inhibitor SulA is involved in plastid division. Plant Cell 16, 1801-1811. Reddy, M.S., Dinkins, R., and Collins, GB. (2002). Overexpression of the Arabidopsis thaliana MinEl bacterial division inhibitor homologue gene alters chloroplast size and morphology in transgenic Arabidopsis and tobacco plants. Planta 215, 167- 176. Reusing, S.A., Kiessling, J., Reski, R., and Decker, E.L. (2004). Diversification of 1152 during early land plant evolution. J Mol Evol 58, 154-162. Reumann, S., and Keegstra, K. (1999). The endosymbiotic origin of the protein import machinery of chloroplastic envelope membranes. Trends Plant Sci 4, 302-307. Reumann, S., Davila-Aponte, J., and Keegstra, K. (1999). The evolutionary origin of the protein-translocating channel of chloroplastic envelope membranes: identification of a cyanobacterial homolog. Proc Natl Acad Sci U S A 96, 784- 789. Rico, A.I., Garcia-Ovalle, M., Mingorance, J ., and Vicente, M. (2004). Role of two essential domains of Escherichia coli FtsA in localization and progression of the division ring. Mol Microbiol 53, 1359-1371. Robertson, E.J., Pyke, K.A., and Leech, RM. (1995). arc6, an extreme chloroplast division mutant of Arabidopsis also alters proplastid proliferation and morphology in shoot and root apices. J Cell Sci 108 ( Pt 9), 2937-2944. Robertson, E.J., Rutherford, S.M., and Leech, RM. (1996). Characterization of chloroplast division using the Arabidopsis mutant arcS. Plant Physiol 112, 149- 159. 46 Romberg, L., and Levin, RA. (2003). Assembly dynamics of the bacterial cell division protein FTSZ: poised at the edge of stability. Annu Rev Microbiol 57, 125-154. Rothfield, L., Justice, 8., and Garcia-Lara, J. (1999). Bacterial cell division. Annu Rev Genet 33, 423-448. Salim, K., Bottomley, M.J., Querfurth, E., Zvelebil, M.J., Gout, 1., Scaife, R., Margolis, R.L., Gigg, R., Smith, C.I., Driscoll, P.C., Waterfield, M.D., and Panayotou, G. (1996). Distinct specificity in the recognition of phosphoinositides by the pleckstrin homology domains of dynamin and Bruton's tyrosine kinase. Embo J 15, 6241-6250. Sanchez, M., Valencia, A., Ferrandiz, M.J., Sander, C., and Vicente, M. (1994). Correlation between the structure and biochemical activities of F tsA, an essential cell division protein of the actin family. Embo J 13, 4919-4925. Saurer, W., and Possingham, J .V. (1970). Studies on the growth of spinach leaves (Spinacea oleracea). J. Exp. Biol. 21, 151-158. Scheffers, D., and Driessen, A.J. (2001). The polymerization mechanism of the bacterial cell division protein FtsZ. FEBS Lett 506, 6-10. Scheffers, D.J., and Driessen, A.J. (2002). Immediate GTP hydrolysis upon FtsZ polymerization. Mol Microbiol 43, 1517-1521. Scheffers, D.J., den Blaauwen, T., and Driessen, A.J. (2000). Non-hydrolysable GTP- gamma-S stabilizes the FtsZ polymer in a GDP-bound state. Mol Microbiol 35, 1211-1219. Scheffers, D.J., de Wit, J .G., den Blaauwen, T., and Driessen, A.J. (2002). GTP hydrolysis of cell division protein FtsZ: evidence that the active site is formed by the association of monomers. Biochemistry 41, 521-529. Schoemaker, J .M., Gayda, R.C., and Markovitz, A. (1984). Regulation of cell division in Escherichia coli: SOS induction and cellular location of the sulA protein, a key to lon-associated filamentation and death. J Bacteriol 158, 551-561. Seong, I.S., Oh, J.Y., Yoo, S.J., Seol, J.H., and Chung, CH. (1999). ATP-dependent degradation of SulA, a cell division inhibitor, by the HslVU protease in Escherichia coli. FEBS Lett 456, 211-214. Sesaki, H., and Jensen, RE. (1999). Division versus fusion: Dnmlp and F zolp antagonistically regulate mitochondrial shape. J Cell Biol 147, 699-706. 47 Sesaki, H., Southard, S.M., Yaffe, M.P., and Jensen, RE. (2003). Mgrnlp, a dynamin- relatcd GTPase, is essential for fusion of the mitochondrial outer membrane. Mol Biol Cell 14, 2342-2356. Shaw, J.M., and Nunnari, J. (2002). Mitochondrial dynamics and division in budding yeast. Trends Cell Biol 12, 178-1 84. Shimada, H., Koizumi, M., Kuroki, K., Mochizuki, M., Fujimoto, H., Ohta, H., Masuda, T., and Takamiya, K. (2004). ARC3, a chloroplast division factor, is a chimera of prokaryotic FtsZ and part of eukaryotic phosphatidylinositol-4- phosphate 5-kinase. Plant Cell Physiol 45, 960-967. Smirnova, E., Shurland, D.L., Newman-Smith, E.D., Pishvaee, B., and van der Bliek, AM. (1999). A model for dynamin self-assembly based on binding between three different protein domains. J Biol Chem 274, 14942-14947. Stokes, K.D., and Osteryoung, K.W. (2003). Early divergence of the Ftle and FtsZ2 plastid division gene families in photosynthetic eukaryotes. Gene 320, 97-108. Stokes, K.D., McAndrew, R.S., Figueroa, R., Vitha, S., and Osteryoung, K.W. (2000). Chloroplast division and morphology are differentially affected by overexpression of Ftle and F tsZZ genes in Arabidopsis. Plant Physiol 124, 1668-1677. Strepp, R., Scholz, S., Kruse, S., Speth, V., and Reski, R. (1998). Plant nuclear gene knockout reveals a role in plastid division for the homolog of the bacterial cell division protein FtsZ, an ancestral tubulin. Proc Natl Acad Sci U S A 95, 4368- 4373. Stricker, J., Maddox, P., Salmon, E.D., and Erickson, H.P. (2002). Rapid assembly dynamics of the Escherichia coli FtsZ-ring demonstrated by fluorescence recovery after photobleaching. PNAS 99, 3171-3175. Suefuji, K., VaIquzi, R., and RayChaudhuri, D. (2002). Dynamic assembly of MinD into filament bundles modulated by ATP, phospholipids, and MinE. Proc Natl Acad Sci U S A 99,16776-16781. in, Q., and Margolin, W. (1998). FtsZ dynamics during the division cycle of live Escherichia coli cells. J Bacteriol 180, 2050-2056. , (2., Yu, X.C., and Margolin, W. (1998). Assembly of the FtsZ ring at the central division site in the absence of the chromosome. Mol Microbiol 29, 491-503. Ltzer, S.M., and H inshaw, J.E. (1998). Dynamin undergoes a GTP-dependent conformational change causing vesiculation. Cell 93, 1021-1029. 48 Takahara, M., Takahashi, H., Matsunaga, S., Miyagishima, S., Takano, H., Sakai, A., Kawano, S., and Kuroiwa, T. (2000). A putative mitochondrial ftsZ gene is present in the unicellular primitive red alga Cyanidioschyzon merolae. Mol Gen Genet 264, 452-460. Takei, K., McPherson, P.S., Schmid, S.L., and De Camilli, P. (1995). Tubular membrane invaginations coated by dynamin rings are induced by GTP-gamma S in nerve terminals. Nature 374, 186-190. Takei, K., Haucke, V., Slepnev, V., Farsad, K., Salazar, M., Chen, H., and De Camilli, P. (1998). Generation of coated intermediates of clathrin-mediated endocytosis on protein-free liposomes. Cell 94, 13 1-141 . Timmis, J.N., Ayliffe, M.A., Huang, C.Y., and Martin, W. (2004). Endosymbiotic gene transfer: organelle genomes forge eukaryotic chromosomes. Nat Rev Genet 5, 123-135. Trusca, D., Scott, S., Thompson, C., and Bramhill, D. (1998). Bacterial SOS checkpoint protein SulA inhibits polymerization of purified F tsZ cell division protein. J Bacteriol 180, 3946-3953. van den Ent, F., Amos, L., and Lowe, J. (2001a). Bacterial ancestry of actin and tubulin. Curr Opin Microbiol 4, 634-638. van den Ent, F., Amos, L.A., and Lowe, J. (2001b). Prokaryotic origin of the actin cytoskeleton. Nature 413, 39-44. van den Ent, F., Moller—Jensen, J., Amos, L.A., Gerdes, K., and Lowe, J. (2002). F - actin-like filaments formed by plasmid segregation protein ParM. Embo J 21, 6935-6943. van der Bliek, A.M., and Meyerowitz, E.M. (1991). Dynamin-like protein encoded by the Drosophila shibire gene associated with vesicular traffic. Nature 351, 411- 41 4. Vitha, S., McAndrew, R.S., and Osteryoung, K.W. (2001). FtsZ ring formation at the chloroplast division site in plants. J Cell Biol 153, 111-120. itha, S., Froehlich, J .E., Koksharova, 0., Pyke, K.A., van Erp, H., and Osteryoung, I(. W. (2003). ARC6 Is a J -Domain Plastid Division Protein and an Evolutionary Descendant of the Cyanobacteria] Cell Division Protein Ftn2. Plant Cell 15, 1918- 1 933. kasugi, T., N agai, T., Kapoor, M., Sugita, M., Ito, M., Ito, S., Tsudzuki, J., Nakashima, K, Tsudzuki, T., Suzuki, Y., Hamada, A., Ohta, T., Inamura, A., Yoshinaga, K, and Sugiura, M. (1997). Complete nucleotide sequence of the 49 chloroplast genome from the green alga Chlorella vulgaris: the existence of genes possibly involved in chloroplast division. Proc Natl Acad Sci U S A 94, S967- 5972. Warnock, D.E., and Schmid, S.L. (1996). Dynamin GTPase, a force—generating molecular switch. Bioessays 18, 885-893. Weiss, D.S., Chen, J.C., Ghigo, J.M., Boyd, D., and Beckwith, J. (1999). Localization of Ftsl (PBP3) to the septal ring requires its membrane anchor, the Z ring, FtsA, FtsQ, and FtsL. J Bacteriol 181, 508-520. Weiss, D.S., Pogliano, K., Carson, M., Guzman, L.M., Fraipont, C., Nguyen- Disteche, M., Losick, R., and Beckwith, J. (1997). Localization of the Escherichia coli cell division protein Ftsl (PBP3) to the division site and cell pole. Mol Microbiol 25, 671-681. Westermann, B. (2003). Mitochondrial membrane fusion. Biochim Biophys Acta 1641, 195-202. Wijsman, H.J., and Koopman, CR. (1976). The relation of the genes envA and ftsA in Escherichia coli. Mol Gen Genet 147, 99-102. Wilsbach, K., and Payne, GS. (1993). Vpslp, a member of the dynamin GTPase family, is necessary for Golgi membrane protein retention in Saccharomyces cerevisiae. Embo J 12, 3049-3059. Wissel, M.C., and Weiss, D.S. (2004). Genetic analysis of the cell division protein Ftsl (PBP3): amino acid substitutions that impair septal localization of Ftsl and recruitment of FtsN. J Bacteriol 186, 490-502. Wong, E.D., Wagner, J.A., Gorsich, S.W., McCaffery, J.M., Shaw, J.M., and Nunnari, J. (2000). The dynamin-related GTPase, Mgrnlp, is an intermembrane space protein required for maintenance of fusion competent mitochondria. J Cell Biol 151, 341-352. Wong, E.D., Wagner, J.A., Scott, S.V., Okreglak, V., Holewinske, T.J., Cassidy— Stone, A., and Nunnari, J. (2003). The intramitochondrial dynamin-related GTPase, Mgrnlp, is a component of a protein complex that mediates mitochondrial fusion. J Cell Biol 160, 303-311. Yamamoto, K., Pyke, K.A., and Kiss, J.Z. (2002). Reduced gravitropism in inflorescence stems and hypocotyls, but not roots, of Arabidopsis mutants with large plastids. Physiol Plant 114, 627-636. 50 Yan, K., Pearce, K.H., and Payne, DJ. (2000). A conserved residue at the extreme C- terrninus of FtsZ is critical for the F tsA-FtsZ interaction in Staphylococcus aureus. Biochem Biophys Res Commun 270, 387-392. Yu, J., Hu, S., Wang, J., Wong, G.K., Li, S., Liu, B., Deng, Y., Dai, L., Zhou, Y., Zhang, X., Cao, M., Liu, J., Sun, J., Tang, J., Chen, Y., Huang, X., Lin, W., Ye, C., Tong, W., Cong, L., Geng, J., Han, Y., Li, L., Li, W., Hu, G., Li, J., Liu, Z., Qi, Q., Li, T., Wang, X., Lu, H., Wu, T., Zhu, M., Ni, P., Han, H., Dong, W., Ren, X., Feng, X., Cui, P., Li, X., Wang, H., Xu, X., Zhai, W., Xu, Z., Zhang, J., He, S., Xu, J., Zhang, K., Zheng, X., Dong, J., Zeng, W., Tao, L., Ye, J., Tan, J., Chen, X., He, J., Liu, D., Tian, W., Tian, C., Xia, H., Bao, Q., Li, G., Gao, H., Cao, T., Zhao, W., Li, P., Chen, W., Zhang, Y., Hu, J., Liu, S., Yang, J., Zhang, G., Xiong, Y., Li, Z., Mao, L., Zhou, C., Zhu, Z., Chen, R., Hao, B., Zheng, W., Chen, S., Guo, W., Tao, M., Zhu, L., Yuan, L., and Yang, H. (2002). A draft sequence of the rice genome (Oryza sativa L. ssp. indica). Science 296, 79-92. Zheng, J., Cahill, S.M., Lemmon, M.A., Fushman, D., Schlessinger, J., and Cowburn, D. (1996). Identification of the binding site for acidic phospholipids on the pH domain of dynamin: implications for stimulation of GTPase activity. J Mol Biol 255, 14-21. 51 Chapter 2 ARC5, a Cytosolic Dynamin-like Protein from Plants, Is Part of the Chloroplast Division Machinery Gao H, Kadirjan-Kalbach D, Froehlich JE, Osteryoung KW. Proceeding of the National Academy of Science of USA 2003 Apr 1; 100 (7):4328-33. Dr. Deena Kadirjan-Kalbach did the cross between arc5 and Col-O wild type and generated an F2 population for mapping the ARC5 gene. The work presented in Fig. 5 was done in collaboration with Dr. John E. Froehlich. 52 Abstract: Chloroplast division in plant cells is orchestrated by a complex macromolecular machine with components positioned on both the inner and outer envelope surfaces. The only plastid division proteins identified to date are of endosymbiotic origin and are localized inside the organelle. Employing positional cloning methods in Arabidopsis in conjunction with a novel strategy for pinpointing the mutant locus, we have identified a gene encoding a new chloroplast division protein, ARC5. Mutants of ARC5 exhibit defects in chloroplast constriction, have enlarged, dumbbell-shaped chloroplasts, and are rescued by a wild-type copy of ARC5 . The ARC5 gene product shares similarity with the dynamin family of GTPases, which mediate endocytosis, mitochondrial division, and other organellar fission and fusion events in eukaryotes. Phylogenetic analysis showed that ARC5 is related to a group of dynamin-like proteins unique to plants. A green fluorescent protein (GFP)-ARC5 fusion protein localizes to a ring at the chloroplast division site. Chloroplast import and protease protection assays indicate that the ARC5 ring is positioned on the outer surface of the chloroplast. Thus, ARC5 is the first cytosolic component of the chloroplast division complex to be identified. ARCS has no obvious counterparts in prokaryotes, suggesting that it evolved from a dynamin-related protein present in the eukaryotic ancestor of plants. These results indicate that the chloroplast division apparatus is of mixed evolutionary origin and that it shares structural and mechanistic similarities with both the cell division machinery of bacteria and the dynamin-mediated organellar fission machineries of eukaryotes. 53 Introduction The chloroplasts of plants and algae are widely believed to have evolved only once from a free-living cyanobacterial endosymbiont (1). Over evolutionary time, many of the genes once present in the endosymbiont have been transferred to the nuclear genome where they have acquired sequences encoding transit peptides that direct their gene products back to the chloroplast (1, 2). This scenario describes the origin of the five previously identified plastid division proteins in plants, all of which evolved fi'om related cell division proteins in cyanobacteria, are encoded in the nucleus, and are localized inside the chloroplast. These include F tle and FtsZZ, tubulin-like proteins that localize to a ring at the site of plastid constriction (3-10), MinD and MinE, which regulate placement of the plastid division site (11-13), and ARTEMIS, which appears to mediate constriction of the envelope membranes (14). Despite localization of the previously identified plastid division proteins inside the chloroplasts in plant cells, ultrastructural studies have shown that plastid division entails the coordinated activity of components localized outside as well as inside the organelle. In plants, the chloroplast division complex comprises electron-dense structures situated both on the stromal surface of the inner envelope membrane and on the cytosolic surface of the outer membrane (15). These structures have been termed the inner and outer plastid-dividing (PD) rings, respectively. A middle PD ring positioned in the intermembrane space has also been described in the red alga Cyanidioschyzon merolae (16), and the dynamics of assembly and disassembly of the three PD rings have been investigated in detail in this organism (1 7, 18). Although it was previously 54 hypothesized that the PD rings might contain F tsZ (4), recent evidence showing that the FtsZ ring assembles prior to and is separable from the PD rings in both C. merolae and plants (19, 20) indicate that this is not the case. Thus, while it is assumed that the PD rings represent multiprotein complexes, their compositions remain unknown. The Arabidopsis mutant arc5 contains an EMS-induced mutation conferring a chloroplast division defect in which chloroplasts initiate but rarely complete constriction (21, 22). As a result, arc5 chloroplasts ofien exhibit a dumbbell shape (Fig. 13). This phenotype suggested that the ARC5 gene product might be a structural component of the chloroplast division complex. Here we show that ARC5 is a member of the dynamin- farnily of GTPases, which have not been shown previously to participate in chloroplast division, and that it localizes to the chloroplast division site in plants. However, in contrast with other chloroplast division proteins, ARC5 is positioned on the cytosolic surface of the organelle and has no obvious homologues in prokaryotes. Our findings reveal that the chloroplast division machinery is an evolutionary hybrid, combining structural and mechanistic features acquired from both the prokaryotic ancestor of chloroplasts and its eukaryotic host. 55 Materials and Methods Plant Material. Arabidopsis thaliana strains Columbia (Col-O) and Landsberg erecta (Ler) were used for all experiments as indicated. The arc5 mutant was identified in the Ler background by Pyke and Leech (21). Plants were grown as described previously (4). Microscopy. Phenotypes were analyzed as previously described (4), except that the images were recorded with a Coolpix 995 digital camera (Nikon Corporation, Tokyo, Japan). For in vivo detection of green fluorescent protein (GF P), fi‘esh leaf tissue was mounted in water and viewed with an L5 filter set (excitation 455 nm to 495 nm, emission 512 to S 75 nm) and a 100X oil immersion objective of a Leica DMR A2 microscope (Leica Microsystems, Wetzlar, Germany) equipped with epifluorescence illumination. Images were captured with a cooled CCD camera (Retiga 1350EX, Qimaging, Burnaby, British Columbia, Canada) and processed with Adobe Photoshop imaging software (Adobe Systems, San Jose, CA). Fine-mapping of ARC5. The arcS mutation was previously mapped between markers ngal62 (20.6 cM) and AtDMCl (32.6 cM) on chromosome 3 (23). To fine map ARC5, an F2 population was generated from a cross between arc5 and Col-O wild type. Out of 7000 F2 plants, 1720 mutants were identified by microscopy. Markers used for polymerase chain reaction (PCR)-based mapping (24) are provided as online supplemenatry material. 56 Identification of a Candidate ARC5 Gene. BAC clones MMB12 and MPN9 were double-digested with HincII and HindIII. The resulting fragments were inserted between the cauliflower mosaic virus (CaMV) 358 promoter and octopine synthase (OCS) terminator in the plasmid SN506 (4) that had been previously digested with Smal and HindIII to remove the insert. Ligation products were amplified in E. coli strain DHSor, transferred to Agrobacterium tumefaciens GV3101, and introduced into wild-type Arabidopsis (Col-0) by floral dipping as described previously (25). T1 transgenic plants were selected by growth on kanamycin and screened for an arc5-like phenotype by microscopy. The T-DNA inserts from the two arc5-like plants identified were amplified by PCR using vector primers flanking the inserts. The PCR products were sequenced to determine which fi'agments from the BAC clones were carried by these plants, as well as their orientation with respect to the promoter. Both plants carried the same HindIII-Hincll fragment from At3g19730 (see Results). To confirm that this fi'agment was the source of the arc5-like phenotype in the T1 transgenics, the fragment was subcloned into SN506 in the antisense orientation as described above and introduced into wild-type Col-O plants. Following selection on kanamycin, the phenotypes of the resulting T1 plants were determined by microscopy. Amplification of ARC5 cDNA. Primers used for RT-PCR were 5’-GAAAAAGGAACGGCGACGAAAAC-3’ and 5’-GCAAACATTGGACCAAAAAGCG-3’. Amplified cDNAs were subcloned into Bluescript KS+ (Stratagene) prior to sequencing. 57 Sequence Alignment and Phylogenetic Analysis. The amino acid sequence of ARC5 was deduced fiom the cDNA sequence. The sequence alignment shown in Fig. 3A was performed with the CLUSTALW multiple alignment program (26) at the Biology Workbench 3.2 website (http://biowb.sdsc.edu/). Protein sequences used for the phylogenetic analysis shown in Fig. 38 were aligned with Clustal X (27) using default settings. The alignment is available upon request. Neighbor joining and maximum parsimony analyses were performed using PAUP version 4.0b10 (28) with default settings except forties being randomly broken. Neighbor-joining and maximum parsimony analyses produced topologically identical trees. Bootstrap analyses were performed on the neighbor-joining and maximum parsimony trees with one thousand replications. GenBank accession numbers for proteins aligned with ARCS (longer form, accession no. AY212885) are as follows: human Dynamin-1 (NP_OO4399), yeast Dnmlp (NP_013100), At1g53140 (NP_175722), rice dynamin like protein (BAB56031), ADL6 (AAF22291), At5g42080 (NP_568602), Glycine phragrnoplastin (AABOS992), tobacco phragrnoplastin (CABS6619), At2g44590 (NP_181987), human Dynamin H (NP_OO4936), ADL2a (NP_567931), ADL2b (NP_565362), rice ADL2-like protein (BAB86118), worm Drp-l (AAL56621) and human Dnmlp/Vpslp-like protein (J C5695). Complementation Analysis. The genomic fragment corresponding to ARC5 (At3g19730 and At3g19720 in the Arabidopsis database, see Results), including 1.9 kb and 1.1 kb of the 5’ and 3’ flanking 58 DNA, respectively, was amplified from MMB12 by PCR using the primers 5’- GGAATTCCGAGTCGAGTTGCTTTGTTG-3 ’ and 5 ’- CGTCTAGAGCTTACCTCAAAGGTACATGGA-3’. The PCR product was digested with EcoRI and ligated into a derivative of the transformation vector pLH7000 (http://www.dainet.de/baz/jb2000/jb_2000direkt.htm) digested with EcoRI and Smal. The construct was transferred to A. tumefaciens GV3101 and introduced into arc5 plants by floral dipping. The phenotypes of the T1 plants were determined by microscopy. GFP-ARC5 Localization. The GFP sequence was amplified from plasmid stS-GFP (29) with the primers 5’-CGGGATCCATGAGTAAAGGAGAAGAACT-3’ and 5’- GCTCTAGATAGTTCATCCATGCCATGT-3’. The PCR product was digested with BamHI and Xbal. The ARC5 coding region and 1.1 kb of the 3' flanking DNA were amplified from the MMB 12 BAC clone with primers 5’- GGACTAGTACGATGGCGGAAGTATCAGC-3’ and 5’- CGGGATCCGCACCGAAGGAGCCTTTAGATT-3’. The PCR product was digested with SpeI and EcoRI. cDNA fragments encoding GF P and ARCS were subcloned into Bluescript KS+ (Stratagene) that had been digested with EcoRI and BamHI to create a GFP-ARC5 fusion construct. The ARC5 promoter was amplified from MMB12 with primers 5’-GACTAGTTGGCTCAACGCTTACCTCAA-3’ and 5’- CGGGATCCGCCATCGTCTCTTACGA-3’, and cloned into Bluescript KS+ (Stratagene) between the SpeI and BamHI sites. The promoter fragment was then subcloned into the plasmid containing the GFP-ARC5 fusion construct at the 5’ end of 59 the fusion. The resulting plasmid was digested with Spel and EcoRI, and the promoter- GFP-ARCS cassette was subcloned into a derivative of the transformation vector pLH7000 (http://www.dainet.de/baz/jb2000/jb_2000direkt.htm). The plasmid was transferred to A. tumefaciens GV3101 and used to transform wild-type A. thaliana plants (Col-0) as described above. The GFP-ARCS localization pattern was visualized by fluorescence microscopy in T1 plants. In Vitro Chloroplast Import and Protease Protection Assays. Transcription/translation reactions, chloroplast isolation, in vitro import reactions, proteolytic treatments, and post-import fractionation and analysis were performed as described (7). The longer ARC5 cDNA, after subcloning into Bluescript KS+ as described above, was used for these experiments. Results Phenotype of arc5. Like other chloroplast division mutants, leaf mesophyll cells in arc5 (Ler) mutants contain fewer and larger chloroplasts than do wild type cells, which have about 120 chloroplasts at maturity (21, 22) (Fig. 1A). The arc5 mutant has been reported to contain an average of 13 chloroplasts in fully expanded mesophyll cells (21, 22), though the phenotype appears somewhat more variable in our hands, with arc5 cells typically containing between 3 and 15 chloroplasts (Fig. 1 B and C). This difference may reflect differences in growth conditions. Chloroplast number is fairly uniform within an 60 Fig. 1. Comparison of chloroplasts in Arabidopsis leaf mesophyll cells. (A) Wild- type (Ler). (B, C) arc5. Cells are from fixed tissue. Bars: A-C, 10 um. 61 individual arc5 plant, and, as shown for other are mutants (30), chloroplast size in arc5 varies inversely with chloroplast number (not shown). As has been reported previously (21, 22), arc5 chloroplasts frequently remain dumbbell shaped (Fig. 13). Fine Mapping of ARC5 and a Novel Antisense Strategy for Identification of an ARC5 Candidate Gene. ARC5 was previously mapped to a region of chromosome 3 flanked by the markers ngal62 (20.6 cM) and AtDMCl (32.6 cM) (23) (Fig. 2A, upper bar). We generated a new mapping population and used standard methods to fine map the arc5 locus to a 92-kb interval corresponding to two overlapping BAC clones, MMB12 and MPN9 (Fig. 2A, lower bar). This interval contained 24 predicted genes, but no clear ARC5 candidate. To identify the gene most likely to represent ARC5, we digested MMB12 and MPN9 with restriction enzymes, and subcloned the resulting fragments between the CaMV 358 promoter and OCS terminator in a derivative of the plant transformation vector pART27 (31), creating a library of fragments spanning the ARC5- containing interval. The library was designed such that at least one fragment fi'om each predicted gene in the interval would be ligated to the vector in the antisense orientation, with the expectation that an antisense fragment from ARC5 would yield an arc5-like phenotype. The library was used to transform wild-type Arabidopsis plants (Col-O), and transgenic plants were screened for chloroplast division defects by microscopy. Among 120 independent T1 individuals examined, two had fewer and larger chloroplasts per cell than wild type (Fig. 2E), and resembled arc5. The inserts carried by 62 Fig. 2. Cloning of ARC5. (A) Fine mapping of ARC5. Triangle indicates the position of ARC5. (B) Open reading frames of ARC5. Black boxes represent exons; solid lines represent introns; gray box indicates the alternatively spliced intron. The mutation in arcS and its position are indicated. (C) Amplification of the transgene inserts in two arcS-like T1 plants produced by transforming wild type plants (Col-0) with a library of sense and antisense fragments derived from MMB12 and MPN9. 100 bp DNA ladder (New England Biolabs) is shown at left. Arrow indicates a HindlII-Hincll fragment from At3g19730 common to both arc5- like transgenic lines. (D-G) Single leaf mesophyll cells from (D) Col-0 wild-type, (E) an arc5-like T1 plant described in C, (F) the arc5 (Ler) parent line used for complementation by ARCS, and (6) an arc5 plant transformed with the ARC5 gene. Bars: D-G, 10 um. 63 £02.; (30 agTTIIIIII—irlill s 4‘ 3mm :98 8rd 2x“ 89d 98d 93.. o hb— thththth PPrhbbPFPP—bbhb—Dbe-hhub—Db — h - —+brh _ r .6 N _ _ _ a _ _ «L .F IT _ _ :5: 3.2! 3.33 opmNS .253 «333 o§§\ 800.3 38.8 308 305 88.3 Iain 3 300.8 ______ __ A _ _____ __ v Bio? 33.... 3a: 5.3. :5: «use: 82. ~28: m o 8:33:3on 0060.0 03 n .50 64 Fig. 2 continued a c .33.. 65 the transgenes in these two transgenic lines were amplified by PCR and sequenced. Two different inserts were detected in both lines (Fig. 2C). One line carried a fragment derived fiom the BAC backbone DNA and a second fragment derived from At3g19730 on MMB12 (Fig. 2C, lane 2). The other line carried a fiagment from At3g19760 on MMBIZ as well as the same fi'agment from At3g19730 (Fig. 2C, arrow). In both plants, the At3g19730 fragment was present in the antisense orientation. To confirm that the arc5 -1ike plastid division phenotype in the two transgenic lines was caused by the shared At3g19730 fragment, we constructed a transgene containing this fragment in the antisense orientation, and introduced it into wild-type Arabidopsis (Col-0). Out of 80 transgenic plants examined under the microscope, 20% had chloroplast division defects similar to those of arc5 and the transgenic line shown in Fig. 2E. These findings indicated that the ARC5 gene corresponded to At3g19730. Sequencing of arc5 and Mutant Complementation. At3g19730 has homology to the dynamin family of GTPases (32). In the Arabidopsis database, At3g19730 and the immediately adjacent sequence, At3g19720, were annotated as separate genes. However, sequence alignments to other dynamin-like proteins as well as EST data from Arabidopsis and other plants strongly suggested they were parts of a single gene, referred to hereafter as At3g19730/At3g19720, and that the annotated start codon for At3g19730 and stop codon for At3g19720 represented the true start and st0p codons of this gene. This was confirmed by subsequent cDNA isolation and analysis (see below). To establish whether arc5 contained a mutation in this region of 66 the genome, we sequenced the At3g19730/At3g19720 locus in both the arc5 mutant and in wild-type Landsberg erecta. The data showed the presence in arc5 of a G-to-A mutation, which altered a tryptophan codon (TGG) to a stop codon (TAG) (Fig. 28). We then introduced a transgene containing the predicted At3g19730 /At3g19720 locus plus 1.9 kb and 1.1 kb of the 5’ and 3’ flanking DNA, respectively, into arc5 mutants to determine whether the wild-type DNA could complement the arc5 mutation. Microscopic analysis of T1 transgenic plants indicated that the chloroplast division defect in the mutant was fully or partially rescued by the wild-type transgene (Fig. 2G'). Differences in gene dosage resulting from position effects probably account for the partial complementation in some individuals (33). Taken together, the point mutation in At3g19730 /At3g19720 in arc5, complementation of the mutant phenotype by the wild- type gene, and ability of a fragment from At3g19730 /At3g19720 to confer an arc5-like phenotype in wild-type plants when expressed in the antisense orientation, indicate that the ARC5 locus and At3g19730 /At3g19720 represent the same gene. Analysis of ARC5 cDNAs and Gene Products. To gain more information on the ARC5 transcription unit and encoded polypeptide, we used RT-PCR and primers spanning from 93 bp upstream to 152 bp downstream of the predicted At3g19730 /At3g19720 start and stop codons, respectively, to amplify the corresponding cDNA. Sequence analysis revealed two distinct cDNA species. The longer cDNA contained a sequence that was spliced out of the shorter cDNA as the 15th intron (Fig. ZB); however, its presence in the longer cDNA did not interrupt the reading frame. More than half the cDNA clones recovered were of this type. This 67 Fig. 3. ARC5 is a dynamin-like protein. (A) Alignment of ARCS with Dynamin-1 from Homo sapiens and Dnm1 p from Saccharomyces cerevisiae. Gray boxes indicate completely conserved residues; yellow boxes are identical residues; cyan boxes are similar residues; dashes indicate gaps. The domain structure is indicated by the lines above the alignment. Red, GTPase domain; green, middle domain; blue, PH domain; lavender, GED domain; black, PR domain. The dotted underline indicates the sequence encoded by the alternatively spliced intron in ARC5. The triangle indicates the position of the arc5 mutation. (B) Phylogenetic analysis of ARC5 with an unrooted neighbor-joining tree. Bootstrap values are shown at selected nodes. The first and second bootstrap values are from the neighbor-joining and parsimony analyses, respectively. Nodes with less than 50% bootstrap support, or not present, are represented by a ‘-’. Accession numbers for the sequences aligned with ARCS are listed in Materials and Methods. Images in this figure are presented in color. (A) is published in Proceeding of the National Academy of Science of USA 2003 Apr 1; 100 (7):4328-33. 68 anhmmflnalaflmhm100u¢m%>0ma4ula~mm>OEAAOhanam‘¢msna>aa‘00#4w°1 liar-oiutroninutia ....... IODQAGMQ tttttttt ladsIIOIJ¥I£I>IIKIIH¢K>MIAINIH IIIIIQIIIIIIIOIIIIIIIIIIIOI IIIIIIIIIIIIIIIIIIIII AH.qu~h“““*>°amu>uau‘ uomm‘mm>‘h‘fllOmhmunkfinflIOGm>nO>Odlunn>ahfilmkn>hhkluloHHQJ‘IIA‘IfiISAIHQK KHBHHDLHandnnlh‘IIJNIlda4mAKa>Halddhulhflal‘m‘dhrluHOOhHOO>A‘N>H 11111111 AdlflhARI“*4:B>AIIO>wGHU¥I>AAUI>OOMHIIKHthfiu>ndz¥HdIUIdlflnlhamni..- IQBIODOUMflAZ‘JAMmhHhIKHZIHIANZHHIQIAOKBHIB>HGIVGD>AZKHPI>O~IAOADIDIIHQQ .................................................u................. JIIIIAHmQJJD‘AISIHBOHmmuaflHimddlmdl>nm¢u¥hGHHO0AA0cK IDiOhhfittIQIKDIFOflmIKILh>OG¢JIu .III 5.! ¥n¥r>280hh lha‘hulla¢3h0lfi>9lfi¥dlo>n IKIIAOIH>> ....... “khan Ilanlballn‘m‘>dlqh>ll0mth0IKOdAN>wHQJJ DODHZImHmmB tttttt OZHOZFOOZNOOQAKmKddintrialuflflflhlxunnlhlwflluouuiHUHIIHHASUKmH>dHIOOSOQHKKM2802n800424hOHthIZHBI 0A0=AhmuLkll<¥‘>(H>U‘H¢muikon=HQI>OUGZ>HIIIHHQ&UIH‘O>dhflhl4l4ulra‘00&fifl l>umxkaoamflfldduuH>IHAINMAKAVK4AI‘QOUKHUHKuIIn.--u.-.IQIHN>JI>UKOWAIARJE S>OIKBKOIKIIHNHF>H~IIHIBA¢QNOOAKKB UOK>Bmo .......... HAImH>IQ>UMJUQIKHKH macaoahu>uGBOOH~laOAkIOIInnm‘>>fluldddmdlhdhdnflhkflufllldlfllndmug-Iu>flad¥u ‘JIn>hdhmlnohm2uH15~>9d>ndlnhhanm8dml°u>llfi~yrH310 OUAIHBZHGDQHOGHQmHhI ‘lOnthOHGHOEHIIH<¥NHHHKQIIIOhIIK>AIhGhBIILHKZHK‘O lemdlkhnmonomvlnfluflhn 4AMHAh>¢lfl¥¢fl~dallah9ldKaHOHuni-OKBAAKOAIIMAmfinonllfldm>¢oxhflaznfifibmaoou >4u48wlzhh00¥ufilxuh A‘lklxdmQaauhlflrlfl>hnflHmaqowOAIZGaOAAFQKHNIHAOOIA>KOdfimHOIIO¢AIshmmlmah hhmwOOAJmmnq¢w4 uuuuuuu mtmdm>fl>0mm0mt0aHlkfifllkm>>HfllmAHhQ>OI>Hl¢kHkflld0HOOOmuZ>>O>IOARAQNIflOmAHDadzHDmnIAnAUHH>OHHIIOO§O>M849> FHDKHDDH9K0m¢2>>0Hl¥0234hJAMZIA>DK‘QHOMOIQDAHHH>GHBIOOOhfl>fl¥‘>flag¢zoa«announce:Han-an”Hua-mouxro>n>uuuuuknenan>ahqxq>nmun>uqznmHuunouo-uo‘uu‘anzn oznuk>uoxaxa¢H0mnHoma--oao>m>uaxoaga>auaxa>uamu>¢qzua>auuouz-hoe>uoaummnnnmoawuq tttttttttttttttttttttttttttttttttttt fihooounhfllfldhufiflflflfl xluuomHlahI03l04llll¥01¥ldmflk0¢lhouHMB>IDI§I>OIDGIIHJGOIAIHIZJOd>dfidIk> HhKflOXUlahM<>fl .......................................... khdl>d0d>ahllh> hBIA4I>J‘IIOQBOOC>>A>40H>QKH4I QO>AHIAHmulOmuOm°§>GAHhann-BGHDmD uuuuuuuuuu NS>DOAK2>HQHADIdtuu¢quuni I0>mlfld>mul°>‘H0manaQ‘zooH‘ iiiiiiiiiiii un4nonm2>amnqnl80flzox ..... moo ova our new nvo Ham com can wan oov onv 0H! 0mm ohm «an or“ am" and mom and mNH «on no no vu a H H nulan d-a«lsn>n Quinn utaulsnao “also a-u«lsa>n manna Hindi-aha naflun Htfldlduha malan ducalsa>n Bulge n-a«las>a Quinn a-d«la:>n Quinn atnqlanhn nflluo a-:«ls:>o manna u-:«lan>n Anlan ntnulaahn mbfld use.» salsa nua‘ use.» nolan muu‘ and-S gala: m0u4 use.» salsa mux‘ oason nulsn mux‘ nan.» salsa mumd use.» gala: muud under aalau mom‘ and.» allflw m0¢¢ use.» ails: nou4 a...» gala: mead use.» salsa < 69 Fig. 3 continued Glycine Phragmoplestin 02107 1001100 AT5942000 ‘°°"°° Tobacco phrangplastin M29445” 1001100 00170\ "mo. 1.— Rice Dynamin-like protein was , ARCS fl ADLC M19102” Hu ' ll 1001100r— man Dynamrn 021- ]— Human Dynamin-1 1001100 ADLZb 1112914120 1001100 1001100 ADL2a AT4933000 Rice ADL2-like protein Yeast Dnmtp Worm DRP-t Human Dnm1 prst p-like protein 7O cDNA encoded a protein of 777 amino acids (Fig. 3A) and 87.2 kDa. The protein can be aligned over its entire length with numerous members of the dynamin family and contains three motifs found in other dynamin-like proteins: a conserved N-terminal GTPase domain, a pleckstrin homology (PH) domain shown in some proteins to mediate membrane association, and a C-terminal GTPase Effector Domain (GED) thought to interact directly with the GTPase domain and to mediate self-assembly (32, 34) (Fig. 3A). The shorter cDNA encoded a protein of 741 amino acids and 83.5 kDa identical to that of the larger gene product except for the absence of 36 amino acids encoded by the sequence of the 15th intron (Fig. ZB and 3A). These results suggest that the ARC5 transcript is alternatively spliced. Alternative splicing of dynamin genes in several other organisms has also been documented (34). Because the shorter gene product lacks a portion of the PH domain (Fig. 3A), the proteins encoded by the two splice variants may have different activities or localization patterns. Phylogenetic analysis was performed to investigate the relationship between ARC5 and other members of the dynamin family of proteins. Only full-length sequences were used, though EST data indicate that related proteins are present in many plants and in green algae (not shown). ARC5 clustered with a group of proteins found in plants, but was in a distinct clade from other dynamin-like proteins in Arabidopsis with functions in cell-plate formation and mitochondrial division (35, 36) (Fig. 3B). Surprisingly, the ARC5-like proteins clustered near ADL6, another Arabidopsis dynamin-like protein involved in vesicle trafficking from the trans-Golgi network to the vacuole in plants (3 7). This suggests that ARC5 and ADL6 could share a common dynamin-like ancestor. 71 Based on the similarity of ARCS to dynamin and its relatives (Fig. 3A), we conclude that ARC5 represents a new class of a dynamin-like proteins that functions specifically in chloroplast division. Localization of ARC5. We expressed a GFP-ARC5 fusion protein in transgenic plants to investigate the subcellular localization of ARC5. Because overexpression of chloroplast FtsZ proteins can result in a dominant-negative phenotype (10), we used the native ARC5 promoter to create the GFP-ARC5 transgene for expression in wild-type plants (Col-0). Fluorescence microscopy showed that the fusion protein was localized in a ring-like pattern at the site of the chloroplast constriction (Fig. 4). This ring could be faintly detected in unconstricted chloroplasts, suggesting that ARC5 may act at an earlier stage of division than previously hypothesized (21, 22). However, ARC5 is not required for FtsZ ring formation, the earliest known event in the assembly of the chloroplast division apparatus (1 8, 19, 40), since the FtsZ ring can be detected in the arc5 mutant (not shown). The GFP-ARCS fusion protein was most obvious in visibly constricted chloroplasts, perhaps as a consequence of ring thickening during constriction. Similar localization patterns have been described for F tle and F tsZZ (10). However, in contrast to the FtsZ rings, which are fairly uniform in fluorescence intensity, the GFP-ARC5 ring was more speckled in appearance (Fig. 4), suggesting that ARC5 localization at the division site may be discontinuous or that the ARC5-containing ring is not of uniform composition. 72 Fig. 4. GFP-ARC5 is localized to the constriction site of dividing chloroplasts. (A) Bright-field; (B) GFP fluorescence. Arrows indicate corresponding positions in the two images. Bars: A-B, 10 um. 73 Fig. 5. ARCS is on the outside surface of chloroplast. Radiolabeled ARCS (upper panel), tp110-110N, the precursor of the inner envelope marker protein 110N (center panel), and p88, the precursor of the stromal marker protein mSS (lower panel), were produced by coupled in vitro transcription/translation, and subsequently incubated with isolated pea chloroplasts (7). Chloroplasts were recovered by centrifugation and incubated with (+) or without (-) therrnolysin. Intact chloroplasts were again recovered and fractionated into membrane (P) and soluble (S) fractions. TP, translation product. 74 _ + 'Thermolysin TP P S" P 5 I _._ «ARC5 I 4-tp110-110N H... 4—110N 4- p88 1 2 3 4 5 75 Even though ARC5 mediates chloroplast division, it is not predicted by subcellular targeting prediction programs to be imported to the chloroplast (results not shown). To further define the topology of the ARC5-containing ring with respect to the chloroplast envelope membranes, we employed in vitro chloroplast import and protease protection assays. A radiolabeled translation product corresponding to the longer ARC5 cDNA was generated by coupled transcription/translation (Fig. 5, top panel, lane 1), then incubated with isolated pea chloroplasts (Fig. 5, top panel, lanes 2-5). Subsequent fractionation of the chloroplasts indicated that the translation product was associated with the membrane fraction, but was not processed (Fig. 5, upper panel, lanes 2 and 3). The binding of the ARC5 translation product to isolated chloroplasts may be effected in part by the PH domain, which has been shown to mediate lipid binding of other dyanamin- like proteins (34, 38). In contrast, two chloroplast-targeted control proteins, one localized to the inner envelope (Fig. 5, center panel) and the other to the stroma (Fig. 5, lower panel), were processed upon import, consistent with the presence of N-terminal transit peptides, and associated with the membrane and soluble chloroplast fractions, respectively (Fig. 5, center and lower panels, lanes 2 and 3). In addition, the two control proteins were both protected from proteolysis by therrnolysin, which does not penetrate the outer envelope (39) (Fig. 5, center and lower panels, lanes 4 and 5), whereas the ARC5 translation product was fully degraded by this protease (Fig 43, upper panel, lanes 4 and 5). These data provide evidence that the ARC5-containing ring represented by the GFP-ARC5 fusion protein is situated on the cytosolic surface of the outer chloroplast envelope membrane. The position of ARCS on the chloroplast surface is topologically 76 equivalent to that of Dnmlp, a dynamin-like protein that mediates mitochondrial division in yeast (40). Discussion Although the arc5 mutation introduces a premature stop codon, the mutant phenotype probably results from a decreased rate of constriction rather than from a complete block in the process. This conjecture is based on the finding that arc5/arc] double mutants have more and smaller dumbbell-shaped chloroplasts than does arc5, indicating that arc5 chloroplasts are capable of completing constriction (21, 23). The slow but continued chloroplast division in arc5 may be due to the presence of a second ARC5 homologue (At1g53140) in a duplicated region of the Arabidopsis genome (41) whose function might overlap to some extent with that of ARC5 . Alternatively, it is conceivable that the arc5 gene product, though severely truncated, could retain partial activity since the mutation occurs just downstream of the GTPase domain. In either case, because total chloroplast volume is maintained as a constant proportion of cell size both in wild-type Arabidopsis plants and in mutants with reduced chloroplast numbers (30), the enlarged size of arc5 chloroplasts can probably be explained by sustained expansion of slowly constricting chloroplasts until the prescribed volume is reached. Previous studies of the arc5 mutant phenotype have suggested that the gene product functions in the constriction of chloroplasts, but not of undifferentiated proplastids (22). This could be the result of reduced gene dosage in the mutant; expression of the ARC5 homologue in arc5 mutants may be sufficient to maintain the 77 division of the few, small proplastids in the meristem, whereas higher levels of ARC5 and its homologue may be required for division of the much larger chloroplasts during leaf expansion. The observation that all the plastid division genes identified thus far are present in only one or two copies in the Arabidopsis genome suggests that the same gene products function in the division of all plastid types. Future analyses of protein levels and accumulation patterns for ARC5, its homologue, and other plastid division proteins will shed light on this issue. Dynamin and its relatives are large GTPases that participate in a variety of organellar fission and fiision events in eukaryotes, including budding of endocytic and Golgi-derived vesicles, mitochondrial fission, mitochondrial fission, and plant cell plate formation (reviewed in (32, 34)). Dynamin has also been shown to regulate actin assembly and organization at membranes (42). ARC5 defines a new class of dynamin- like proteins that function specifically in plastid division, and its identification extends the range of cellular processes in which dynamin-like proteins participate. The molecular mechanisms by which this large group of GTPases function is still uncertain. Recent structural data (43, 44), along with studies showing that dynamin self-assembles into rings (45) and can tubulate and vesiculate liposomes in vitro (46, 47), support proposals that dynamin forms a collar around the neck of budding vesicles during endocytosis and acts as a GTP-stimulated, force-generating “constrictase.” However, other studies provide evidence that dynamin family members function as molecular switches, analogous to classical signaling GTPases (48). Functional investigations of dynamin and 78 its relatives will clearly be relevant to future studies aimed at determining the biochemical mechanism underlying ARC5 activity during chloroplast division. The localization of ARC5 on the outer envelope surface at the site of chloroplast constriction parallels that of the yeast dynamin-related protein Dnmlp and its orthologues in animals and plants, which localize to the mitochondrial division site on the cytosolic side of the outer mitochondrial membrane (32, 34, 36, 40). This suggests that ARC5 is the chloroplastic counterpart of these mitochondrial division proteins. However, mitochondrial and chloroplast division are not equivalent processes. Chloroplast division requires the activity of several prokaryotically derived proteins in the chloroplast stroma, including F tle, FtsZZ, MinD, MinE and ARTEMIS (6, 7, 11-13, 49), whereas related molecules are lacking in the mitochondria of animals, plants, and fiingi, and no matrix- localized mitochondrial division proteins have been identified in these organisms. Interestingly, the topology of the ARCS-containing ring is similar to that of the outer PD ring (15, 50). Like ARC5, which lacks homologues in cyanobacteria and was probably derived from the eukaryotic host cell, the outer PD ring has been proposed to be of eukaryotic origin (19). Thus, ARC5 could be an outer PD ring constituent. Further, the outer PD ring is composed of filaments about 5 nm in diameter (50), which is close to the diameter of previously observed dynamin strands (51). These similarities suggest that the outer PD ring filaments could be composed of ARC5. However, it has been suggested that the filament protein is an unidentified polypeptide of 56 kDa (50), which is considerably smaller than ARC5. Irrespective of the relationship between ARC5 and 79 the outer PD ring, our finding that a dynamin-related protein functions in chloroplast division indicates that the chloroplast division machinery is partly of prokaryotic and partly of eukaryotic origin, and shares at least one component in common with the machineries regulating fission of other eukaryotic organelles. Identification of this new chloroplast division protein will facilitate studies aimed both at further dissection of the plastid division machinery and at understanding how the activities of protein complexes separated by the two envelope membranes are coordinated to achieve constriction of the organelle. Acknowledgement We thank Kevin Pyke, University of Nottingham, and the Arabidopsis Biological Resource Center, Ohio State University, for arc5 seeds, StanislavVitha for advice on microscopy, Kevin Stokes for help with phylogenetic analysis, Magali Tshiamala for technical assistance, and all the members of the laboratory for helpful comments. Supported by a grant from the Michigan State University Intramural Research Grants Program. 80 Online 51 Table 1. BAC clone MDC 8 .‘vlCBZZ MYI l l MLD 1 4 i BM 1 8 .\-f.\lB l.‘ F16J l 4 1.18.46 MAL21 MPN9 m"11312 MPN9 MP.\'9 Online supporting Information: Table 1. PCR markers used for fine mapping ARC5 BAC clone MDC 8 MCBZZ MVIll MLD14 T31J18 MMB12 F16Jl4 MSA6 MAL21 MPN9 MMBlZ MPN9 MPN9 Primer sequences for PCR 5 ’-GATTAATGAGACTATATATGAGAG-3 ’ and 5 ’- ATCTGCATAACTTCAATTGAACTG-3 ’ 5’-GAACCCCCAGAATATCAACATC and 5 ’- GCTCTGATGGTGATTCTGGTAAC-3 ’ 5’-GTAGCATTCTTTAGAGATTGATCTAG-3’ and 5 ’ -TATTCGAGTTTGAAATTATGATTTATGC-3 ’ 5 ’-GCTACAGTTCTCAACCGGTAAATC-3 ’ and 5 ’ - CATAAGCTTTTATGCTCCAAAATAGTCTC-3’ 5 ’-CTTGATCTTGTGTTCTGACATCTC-3 ’ and 5 ’- CTAAACTATTCACAAATGCCATAGACG-3’ 5 ’-AGCCGTCTTGTCCCATCATTAAAG-3 ’ and 5 ’ - GCACAAACAAACAGGGTCAATAGTTA-3 ’ * 5 ’ -TTAAAGTGAAGCTTAAGCAGAGG-3 ’ and 5 ’ - CATTGTTAGAAAGTCAACACTTTG-3 ’ 5 ’-GCAAGACATAACCAATGAACAAG-3 ’ and 5 ’- GACACGTATGCGTTTCTAAGAG-3 ’ 5’-CTCCAACTTCAAGCAAAACGGATG-3’ and 5’- C TC TGTTTTTTGGGCTAGTGATGG-3 ’ 5 ’-GCATACCCAATATCCTTTGTGC-3 ’ and 5 ’ - GATAGTATAAC C AGAGGTTGGAG-3 ’ * 5’-GAATCTTCTCAAACTGAAATCCACC-3’ and S’-TCGAAAGGAAGATCGGTGAACC-3’§ 5’-GATTGTGCTATGGTTCAGGAGTTC-3’ and 5’- CATCAGCTATAACCTCCTCAGTG-3’§ 5 ’-ACTGACTATAAGGACCCCTCAAAC-3 ’ and 5 ’ - GTTGACCATAATTCATCCACCACTATTA-3 ’§ 81 Marker type, enzyme INDEL“ INDEL INDEL INDEL CAPS“, DraI CAPS, EcoRV INDEL INDEL INDEL CAPS, T sp509I CAPS, T an CAPS, Ach INDEL (cut by HindIII) BAC, b *lnserth +Cleavet :These t "No reco BAC, bacterial artificial chromosome. *lnsertion-deletion. lCleaved-amplified polymorphic sequence. :These two markers defined the final interval containing arc5. §No recombination between these markers and arc5 was observed. 82 A c, 1121 Mi} (19' Mi} 207 10. 11. 12. l3. 14. 15. l6. 17. 18. Cavalier-Smith, T. (2000) Trends Plant Sci 5, 174-82. Martin, W., Stoebe, B., Goremykin, V., Hansmann, S., Hasegawa, M. & Kowallik, K. V. (1998) Nature 393, 162-165. Osteryoung, K. W. & Vierling, E. (1995) Nature 376, 473-474. Osteryoung, K. W., Stokes, K. D., Rutherford, S. M., Percival, A. L. & Lee, W. Y. (1998) Plant Cell 10, 1991-2004. Strepp, R., Scholz, S., Kruse, S., Speth, V. & Reski, R. (1998) Proc Natl Acad Sci USA 95, 4368-4373. Fujiwara, M. & Yoshida, S. (2001) Biochem Biophys Res Commun 287, 462-7. McAndrew, R. S., Froehlich, J. E., Vitha, S., Stokes, K. D. & Osteryoung, K. W. (2001) Plant Physiol 127, 1656-1666. Mori, T., Kuroiwa, H., Takahara, M., Miyagishima, S. & Kuroiwa, T. (2001) Plant Cell Physiol 42, 555-9. Osteryoung, K. W. & McAndrew, R. S. (2001) Annu Rev Plant Physiol Plant Mol Biol 52, 315-333. Vitha, S., McAndrew, R. S. & Osteryoung, K. W. (2001) J Cell Biol 153, 111- 119. Colletti, K. S., Tattersall, E. A., Pyke, K. A., Froelich, J. E., Stokes, K. D. & Osteryoung, K. W. (2000) Curr Biol 10, 507-516. Itoh, R., Fujiwara, M., Nagata, N. & Yoshida, S. (2001) Plant Physiol 127, 1644- 55. Maple, J., Chua, N. H. & Moller, S. G. (2002) Plant J31, 269-77. Fulgosi, H., Gerdes, L., Westphal, S., Glockmann, C. & 8011, J. (2002) Proc Natl Acad Sci USA 99, 11501-6. Kuroiwa, T., Kuroiwa, H., Sakai, A., Takahashi, H., Toda, K. & Itoh, R. (1998) Int Rev Cytol 181, 1-41. Miyagishima, S., Itoh, R., Toda, K., Takahashi, H., Kuroiwa, H. & Kuroiwa, T. (1998) J Electron Microscopy 47, 269-272. Miyagishima, S., Kuroiwa, H. & Kuroiwa, T. (2001)P1anta 212, 517-528. Miyagishima, S., Itoh, R., Toda, K., Kuroiwa, H. & Kuroiwa, T. (1999) Planta 207, 343-353. 83 19. (J) ’1’) 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. Miyagishima, S., Takahara , M., Mori, T., Kuroiwa, H., Higashiyama, T. & Kuroiwa, T. (2001) Plant Cell 13, 2257-2268. Kuroiwa, H., Mori, T., Takahara, M., Miyagishima, S. Y. & Kuroiwa, T. (2002) Planta 215, 185-90. Pyke, K. A. & Leech, R. M. (1994) Plant Physiol 104, 201-207. Robertson, E. J ., Rutherford, S. M. & Leech, R. M. (1996) Plant Physiol 112, 149-159. Marrison, J. L., Rutherford, S. M., Robertson, E. J ., Lister, C., Dean, C. & Leech, R. M. (1999) Plant J 18, 651-662. Jander, G., Norris, S. R., Rounsley, S. D., Bush, D. P., Levin, I. M. & Last, R. L. (2002) Plant Physiol 129, 440-50. Osteryoung, K. w. (2000) Plant Physi01123, 1213-1216. Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994) Nucleic Acids Res 22, 4673-4680. Thompson, J. D., Gibson, T. J., Plewniak, F., J eanmougin, F. & Higgins, D. G. (1997) Nucleic Acids Res 25, 4876-82. Swofford, D. L. (1998) PA UP *. Phylogenetic Analysis Using Parsimony (*and Other Methods). Version 4. 0b10 (Sinauer Associates, Sunderland, Massachusetts). Davis, S. J. & Vierstra, R. D. (1998) Plant Mol Biol 36, 521-8. Pyke, K. A. (1999) Plant Cell 11, 549-556. Gleave, A. P. (1992) Plant Mol Biol 20, 1203-1207. Danino, D. & Hinshaw, J. E. (2001) Curr Opin Cell Biol 13, 454-60. Hooykaas, P. J. & Schilperoort, R. A. (1992) Plant Mol Biol 19, 15-38. Hinshaw, J. E. (2000) Annu Rev Cell Dev Biol 16, 483-519. Gu, X. & Verma, D. P. (1996) Embo J 15, 695-704. Arimura, S.-i. & Tsutsumi, N. (2002) Proc Natl Acad Sci U S A 99, 5727-5731. Jin, J. B., Kim, Y. A., Kim, S. J., Lee, S. H., Kim, D. H., Cheong, G. W. & Hwang, I. (2001) Plant Cell 13, 1511-26. 84 40. 41. 47. 48. 49. 50. 38. 39. 40. 41. 42. 43. 45. 46. 47. 48. 49. 50. 51. Lee, S. H., Jin, J. B., Song, J., Min, M. K., Park, D. S., Kim, Y. W. & Hwang, I. (2002) J Biol Chem 277, 31842-9. Cline, K., Wemer-Washburne, M., Andrews, J. & Keegstra, K. (1984) Plant Physiol 75, 675-678. Bleazard, W., McCaffery, J. M., King, E. J ., Bale, S., Mozdy, A., Tieu, Q., Nunnari, J. & Shaw, J. M. (1999) Nature Cell Biol 1, 298-304. Arabidopsis Genome Initiative (2000) Nature 408, 796-815. Schafer, D. A., Weed, S. A., Binns, D., Karginov, A. V., Parsons, J. T. & Cooper, J. A. (2002) Curr Biol 12, 1852-7. Niemann, H. H., Knetsch, M. L., Scherer, A., Manstein, D. J. & Kull, F. J. (2001) Embo J20, 5813-21. Zhang, P. & Hinshaw, J. E. (2001) Nat Cell Biol 3, 922-6. Hinshaw, J. E. & Schmid, S. L. (1995) Nature 374, 190-192. Takel, K., McPherson, P. S., Schmid, S. L. & De Camilli, P. (1995) Nature 374, 186-190. Sweitzer, S. M. & Hinshaw, J. E. (1998) Cell 93, 1021-1029. Sever, S. (2002) Current Opinion in Cell Biology 14, 463-467. Osteryoung, K. W. (2001) Curr Opin Microbiol 4, 639-46. Miyagishima, S., Takahara , M. & Kuroiwa, T. (2001) Plant Cell 13, 707-721. Klockow, B., Tichelaar, W., Madden, D. R., Niemann, H. H., Akiba, T., Hirose, K. & Manstein, D. J. (2002) Embo J21, 240-50. 85 Chapter 3 ARC5H, a Homolog of ARC5, is not Involved in Chloroplast Division 86 Abstract 6 mutation l Neverthel: One poten: ArabidOps; arci-"arcj/z IranSgene h ARC5H-G] These data . ARC5 is im from the Eu] Othe ChiOn Abstract arc5 is a chloroplast division mutant with 1-10 chloroplasts per cell. The mutation in the arc5 mutant in Arabidopsis causes a stop codon in the middle of the gene. Nevertheless, chloroplasts divide in arc5 albeit at a lower fiequency than in wild type. One potential explanation for the chloroplast division defect in arc5 is that an Arabidopsis homolog of ARC5 , ARC5H, could be redundant to ARC5. However, an arc5/arc5h double mutant showed the same phenotype as arc5. Further, a 35S-ARC5H transgene had no effect on chloroplast division either in arc5 or in wild-type plants. ARC5H-GF P is localized to speckles in the nuclei and is not associated with chloroplasts. These data strongly suggest that ARC5H plays no role in chloroplast division. Therefore, ARC5 is important but not essential for chloroplast division, and it may have evolved from the eukaryotic host during evolution to generate force and facilitate the constriction of the chloroplast division furrow. 87 Introdl l chloropl cell in vs chloropl: division 1 cell divis 2001; Shi division p protein of are localiz do their ho interferes v ChiOl‘OpIast de Boer et 2 et al., 2000; ARC6, a D) rESponsl‘b] e : to the 931050 ”Mam has 1 {Pl’ke and Le mutants and t of chlorop 138 t. Introduction Chloroplasts carry out photosynthesis in plant cells. It is widely believed that chloroplasts originated from cyanobacteria as endosymbionts. In the mature mesophyll cell in wild-type Arabidopsis plants, there are about 100 chloroplasts, suggesting that chloroplast division occurs (Osteryoung and McAndrew, 2001). Known chloroplast division proteins include: F tle, F tsZZ, ARC3, MinD and MinE, homologues of bacterial cell division proteins (Colletti et al., 2000; Itoh et al., 2001; Osteryoung and McAndrew, 2001; Shimada et al., 2004); ARC6, a homologue of the cyanobacteria-specific cell division protein Ftn2 (Vitha et al., 2003); and ARC5/AtDRPSB, a dynamin-related protein of apparent eukaryotic origin (Gao et al., 2003; Hong et al., 2003). F tsZ proteins are localized to and probably mark the division site in chloroplasts (V itha et al., 2001) as do their homologues in bacteria (Errington et al., 2003). Improper localization of FtsZ interferes with normal chloroplast division (Vitha et al., 2003). FtsZ localization in chloroplasts is regulated by MinD and MinE, which, as in bacteria (de Boer et al., 1991; de Boer et al., 1992), probably prevent FtsZ ring formation at non-division sites (Colletti et al., 2000; Itoh et al., 2001; Margolin, 2001; Maple et al., 2002; Vitha etal., 2003). ARC6, a DNA-J-like protein localized to the chloroplast division site, probably is responsible for F tsZ ring stability or maintenance (Vitha et al., 2003). ARC5 is localized to the cytosolic surface of the chloroplast division site (Gao et al., 2003). The arc5 mutant has 1-10 enlarged chloroplasts per cell that frequently have a dumbbell shape (Pyke and Leech, 1994; Robertson et al., 1996; Gao et al., 2003). The phenotype of arc5 mutants and the localization of ARC5 indicate that ARC5 is involved in the constriction of chloroplasts during chloroplast division (Gao et al., 2003). 88 complt chlorol At 1 gSE search . Among ARC 5, suggest chlorOp should I even be The phenotype of arc5 mutant also suggests that chloroplast division is not completely blocked. Since the arc5 is a null allele, ARC5 is either not essential for chloroplast division or is redundant to some other gene or genes. A homolog of ARC5, At1g53140 (hereafter ARC5H), was found in the Arabidopsis genome by a BLAST search of the protein database of Arabidopsis (http://www.arabidopsis.org/Blast/). Among all the dynamin-related proteins in Arabidopsis, ARC5H is not only closest to ARC5, but also can be aligned with ARC5 along its entire length (Figure 1A). This suggests that ARC5H may be redundant to ARC5 and responsible for the continued chloroplast division in the arc5 mutant. If this is true, an arc5/arc5h double mutant should have a more severe chloroplast division phenotype than that of arc5 and could even be lethal. To test the above hypothesis, an arc5/arc5h double mutant was made. It showed a phenotype similar to that of arc5. Furthermore, an ARC5H gene driven by the 35 S promoter could neither fully nor partially rescue the mutant phenotype of arc5. This transgene does not cause any chloroplast division phenotype in wild-type plants. Instead of being associated with chloroplasts, ARC5H-GFP is localized to a cluster of speckles outside the organelle. All these data strongly suggest that ARC5H is not redundant with ARCS but has a function unrelated to chloroplast division. 89 Figure 1 ARCSH is a homolog of ARCS and the closest relative of ARCS in plants. (A) Sequence alignment of ARC5 and ARCSH-AT195314O in Arabidopsis. The domain structure is indicated by the lines above the alignment. Red, GTPase domain; green, middle domain; blue, PH domain; lavender, GTPase effector domain. “*” indicates fully conserved residues; indicates residues with strong conservation; indicates residues with weak conservation; “-” indicates gaps. (B) Phylogenetic analysis of dynamin-related proteins in Arabidopsis with a rooted neighbor-joining tree. Bootstrap values from the neighbor-joining analysis are shown at nodes with >50% bootstrap support. The functions of the proteins are indicted on the left side. (C) Phylogenetic analysis ARCS and ARCSH in Arabidopsis, their homologs in rice and Chlamydomonas, and some other dynamin-related proteins with a rooted neighbor-joining tree. At4933650, At2914120, At1g59610 and At1g10290 have functions in mitochondrial division and trans-Golgi vesicle trafficking and are used as outgroups here. Bootstrap values from the neighbor-joining analysis are shown at nodes with >50% bootstrap support. Images in this figure are presented in color. 90 .au‘a [K AT3gl9720—ARCS AtlgS3140—ARC5H AT3gl9720-ARC5 Atlg53l40-ARC5H AT3gl9720—ARC5 Atlg53l40—ARC5H AT3gl9720—ARC5 Atlg53l40—ARC5H AT3gl9720-ARC5 AtlgS3l40—ARC5H AT3gl9720—ARC5 AtlgS3l40—ARC5H AT3gl9720—ARC5 AtlgS3l40-ARC5H AT3ng720—ARCS AtlgS3l40—ARC5H AT3gl9720—ARCS AtlgS3140—ARC5H AT3gl9720—ARC5 AtlgS3l40—ARC5H AT3919720—ARC5 Atlg53l40—ARC5H AT3gl9720—ARCS Atlg53140—ARC5H AT3gl9720—ARC5 AtlgS3l40—ARC5H AT3919720—ARC5 AtlgS3l40—ARC5H —————————————— MAEVSAKSVTVEEMAEEDDAAIEERWSLYEAYNELHALAQELETPF MANSNTYLTTPTKTPSSRRNQQSQSKMQSHSKDPINAESRSRFEAYNRLQAAAVAFGEKL * * .‘k‘kit *.* * EAPAVLVVGQQTDGKSALVEALMGFQFNHVGGGTKTRRPITLHMKYDPQCQFPLCHLGSD PIPEIVAIGGQSDGKSSLLEALLGFRFNVREVEMGTRRPLILQMVHDLSALEPRCRFQDE * .. .~k t.**~ki.*.x*ir.k*.~kir *ii. *.~k .* * ir.. DDPSVSLPKS- -LSQIHAYIEAENMRLEQEPCSPFSAKEIIVKVQYKYCPNLTIIDTPGLI DSEEYGSPIVSATAVADVIRSRTEALLKKTKTAVSPKPIVMRAEYAHCPNLTIIDTPGFV * 1* u i . 1’ .. . 'k * '1... .‘k .***********-. APAPGLKNRALQVQARAVEALVRAKMQHKEFIILCLEDS—SDWSIATTRRIVMQVDPELS LKAK---KGEPETTPDEILSMVKSLASPPHRILLFLQQbDVbHLbbLWLUAVKLlDbbbK I’ u . . .-*.. *.* *..* .i . t ..‘k u . .. . . .. .. . . . .. . . . .. ... KTIVVbTKLDTKIBQrbLbbUvarLSPPASALDSSLLGDSPFFTSVPSGRVGYGQDSVY RTIVVVSKFDNRLKEFSDRGEVDRYLSASGYLGEN————TRPYFVALP ——————— KDRST ***** .*.1\’ .. .ir’k .‘k. .‘k* '11.)? ..* .* KSNDEFKQAVSLREMEDIASLEKKLG— RLLTKQEKSRIGISKLRLFLEELLWKRYKESVP ISNDEFRRQISQVDTEVIRHLREGVKGGFDEEKFRSCIGFGSLRDFLESELQKRYKEAAP *****.. u . i i .i’ *i. ** *** t tt‘k‘k‘k. 'h LIIPLLGKEYRSTVRKLDTVSKELSS—LDEAKLKERGRTFHDLFLTKLSLLLKGTVVAPP ATLALLEERCSEVTDDMLRMDMKIQATSDVAHLRKAAMLYTASISNHVGALIDGAANPAP . *‘k . . . .. . i *.t-- u - u. *a u ‘k DKFGETLQDERTQGGAFVGTDGLQFSHKLIQNAGMRLYGGAQYHRAMAEFRFLVGAIKCP EQWGKTTEEERGESG-—IGSWPGVSVDIKPPNAVLKLYGGAAFERVIHEFRCAAYSIECP a n‘k-i ..** u if -*n *‘k . *ti‘ ‘kki .*. ti PITREEIVNACGVEDIHDG- -TNYSRTACVIAVAKARETFEPFLHQLGARLLHILKRLLPI PVSREKVANILLAHAGRGGGRGVTEASAEIARTAARSWLAPLLDTACDRLAFVLGSLFEI *..**.. * u .* t. ‘A’ VY ——————— J.LOKF‘GF‘VLQCHF‘VFT KPVA QAF‘NSFVF‘Q'T‘F‘KQCRDKCMF‘DT .AQTTRYVT ALERNLNQNSEYEKKTENMDGYVGFHAAVRNCYSRFVKNLAKQCKQLVRHHLDSVTSPYS - u u ‘k - ‘k . ‘k 'k 1' . WSLHN'K ”‘3 7”“ RQFLL/or UL: J. an I 1. autunIGFSLPQDALGGTTDT MACYENNYHQGGAFGAYNKFNQASPNbrLbLLbUlSKULPMKUQLNIEELKNNUQLIlyb . . . . t . i * . i . . . . . . * t KS— RSDVKLSHLASN —————————— IDSGSSIQTTEMRLADLLDSTLWNRKLAPSSER—- KGGESHITVPETPSPDQPCEIVYGLVKKEIGNGPDGVCARV lerKVQNb * ‘k _IGYALvébifibaikaiiiELAsAiikrNéEiifibEvbkiéXiifiégiéfixiéBBEBEEE GLMFANADNGMKSSSAYSEICSSAAQHFARIREVLVERSVTSTLNSGFLTPCRDRLVVAL nu-‘k a. u. i - u*‘k u" u s u u - * a * * - birnrfiééifidkkfisnfiféififiokfiikéfiéfiéfinsirmsHEFAQNLKAPSVQH GLDLFAVNDDKFMDMFVAPGAIVVLQNERQQLQKRQKILQSCLTEFKTVARSL . a *. f .X. I. s n - .u*- u 91 Figure 1 continued B 1000’ AT3919720 — ARCS At1953140 — ARCSH 1oooF'At1959510 Trans-Golgi 1 AH 91 0290 vesicle trafficking 757|-—-: ATCSg 601 90 Phragmoplast - formation '+\ IMEATSngZOBO 852 ATISg 6 l 7 60 wool—— “4933550 I Mitochondrial l—- At2g 1 41 20 “MSW" M t i |_Atlg€50500 xproen 92 Figure 1 continued 1000I—AMgsseso '———At2g1 41 20 1ooo|---At1959610 1At1g10290 1000 Rice-ARC5 "' rooo AT3919720—ARC5 Chlomy-ARCS rooo Rice—ARCSH 1000 At1953140—ARC5H Chlomy-ARCSH , 1000 93 Resul ARC 5 phylog— As sho ARC 5] all the 1 their G was pre amino 2 the reliz ARC5H GTPaSQ (Figure é and that . algae_ Th “Och-‘0' Tc Results: ARC5H is a homolog of ARC5 in Arabidopsis To better understand the evolutionary relationship between ARC5 and ARC5H, a phylogenetic analysis of all the dynamin-related proteins in Arabidopsis was performed. As shown in Figure 1B, ARC5H is closely related to ARC5. The protein sequence of ARC5H can be aligned with that of ARC5 along its full length in a BLAST aligment, but all the other dynamin-related proteins can only be aligned with ARC5 in the regions of their GTPase and Conserved Middle domains (Figure 3 in Chapter 2). Although ARC5H was predicted to have a chloroplast transit peptide, the predicted transit peptide is only 36 amino acids and the score is 0.674 (http://mips.gsfide/proj/thal/db/index.html), suggesting the reliability of this prediction is not high. The above data suggested that ARC5 and ARC5H have a close evolutionary relationship and may have similar function. However, the sequences of ARC5 and ARC5H are not well conserved beyond the GTPase and Conserved Middle domains (Figure 1A). Further, separate orthologs of ARC5 and ARC5H exist not only in rice but also in the green alga Chlamydomonas (Figure 4). Phylogenetic analysis indicated that ARC5 and ARC5H are in different clades and that the two clades may have diverged before the split between plants and green algae. These data suggest that the function of ARC5 and ARC5H may not be identical. Knock-out of ARC5H has no effect on chloroplast division To test whether ARC5H is also involved in chloroplast division, the phenotypes of ARC5H knockout lines were checked. Two T-DNA insertion lines, Sa1k__065118 and 94 Salk} contin yields region: insertic http S : only 8 1 2A). Tl: ARCSH ARC 5H arnplifie Salk_06. knocked Ofarcjlz “'38 foun and ARC ”PCS/arc5 If. divistn p. CFOSSed Wi ChlomplasI pie-DIS “Ere Salk_066401, were ordered from ABRC. The homozygous knockout lines were confirmed by PCR (Figure ZB). Because the template DNA was from crude extracts, the yields of the PCR products varied in different samples. By sequencing the junction regions between the T-DNA lefi border sequences and the flanking genomic DNA, the insertion sites were confirmed to be the same as shown on the website http://signal.salk.edu/cgi-bin/tdnaexpress. The insertion sites in the two different lines are only 8 base pairs apart. In both lines, T-DNAs are inserted into the third intron (Figure 2A). The third and fourth exons are part of the coding regions for the GTPase domain in ARC5H. RT-PCR was done to detect ARC5H mRNA in Salk_065118 and no detectable ARC5H mRNA was found (Figure 2C). As controls, the cDNA of ARC5H was well amplified in wild-type plants and the cDNA of ARC5 was well amplified in both Salk_065118 and wild type (Figure 2C). This means that the ARC5H gene is completely knocked out in Salk_065118 (hereafter named arc5h). The chloroplast sizes and numbers of arc5h plants were compared with that of wild-type plants and no obvious difference was found (Figure 2D and E). However, this may be due to redundancy between ARC5 and ARC5H. arc5/arc5h double mutant has a similar phenotype to arc5 If ARC5H is redundant with ARC5, we should observe a more severe chloroplast division phenotype in arc5/arc5h double mutants. To test this hypothesis, arc5 was crossed with arc5h plants. In an F2 segregation group of 70 plants, 16 plants with large chloroplasts were identified by phenotyping. The T-DNA insertions in ARC5H in 15 plants were checked by PCR and 5 plants were further identified to be homozygous 95 Figure 2 ARC5H is not involved in chloroplast division. (A) Gene structure of ARC5H. The T-DNA insertion sites in the two Salk lines are indicated by arrows. (B) PCR identification of the T-DNA insertion in Salk_0651 18. The first lane from the left is a 100bp DNA ladder; lane 1, homozygous insertion; lanes 2 and 4, wild type; lane 3, heterzygous insertion. (C) RT-PCR of ARC5 and ARC5H in Salk_065118 and a wild-type plant. The first lane from the left is a 1Kb DNA ladder; lane1, RT-PCR of ARC5H in SALK_O65118, arcSh; Iane2, RT-PCR of ARC5H in a wild-type plant; lane3, RT-PCR of ARC5 in SALK_O65118, arc5h; lane4, RT-PCR of ARC5 in a wild-type plant. (D-G) Comparison of the chloroplasts in Arabidopsis leaf mesophyll cells. Cells are from fixed 3-week-old leaf tissue. Scale bar represents 10 pm in D—G. (D) Wild type (Col). (E) Homozygous arc5h knockout line—SALK_065118. (F) arc5. (G) arc5/arc5h double mutant. (H) PCR identification of the T-DNA insertion in the F2 group of the arc5 X arc5h cross. Lanes with homozygous T-DNA insertions are indicated by arrows. (l) Verification of ar05 mutation by PCR amplification and subsequent Xbal and Hindlll digestion. Images in this figure are presented in color. 96 Salk_0651” Salk_066401 TM ATG 97 Figure 3 35S-ARC5H has no effect on chloroplast division. (A) 35S—ARC5H in arc5. (B) 35S—ARC5H in wild type. Cells are from fixed 3-week-old leaf tissue. Scale bar represents 10 pm in A and B. Images in this figure are presented in color. 98 99 knockout at the arc5h locus (Figure 2H). Those 5 plants are all homozygous arc5 mutant (Figure 21). The chloroplast sizes and numbers in the arc5/arc5h double mutants were compared with those in the arc5 mutant and no obvious difference was found (Figure 2F and G). This suggests ARC5H is not involved in chloroplast division. 3SS-ARC5H has no effect on chloroplast division in arc5 or wild-type plants I- To further test whether ARC5H could be redundant with ARC5, a 35S-ARC5H transgene was constructed and introduced into arc5 to determine whether overexpression of ARC5H could complement the arc5 mutant phenotype. Even in the case of partial redundancy, it might be expected that overexpression of ARC5H would increase chloroplast number and decrease chloroplast size in the arc5 background. About 100 arc5 mutant plants transformed with the 35S-ARC5H transgene were checked by microscopy. Their phenotypes were found to be the same as that of arc5 (Figure 3A and 2F). The 35S-ARC5H transgene also caused no phenotype when expressed in wild-type plants (Figure 3B and 2D). These data further support the conclusion that ARC5H is not a chloroplast division gene. ARC5-GFP is localized to speckles in the nucleus To determine the localization of ARC5H, an ARC5H-GFP fusion gene with the native promoter was made and transformed into both the wild-type and arc5h plants. In the transgenic plants, ARC5H-GFP is localized to a cluster of speckles in the cell, instead of being localized to the division site of chloroplasts like ARC5 (Figure 4A and 4B). 100 Figure 4 ARCSH-CF P is localized to a cluster of punctate structures in the nucleus. (A-B) AR05H-GFP in mesophyll cells of the cotyledons of 6-day-old plants. (A) arc5. (B) wild type. Arrows indicate GFP signals in the two images. Scale bar represents 10 pm in A and B. (C-E) ARC5H-GFP in root-tip cells of a wild-type plant. (C) ARC5H-GFP; (E) DAPI staining of DNA; (D) overlay of C and E. Images in this figure are presented in color. 101 102 DAPI staining of the root tip cells indicated that ARC5H-GFP-labeled speckles are in the nucleus (Figure 4C, 4D and 4E). Like 35S-ARC5H, ARC5H-GFP neither rescues the mutant phenotype of arc5 nor affects chloroplast division in wild-type plant (Figure 4A and B). Therefore, the function of ARC5H is not related to chloroplast division. Discussion: Because the mutation in arc5 creates a stop codon in the middle of the gene, the mRNA should be degraded by non-sense mediated decay and there should be no gene product (Baker and Parker, 2004; Maquat, 2004; Neu-Yilik et al., 2004). However, there are still 1-10 enlarged chloroplasts in the leaf cells of the arc5 mutant (Figure 2F), indicating that plastid division still occurs to some degree in this null arc5 background. It was thought that the homolog of ARC5 , ARC5H might be responsible for chloroplast division in arc5. However, neither the arc5/arc5h double mutant phenotype is more severe than that of arc5 (Figure 2G) nor can the overexpression of ARC5H rescue the phenotype of arc5 (Figure 3A). Furthermore, the ARC5H-GFP fusion protein is not localized to chloroplasts (Figure 4). These data strongly suggest that ARC5H does not function in chloroplast division and consequently is not redundant with ARC5. Therefore, ARC5 is not essential for chloroplast division. Possibly, the chloroplast division proteins with cyanobacterial origin are competent for the first several rounds of chloroplast division in the very young tissue in arc5. 103 One can image that in the very early stage of the endosybiosis, ARC5 was not involved in the division of the cyanobacterial cell, but that the cyanobacterium could still divide. During chloroplast evolution, the outer membrane of the chloroplast acquired some eukaryotic properties (Douce and J oyard, 1990; Dyall et al., 2004). The cyanobacterial cell division machinery must have been modified to adapt to this change during evolution. This may explain why some bacterial cell division genes were lost in plants and new chloroplast division genes, like ARC5, evolved. A similar scenario also happened in mitochondria. However, mitochondria are about one billion years older than chloroplasts and the F tsZ gene for mitochondrial division was lost in most eukaryotes (Beech et al., 2000), leaving the dynamin-related protein Dnm1 as the only possible force-generating protein for mitochondrial division (Otsuga et al., 1998; Sesaki and Jensen, 1999; Arimura and Tsutsumi, 2002). ARC5 may generate force to facilitate the division of chloroplasts or overcome the problems caused by the loss of some cyanobacterial cell division proteins. It has been observed by electron microscopy that there are electron-dense materials on both the inner envelope and outer envelope at the chloroplast division site in the red alga Cyanidioschyzon merolae and higher plant Pelargonium zonale, which are named inner and outer plastid-dividing (PD) rings (Kuroiwa, 2000; Miyagishima et al., 2001; Kuroiwa et al., 2002). The PD rings are potential components of chloroplast division machinery and are proposed to be involved in chloroplast division (Kuroiwa, 2000). Especially, the outer PD ring may be responsible for constriction of the division furrow from the outside of chloroplast in the arc5 mutant. However, there is no molecular 104 evidence to support the existence of the inner and outer PD rings. F tsZ was shown not to be in the inner PD ring (Kuroiwa et al., 2002) and the homolog of ARC5,CmDnm2 in C. merolae, was shown not to be in the outer PD ring (Miyagishima et al., 2003). ARC6 is also localized to the chloroplast division site and spans the inner membrane with its N- terrninal region in the stroma and C-terminal region in the intermembrane space (Vitha et al., 2003). However, ARC6 is not found to have a homolog in C. merolae (Matsuzaki et al., 2004). So, it is unclear whether PD rings exist in vivo or not. Our data suggest that ARC5 and ARC5H have different functions although they are closely related. A phylogenetic analysis indicated that ARC5 and ARC5H may have diverged earlier than the split between the plant and green algal lineages (Figure 1C). Analysis of the Arabidospis genome has shown that ARC5 and ARC5H belong to two ancient duplicated regions that have some degree of synteny (Arabidopsis_Genome_Initiative, 2000). There were probably three large-scale genome duplications in Arabidopsis (Arabidopsis_Genome_Initiative, 2000; Ku et al., 2000; Simillion et al., 2002; Blane et al., 2003; Errnolaeva et al., 2003): the recent one happened before the Arabidopsis/Brassica split and probably during the early emergence of the crucifer family; one happened after the monocot/dicot divergence; another one is even more ancient. It is generally accepted that genome duplication is an important driving force for evolution and that one of a pair of duplicated genes may be lost or acquire new function after the duplication (Blanc and Wolfe, 2004; Taylor and Raes, 2004). In the case of ARC5 and ARC5H, although it is unclear which has originated earlier, at least one of them has apparently acquired a new firnction after the duplication. 105 n 'u my»..- Ill - -.-- Material and methods Plant material Arabidopsis thaliana ecotype Columbia (Col-0) was used as wild type for all experiments. T-DNA insertion lines of ARC5H in the Col-0 background were ordered from the Arabidopsis Biological Resource Center (ABRC).The arc5h mutant was ‘* identified in the segregation groups from the Salk line: Salk_065118 and Salk_066401. Plants were grown as described (Osteryoung et al., 1998). ii— I Sequence Alignment and Phylogenetic Analysis. Full-length protein sequences were used for phylogenetic analysis and aligned with CLUSTALW (Thompson et al., 1994) using default settings. The phylogenetic tree was drawn by using the program DRAWTREE at the Biology Workbench 3.2 website (http://biowb.sdsc.edu/). Neighbor-joining analysis was performed by using the program CLUSTALTREE at the same website. Bootstrap analysis was performed on the neighbor- joining tree with one thousand replications. The protein sequences of all the dynamin-like proteins in Arabidopsis were obtained from TAIR (http://www.arabidopsis.org). The sequences of the orthologs of ARC5 and ARC5H in Chlamydomonas were obtained from a BLAST search at the J GI website (http://genome.jgi-psforg/ehlre2/chlre2.home.html). The Genebank accession number for rice-ARC5H is BAD86966. The protein sequence of rice-ARC5 is derived from the cDNA sequence AK072318. 106 Microscopy Phenotypes were analyzed as previously described (Gao et al., 2003), except that the images were recorded with a Coolpix 4500 digital camera (Nikon Corporation, Tokyo). For in vivo detection of GP P by conventional fluorescence microscopy, flesh leaf tissue or root tissue from young seedlings was mounted in water and viewed with an L5 filter set (excitation 460-500 nm, emission 512-542 nm) and 40X objective lense of a Leica DMR A2 microscope (Leica, Wetzlar, Germany) equipped with epifluorescenee illumination. For DNA staining, roots were mounted in 1 rig/ml DAPI solution and viewed with an A4 filter set (excitation 340-3 80 nm, emission 450-490 nm) and 40X objective lense. Images were captured with a cooled charge-coupled device camera (Retiga 1350EX, Qimaging, Burnaby, British Columbia, Canada). All images were processed with PHOTOSHOP imaging software (Adobe Systems, San Jose, CA). PCR identification of the Salk lines and verification of the arc5 mutation Three primers 5'-CGGAGGACTATTGTTGTTGTGTCCA-3' (ARC5H-L), 5'- GGCACTCGATTGAGTAGGCTGC-B' (ARC5H-R) and 5'- GCGTGGACCGCTTGCTGCAACT-3' (LBbl) were used for verifying T-DNA insertion sites in the two Salk lines. ARC5H-L and ARC5H-R will give a band of 932 bp in wild- type plants. In T-DNA insertion lines, LBbl and ARC5H-R will give a band of about 480 bp that corresponds to the junction region between the T-DNA left border and genomic DNA. Two bands will be amplified in heterozygous insertion lines. The mutation in arc5 creates an Xbal cutting site. When the PCR products amplified by 5'- 107 CTGGAAAGAGAAGGGCATCAAG-3' and 5'- CTGGGAAGATTTTGACGAATGTTG-B' are cut by Hindlll and Xbal, DNA from wild type will give two bands with the sizes of 480bp and 608bp, DNA from arc5 will give three bands of 98bp, 382bp and 608bp. 100bp DNA ladder is from Invitrogen (Carlsbad, California). RT-PCR Primers used for RT-PCR of ARC5H were 5'-TGTCGATTCCAGGATGAAGAT TCTG- 3' and 5'-TTGGGAATA AAACACCAGCCATG-B'. Primers used for RT-PCR of ARC5 were 5'-GAAAAAGGAACGGCGACGAAAAC-3' and 5'- GCAAACATTGGACCAAAAAGCG-3'. RT-PCR was done with Qiagene OneStep RT- PCR kit. The 1Kb DNA ladder is from Invitrogen (Carlsbad, California). Overexpression of ARC5H The genomic DNA of ARC5H was amplified by Pfu DNA polymerase using the primers 5'-TCGTCAATGGCGAATTCAAACAC-3' and 5'- GCTAGGATCCATTATAGAGAACGAGCCACCGT-3'. The PCR product was digested by BamHI and cloned between the 355 promoter and OCS terminator in the transformation vector pFGC5941 (Genebank accession number AY310901), which was cut by SwaI and BamHI. The construct was transferred to Agrobacterium tumefaciens GV3101 and introduced into Arabidopsis plants by floral dipping. 108 ARC5H-GFP Localization To make an ARC5H-GFP fusion gene, the genomic DNA of ARC5H with ~1.8 kb of the promoter region was amplified by Pfu DNA polymerase using the primers 5'- AGGTTGTCTTGGTTGTAAATCCTCTC-3' and 5'- GCATTGATCAGAACGAGCCACCGTTTTAAACA-3', digested by BgIII, and then cloned into a derivative of the transformation vector pCAMBIA-1302 (CAMBIA, Canberra, Australia), pCAMBIA-1302-bar (the hptII gene was replaced with the bar gene from pCAMBIA-3300), between the Smal and BglII cutting sites. The construct was introduced into Arabidopsis plants as described above. Green fluorescence from GF P of the transgenic plants was detected by fluorescence microscopy. 109 Literature cited: Arabidopsis_Genome_Initiative. (2000). Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408, 796-815. Arimura, S., and Tsutsumi, N. (2002). A dynamin-like protein (ADL2b), rather than FtsZ, is involved in Arabidopsis mitochondrial division. Proc Natl Acad Sci U S A 99, 5727-5731. Baker, K.E., and Parker, R. (2004). Nonsense-mediated mRNA decay: terminating i erroneous gene expression. Curr Opin Cell Biol 16, 293-299. Beech, P.L., Nheu, T., Schultz, T., Herbert, S., Lithgow, T., Gilson, RR, and McFadden, G.I. (2000). Mitochondrial FtsZ in a chromophyte alga. Science 287, 1276-1279. Blane, G., and Wolfe, KB. (2004). Functional divergence of duplicated genes formed by polyploidy during Arabidopsis evolution. Plant Cell 16, 1679-1691. Blane, G., Hokamp, K., and Wolfe, K.H. (2003). A recent polyploidy superimposed on older large-scale duplications in the Arabidopsis genome. Genome Res 13, 137- 144. Colletti, K.S., Tattersall, E.A., Pyke, K.A., Froelich, J.E., Stokes, K.D., and Osteryoung, K.W. (2000). A homologue of the bacterial cell division site- determining factor MinD mediates placement of the chloroplast division apparatus. Curr Biol 10, 507-516. de Boer, P.A., Crossley, R.E., and Rothfield, LJ. (1992). Roles of MinC and MinD in the site-specific septation block mediated by the MinCDE system of Escherichia coli. J Bacteriol 174, 63-70. de Boer, P.A., Crossley, R.E., Hand, A.R., and Rothfield, LJ. (1991). The MinD protein is a membrane ATPase required for the correct placement of the Escherichia coli division site. Embo J 10, 4371-4380. Deuce, R., and J oyard, J. (1990). Biochemistry and function of the plastid envelope. Annu Rev Cell Biol 6, 173-216. Dyall, S.D., Brown, M.T., and Johnson, P.J. (2004). Ancient invasions: from endosymbionts to organelles. Science 304, 253-25 7. Ermolaeva, M.D., Wu, M., Eisen, J.A., and Salzberg, S.L. (2003). The age of the Arabidopsis thaliana genome duplication. Plant Mol Biol 51, 859-866. 110 Errington, J., Daniel, R.A., and Scheffers, DJ. (2003). Cytokinesis in bacteria. Microbiol Mol Biol Rev 67, 52-65, table of contents. Gao, H., Kadirjan-Kalbach, D., Froehlich, J.E., and Osteryoung, K.W. (2003). ARC5, a cytosolic dynamin-like protein from plants, is part of the chloroplast division machinery. Proc Natl Acad Sci U S A 100, 4328-4333. Hong, Z., Bednarek, S.Y., Blumwald, E., Hwang, 1., Jurgens, G., Menzel, D., Osteryoung, K.W., Raikhel, N.V., Shinozaki, K., Tsutsumi, N., and Verma, D.P. (2003). A unified nomenclature for Arabidopsis dynamin-related large GTPases based on homology and possible functions. Plant Mol Biol 53, 261-265. I if Itoh, R., Fujiwara, M., Nagata, N., and Yoshida, S. (2001). A chloroplast protein homologous to the eubacterial topological specificity factor minE plays a role in chloroplast division. Plant Physiol 127, 1644-1655. b Ku, H.M., Vision, T., Liu, J., and Tanksley, SD. (2000). Comparing sequenced segments of the tomato and Arabidopsis genomes: large-scale duplication followed by selective gene loss creates a network of synteny. Proc Natl Acad Sci U S A 97, 9121-9126. Kuroiwa, H., Mori, T., Takahara, M., Miyagishima, S.Y., and Kuroiwa, T. (2002). Chloroplast division machinery as revealed by immunofluorescence and electron microscopy. Planta 215, 185-190. Kuroiwa, T. (2000). The discovery of the division apparatus of plastids and mitochondria. J Electron Microsc (Tokyo) 49, 123-134. Maple, J., Chua, N.-H., and Moller, S.G. (2002). The topological specificity factor AtMinEl is essential for correct plastid division site placement in Arabidopsis. Plant J 31, 269-27 7. Maquat, LE. (2004). Nonsense-mediated mRN A decay: splicing, translation and mRNP dynamics. Nat Rev Mol Cell Biol 5, 89-99. Margolin, W. (2001). Spatial regulation of cytokinesis in bacteria. Curr Opin Microbiol 4, 647-652. Matsuzaki, M., Misumi, 0., Shin, I.T., Maruyama, S., Takahara, M., Miyagishima, S.Y., Mori, T., Nishida, K., Yagisawa, F., Yoshida, Y., N ishimura, Y., Nakao, S., Kobayashi, T., Momoyama, Y., Higashiyama, T., Minoda, A., Sane, M., Nomoto, H., Oishi, K., Hayashi, H., Ohta, F., Nishizaka, S., Haga, S., Miura, S., Morishita, T., Kabeya, Y., Terasawa, K., Suzuki, Y., Ishii, Y., Asakawa, S., Takano, H., Ohta, N ., Kuroiwa, H., Tanaka, K., Shimizu, N., Sugano, S., Sato, N., Nozaki, H., Ogasawara, N., Kohara, Y., and Kuroiwa, T. (2004). 111 Genome sequence of the ultrasmall unicellular red alga Cyanidioschyzon merolae 10D. Nature 428, 653-657. Miyagishima, S., Takahara, M., and Kuroiwa, T. (2001). Novel filaments 5 nm in diameter constitute the cytosolic ring of the plastid division apparatus. Plant Cell 13, 707-721. Miyagishima, S.Y., Nishida, K., Mori, T., Matsuzaki, M., Higashiyama, T., Kuroiwa, H., and Kuroiwa, T. (2003). A plant-specific dynamin-related protein forms a ring at the chloroplast division site. Plant Cell 15, 655-665. ‘ Neu-Yilik, G., Gehring, N.H., Hentze, M.W., and Kulozik, A.E. (2004). Nonsense- mediated mRN A decay: from vacuum cleaner to Swiss army knife. Genome Biol 5, 218. Osteryoung, K.W., and McAndrew, RS. (2001). The Plastid Division Machine. Annu Rev Plant Physiol Plant Mol Biol 52, 315-333. Osteryoung, K.W., Stokes, K.D., Rutherford, S.M., Percival, A.L., and Lee, W.Y. (1998). Chloroplast division in higher plants requires members of two functionally divergent gene families with homology to bacterial fisZ. Plant Cell 10, 1991-2004. Otsuga, D., Keegan, B.R., Brisch, E., Thatcher, J .W., Hermann, C.J., Bleazard, W., and Shaw, J .M. (1998). The dynamin-related GTPase, Dnmlp, controls mitochondrial morphology in yeast. J Cell Biol 143, 333-349. Pyke, K.A., and Leech, RM. (1994). A Genetic Analysis of Chloroplast Division and Expansion in Arabidopsis thaliana. Plant Physiol 104, 201-207. Robertson, E.J., Rutherford, S.M., and Leech, RM. (1996). Characterization of chloroplast division using the Arabidopsis mutant arc5. Plant Physiol 112, 149- 159. Sesaki, H., and Jensen, RE. (1999). Division versus fusion: Dnmlp and F zolp antagonistically regulate mitochondrial shape. J Cell Biol 147, 699-706. Shimada, H., Koizumi, M., Kuroki, K, Mochizuki, M., Fujimoto, H., Ohta, H., Masuda, T., and Takamiya, K. (2004). ARC3, a chloroplast division factor, is a chimera of prokaryotic FtsZ and part of eukaryotic phosphatidylinositol-4— phosphate 5-kinase. Plant Cell Physiol 45, 960-967. Simillion, C., Vandepoele, K., Van Montagu, M.C., Zabeau, M., and Van de Peer, Y. (2002). The hidden duplication past of Arabidopsis thaliana. Proc Natl Acad Sci U S A 99, 13627-13632. 112 Taylor, J.S., and Raes, J. (2004). Duplication and divergence: the evolution of new genes and old ideas. Annu Rev Genet 38, 615-643. Thompson, J.D., Higgins, D.G., and Gibson, T.J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673-4680. Vitha, S., McAndrew, R.S., and Osteryoung, K.W. (2001). FtsZ ring formation at the chloroplast division site in plants. J Cell Biol 153, 111-120. Vitha, S., Froehlich, J.E., Koksharova, 0., Pyke, K.A., van Erp, H., and Osteryoung, K.W. (2003). ARC6 is a J-domain plastid division protein and an evolutionary descendant of the cyanobacterial cell division protein Ftn2. Plant Cell 15, 1918- 1933. 113 Chapter 4 FZL, an FZO—like Protein in Plants, Links Thylakoid Morphogenesis to Chloroplast Division 114 Abstract FZO is a mitochondrial fusion protein in fungi and animals. We identified an FZO-like protein in plants, FZL, that was shown to have a chloroplast transit peptide. Knock-out of F ZL caused a chloroplast division defect and disorganized stacking of thylakoids. The mutant phenotype was rescued by an FZL-GFP transgene. Overexpression of FZL-GFP caused very long thylakoid sacs with considerably less stacking than in wild type. These data indicate that FZL is involved in the morphogenesis . of thylakoids. Fluorescence microscopy showed that F ZL-GFP was localized to punctate structures associated with chloroplasts and in the cytosol. Chloroplast fractionation indicated that FZL-GFP is associated with both the thylakoid and inner envelope membranes. Irnmunogold labeling indicated F ZL-GFP is localized to thylakoids, and to vesicle-like structures found underneath the inner envelope, protruding from chloroplasts, and in the cytosol. The level of F ZL—GFP expression correlates with the number of punctate structures and vesicle-like structures on the surface of the chloroplast. These data suggest that the punctate structures and vesicle-like structures are coincident and that F ZL may be involved in their formation. Mutation of a conserved lysine residue in the F ZL GTPase domain abolished the punctate pattern of FZL-GFP localization and the ability to complement the knock-out mutant phenotype. Thus, F ZL seems to be involved in vesicle formation and trafficking process from the thylakoid to the outside of chloroplasts, and blocking of this process affects both the thylakoid morphology and chloroplast division. 115 Introduction Chloroplasts carry out photosynthesis and the biosynthesis of amino acids, lipids, starch and many other compounds in plant cells. The chloroplast envelope contains a permeable outer membrane and a less permeable inner membrane. Both membranes contain many proteins that regulate the transport of small molecules and proteins into and out of the chloroplast (N euhaus and Wagner, 2000; Schleiff and S011, 2000). Vesicle-like structures have been observed budding from or associated with the inner and outer membrane and may be involved in some transport processes into and (or) out of chloroplasts (Westphal et al., 2001a, 2003). Vesicle-like structures with double membranes were also observed containing stromal protein in the cytoplasm and in the vacuole (Chiba et al., 2003). The biological roles of these vesicles and the molecular mechanisms of their formation are unknown. Localted within the chloroplast is a system of interconnecting, disk-shaped sacs called thylakoids (Trissl and Wilhelm, 1993). Stacked thylakoid sacs called grana are connected by non-stacked thylakoid sacs called lamellae. Generally, the sacs in all the grana of a chloroplast are similar in size and stacked in arrays. The thylakoid membrane is the location for light absorption and ATP synthesis. Photosystem (PS) 11 and LHC (light-harvesting complex) 11 are mainly located on the membrane of stacked thylakoids (Andersson and Anderson, 1980; Chow, 1984; Borodich et al., 2003). Most of the PS I complexes and the ATP synthase are located on the lamellae (Andersson and Anderson, 1980; Chow, 1984; Borodich et al., 2003). The formation of grana and the lateral heterogeneity of the protein complexes in the thylakoid membrane separate the slow (PS 116 II) and the fast (PS I) photosystems and makes photosynthesis more efficient (Trissl and Wilhelm, 1993; Westphal et al., 2003). The mechanisms controlling the organization of the thylakoid membranes and the differentiation of grana and lamellae are still unclear. F20 and its homologues in fungi and metazoa are dynamin-like outer mitochondrial membrane proteins required for mitochondrial fusion (Hermann et al., 1998; Santel et al., 2003). F20 has a GTPase domain, two transmembrane domains and two coiled-coil domains (Hales and Fuller, 1997; Mozdy and Shaw, 2003; Westermann, 2003). The GTPase domain and the two coiled-coil domains of the FZO protein are exposed to the cytosol. Mutations in FZO block mitochondrial fusion and result in a fragmented mitochondrial morphology and loss of mitochondrial DNA (Hermann et al., 1998; Rapaport et al., 1998). Detailed mutational analysis has shown that the GTPase domain of FZO is important for its function, and that its transmembrane domains and coiled-coil domains are important for its localization to the outer membrane (Santel and Fuller, 2001; Rojo et al., 2002). Overexpression of mitofusin 1 (anl), a human homologue of FZO, can increase mitochondrial length and interconnectivity (Legros et al., 2002). Interactions between the coiled-coil domains of F 20 molecules were proposed to mediate the tethering between membranes from different mitochondria (Rojo et al., 2002; Westermann, 2003; Koshiba et al., 2004). Hydrolysis of GTP may mediate the fusion of mitochondrial outer membranes by generating force for the movement of proteins and opening the attached membranes on the other side (Westermann, 2003; Meeusen et al., 2004). The fusion of the mitochondrial inner membranes may be mediated by the Mgml, which is located in the intermembrane space and resides on the 117 inner membrane of mitochondria (Wong et al., 2000; McQuibban et al., 2003; Wong et al., 2003). In fungi, Ugolp links onlp and Mgrnlp for mitochondrial fusion (Sesaki and Jensen, 2001, 2004). We identified an F ZO-like protein in Arabidopsis, FZL, which is similar to FZO in both sequence and domain arrangement. Phylogenetic analysis indicated that F ZL and F ZO may have a common ancestor before the divergence of plants and animals; however, FZL is predicted to be targeted to chloroplasts with high probability. For this reason, we hypothesized that F ZL might have a role in a chloroplast process requiring membrane fusion activity. To investigate the role of FZL, we cloned the F ZL gene and characterized the function of its gene product. FZL was confirmed to be a chloroplast protein. Loss of fimction of F ZL results in disorganized thylakoid stacks and heterogeneity of chloroplast size and number. Overexpression of FZL causes very long thylakoid sacs with much less stacking. Thus, FZL links the morphogenesis of thylakoids to the division of chloroplasts. Results FZL is an FZO-like protein in plants FZL (At1g03 160) was identified by a BLAST search of the Arabidopsis protein database (http://www.arabidopsis.org/Blast/) using FZO as a query sequence. FZL is conserved in many photosynthetic eukaryotes, including tomato, rice and 118 ll Figure 1 FZL is an FZO-like protein involved in chloroplast division. (a) Predicted domains of FZL. (b) Phylogenetic analysis of FZL based on aligment of the GTPase domains with an unrooted neighbor-joining tree. Bootstrap values from the neighbor-joining analysis are shown at nodes with >50% bootstrap support. “At” represents Arabidopsis thaliana. Accession numbers for the sequences aligned with FZL are listed in Materials and Methods. (c) Sequence alignment of the GTPase domains of FZL and F20 (GenBank accession number: AAF56110). Green boxes are identical residues; cyan boxes are similar residues; dashes indicate gaps. Arrow indicates K362, which is conserved in all the dynamin-related proteins and is changed to M in the FZL(K362M)-GFP mutant. (d) fzI mutant plants look pale and flower later than wild-type plants. Left, wild type plants; middle, le mutant plants; right, le mutant plants complemented by FZL-GFP. (e) Gene structure of FZL. Exons are represented by black boxes; introns are represented by grey boxes. The sites of the insertions in the two T-DNA insertion lines are indicated by arrows. (f—i): Chloroplast division phenotype and complementation from 4 week old fully expanded leaves. (f) Single cell from wild type plant. Cells in (g and h) are from le mutant plants. (i) Single cell from le mutant plants complemented by FZL- GFP. Scale bar represents 10 pm in f—i. Images in this figure are presented in color. 119 a GTPase domain Transmembrane domains i + II Chloroplast transit peptide Coiled coil domains I no an we I I an 0 sc 0 Alesulm rzr. v: ms x :6 r CYSDLIS e no our“ squsnspeux 'r xxaxurtcsnn :21. up any x ------------- P ------------- no LDDC - slsrrn r r. s'r'nvlsq r'rw's': nr. Ir. con i: p r. s n ' us All. -Y Q no 3': rum 3 var. r: r ---- n 120 Figure 1 continued \r if” DrOSOphila no t SALK_009051 121 Figure 1 continued 122 Chlamydomonas (data not shown). The cDNA of F ZL in Arabidopsis was amplified by RT-PCR and the resulting sequence showed that the annotation of the coding region lacked 270 residues at the C-terminal end of the protein. A BLAST search of the NCBI database (http://www.ncbi.nlm.nih.gov/BLAST/) showed that FZL is similar to a cluster of proteins involved in mitochondrial fusion. The putative GTPase domain of F ZL can be aligned with that of FZO (Figure 1c). Interestingly, FZL was predicted by TargetP (Emanuelsson et al., 2000) (http://www.cbs.dtu.dk/services/TargetP/) to have a chloroplast transit peptide (score, 0.871; reliability class, 2.) comprising the first 54 amino acid residues at the N-terminus (Figure 1a). This indicates that FZL is very likely to be targeted to the chloroplast in vivo. Phylogenetic analysis was performed to investigate the relationship between FZL and other members of the dynamin family of proteins (Figure 1b). Because the sequences of the members of dynamin family are very divergent and the GTPase domain is the most conserved region among these proteins, only the GTPase domains were used for the phylogenetic analysis. FZL clustered in the same branch as the mitochondrial fusion protein FZO (Figure lb). Further analysis of the FZL protein sequence predicted two transmembrane domains situated close to each other, and two coiled-coil domains flanking the predicted transmembrane domains (Figure 1a). This domain arrangement is very similar to that of mitochondrial FZO (Westermann, 2003). These data suggest that FZL and FZO may have arisen and diverged from a same ancestral gene in the common ancestor of plants and animals and that F ZL may play a role in a membrane remodeling process in the chloroplast similar to that of FZO in mitochondria. 123 FZL has a chloroplast transit peptide To test the fimction of the predicted chloroplast transit peptide of FZL, the coding sequence from the third to the 67th amino acid residue was amplified by PCR and inserted into the plant transformation vector pCAMBIA-1302-BAR to make a 358 promoter-FZL transit peptide-GFP-NOS terminator construct (Figure 2j). This construct was transformed into wild type Arabidopsis plants by floral dipping (Clough and Bent, 1998). In mesophyll cells of transgenic plants, the GFP signal was coincident with chlorophyll autofluorescence, indicating its association with chloroplasts (Figure 2a-c). The GF P looks to be present in the stroma, not the chloroplast envelope (Figure 2a, 0). In the GFP fusion genes, the second codon for methionine is located about one third of the way into the GFP coding sequence. If this codon is used as the start codon, the gene product will lack the predicted chloroplast transit peptide and the N-terminal one-third of the GFP and presumably will not fluoresce. As a control, the GFP gene without the coding sequence of the chloroplast transit peptide was transformed into wild type plants using pCAMBIA-1302-BAR (Figure 2k) and GFP remained in the cytosol of mesophyll cells (Figure 2d-f). In wild-type plants, no GF P signal could be detected and the very dim fluorescence was from chlorophyll autofluorescence (Figure 2 g-h). These results confirm the prediction that FZL has a chloroplast transit peptide. 124 Figure 2 FZL has a chloroplast targeted transit peptide. (a—c) A single cell from a wild type plant transformed by CaMV35S promoter-FZL transit peptide- GFP-nopaline synthase (NOS) terminator. (d—f) A single cell from a wild type plant transformed by CaMV35S promoter-GFP-NOS terminator. (g—i) A single cell from a wild type plant. (j) The GF P construct in a—c. (k) The GFP construct in d—f. Scale bar represents 10 pm in a—i. Images in this figure are presented in color. 125 GFP Chlorophyll Overlay ' m 3‘” ---- NOS 35S promoter t_tr_anslt E iii-151?» GFP 1 terminator . ' '1 358 promoter 3 GFP ; NOS h .. terminator 126 To test whether FZL also functions in mitochondria, we crossed le mutant plants with plants that have a mitochondria-targeted GFP (Kohler et al., 1997a). We compared the mitochondria in f2] and wild type plants by visual detection using fluorescence microscopy and found no obvious differences in size, morphology, motility, fluorescence intensity etc. (data not shown). This further suggests that F ZL is only targeted to chloroplasts. This result is consistent with the observation that plants lack Mgrnlp and Ugolp (Wong et al., 2000; Sesaki and Jensen, 2001), which in yeast work in a complex with FZO during mitochondrial fusion (Sesaki and Jensen, 2004). These findings together suggest that F ZL does not have the same function as mitochondrial FZO, but instead has a unique function in chloroplasts. Loss of FZL function alters chloroplast size and morphology To understand the function of F ZL, T-DNA insertion lines of the F ZL gene were obtained from the Arabidopsis Biological Resource Center (Columbus, Ohio, USA). The Salk_033745 line has an insertion in the region encoding the GTPase domain (Figure 1c). The Salk_009051 line has two tandem insertions: one in the intron between the two exons encoding the second transmembrane domain and a second in the 3’-UTR (Figure 1e). Both mutant lines had the same phenotype: leaves were visibly pale and flowering was delayed by several days compared with wild type (Figure 1d). Light microscopy showed that mature mesophyll cells in the le knockout lines contained only 1-20 chloroplasts per cell (Figure 1 g, h), whereas wild-type plants had approximately 120 chloroplasts per cell (Figure 1f). Chloroplasts in the fil mutants were heterogeneous in size. The heterogeneity was observed both within a single cell and among different cells (Figure lg, h). 127 To confirm that the mutant phenotype resulted from the disruption of the F ZL gene, the coding region of F ZL, including ~0.8 kb upstream of the start codon, was amplified by PCR, inserted into the plant transformation vector pCAMBIA-1302-BAR with a GFP gene fused to the 3’-end, and transformed into le mutant plants by floral dipping (Clough and Bent, 1998). The mutant phenotypes, including enlarged chloroplasts with heterogeneous size, pale leaves and late flowering, were rescued by the transgene (Figure 1d, i). Expression levels higher than that needed to complement the mutant phenotype had no obvious effect on chloroplast number and size (Figure 3 and 7). This is in contrast to the overexpression of some other chloroplast division proteins in plants (Osteryoung et al., 1998; Colletti et al., 2000; Itoh et al., 2001) and to the overexpression of some bacterial cell division proteins in bacteria, which often have a dominant-negative effects (Dai and Lutkenhaus, 1992; de Boer et al., 1992). FZL level affects the structure and the morphology of the thylakoid To further investigate the effect of FZL on the ultrastructure of chloroplasts,le mutant, wild type and F ZL-GF P over-expression plants were examined by transmission electron microscopy (TEM). The results showed that thylakoid structure was affected by both loss of function of FZL and overexpression of F ZL-GFP. In wild type plants, unstacked lamellae and stacked grana are well organized inside the chloroplast (Figure 3a, b) and the sacs in each granum are similar in size and connected by stroma lamellae. In fizl mutant plants, the thylakoid sacs appear thinner and 128 Figure 3 FZL level is related to the structure of thylakoid. TEM of the thylakoid structure in the plants with different levels of FZL. (a and b) Thylakoid structure of wild type plants. (c and d) Thylakoid structure of f2! mutant plants. (e and f) Thylakoid structure of f2] mutant plants complemented by normal levels of FZL-GF P. (g and h) Thylakoid structure of f2! mutant plants over-expressing FZL-GF P. Scale bar represents 2 pm in a, c, e and 9. Scale bar represents 500 nm in b, d, fand h. 129 one 25> ‘Nh umEoEcano cemwcaxccg 130 less organized and lack the lamellar thylakoid sacs seen in wildtype plants (Figure 3c, (1). Furthermore, the thylakoid stacks are more compact and appear more variable in size (Figure 3d). In most of the le plants complemented by FZL-GFP with native promoter, each chloroplast has on average 1 or 2 GFP-labeled structures (Figure 5 a, b and e; Figure 7d) and the thylakoid structure looks similar to that in wild-type plants (Figure 3e, f; Figure 70. In some of the fil plants complemented by FZL-GFP expressed from the native promoter, FZL-GFP levels are much higher (Figure 7 g-k) and there is considerably less stacking of thylakoid sacs and the unstacked stroma lamellae appear very long and swollen (Figure 3g and h; Figure 7i). However, unstacked sacs of stroma lamellae are infrequent in wild-type plants. The complementation of the defects of f2! in chloroplast division and thylakoid morphology firrther suggests that FZL-GFP is firnctional. The abnormal morphologies in both the le and the plants overexpressing FZL-GFP indicate that the level of FZL influences the morphology and structure of thylakoids. FZL seems to have a role in promoting the formation of stroma lamellae and (or) preventing the stacking of thylakoid sacs into grana. Topology of FZL To understand the function of F ZL, we studied the topology of F ZL by a protease protection assay with isolated chloroplasts from the F ZL-GFP complemented mutant plants. Therrnolysin, which cannot penetrate the outer chloroplast envelope membrane (McAndrew et al., 2001), and trypsin, which can penetrate the outer but not the inner membrane (McAndrew et al., 2001), were the proteases used for this assay. FZL-GFP was protected when the chloroplasts were treated with either thermolysin or trypsin 131 Figure 4 FZL is a chloroplast-targeted membrane protein. (a) FZL-GFP is protected from thermolysin and trypsin digestion. Isolated intact chloroplasts were incubated with thermolysin and trypsin (left). Lysed chloroplasts were incubated with thermolysin and trypsin. (b) FZL-GFP is a membrane protein. Isolated intact chloroplasts were lysed in hypotonic solution and separated into pellet fraction and supernant fraction (left). FZL-GF P is solubilized by 1% TritonX-100 (right). After centrifugation, FZL-GFP is present in soluble part. (c) FZL-GFP is present in both thylakoid and envelope of chloroplast. Isolated intact chloroplasts were lysed in hypotonic solution and separated into thylakoid and envelope fractions. Crude proteins representing approximately equivalent proportion of the chloroplast from each fraction were analyzed by SDS-PAGE and immunoblotting with antibodies against GFP, Tic110, FtsZ1 and LHCb. cp, chloroplast. 132 FZL-GFP Tic110 1.3m- ;:-. Hana-.- in .1“— 1" S 1:912:11. -- 415. . rm. ARCB4NW’Hflbnuo mm were . m. . c grow *8 330$ ow bckvccx cc a FZL-GFP Iii; T1ct10 1‘“ FtsZ1 1" - ' ‘4III (III FZL-GFP FZL-GFP - an. .- LHCb . Ixhlhfunfi It" . 3" a. 4.. “Jam = "Whit- §Fm lad—a; mn- _ nu : M m m. Tic110 133 unless the chloroplasts were osmotically lysed prior to protease treatment (Figure 4a). The control protein Ticl 10, an inner membrane protein facing the chloroplast stroma (Jackson et al., 1998), was also protected from digestion by thermolysin and trypsin. As a control for the intermembrane space, we used a C-terminal GFP fusion to ARC6, a protein whose C-terminus resides in the intermembrane space (Vitha et al., 2003). This ARC6-GFP was protected from digestion by thermolysin but not trypsin (Figure 4a). These data indicate that FZL is targeted to the stromal side of the chloroplast envelope. The pellet and soluble fractions of the isolated chloroplasts were separated by centrifugation afier chloroplast lysis by hypotonic solution. FZL-GFP and the inner envelope protein Ticl 10 (Jackson et al., 1998) were found in the pellet fraction, but not in the soluble fraction (Figure 4b). Ftle , a soluble stromal protein (McAndrew et al., 2001), was found mainly in the soluble fraction (Figure 4b). FZL-GFP was detected in the supernatant fraction after the chloroplasts were incubated in 1% TritonX-lOO (Figure 4b). These data confirm that FZL is a membrane protein as predicted by sequence analysis. To further determine the localization FZL-GFP, chloroplast membranes from hypotonically lysed chloroplasts were fractionated into thylakoid and envelope fractions by sucrose gradient centrifugation (Block et al., 2002). Irnmuno blot analysis indicated that FZL-GF P was present in both thylakoid and envelope fractions (Figure 4c). As controls, LHCb and Tic110 were detected only in the thylakoid and envelope fractions respectively (Figure 4c). 134 Localization of FZL by microscopy In le plants complemented by FZL-GFP, F ZL-GFP usually exhibited a punctate pattern of localization. Punctate structures were often associated with the periphery of the chloroplasts (Figure 5a, b). Less frequently, punctate structures were observed in the stroma (Figure 5c, (1). When the expression levels were low, the GF P signal also appears to be present in the cytosol as well as the chloroplasts (Figure 5e, indicated by arrows). The same pattern of GFP localization is observed in wild type plants transformed with F ZL-GFP (data not shown). To further investigate the localization of F ZL-GFP, we performed immunogold labeling with GFP antibodies in the F ZL-GFP complemented plants. FZL-GF P was detected in thylakoids (Figure 6b), vesicle-like structures associated with the inner chloroplast envelope (Figure 6a), membrane protrusions from chloroplasts (Figure 6b, c), vesicle-like structures close to chloroplasts (Figure 6d) and vesicle-like structures in the cytosol (Figure 6e). These data are consistent with the observations made with fluorescence microscopy. The presence of F ZL-GFP-labeled punctate or vesicle-like structures on the stromal and cytosolic sides of the envelope, and in the cytosol suggests that FZL-GFP or these structures are being trafficked. 135 Figure 5 FZL-GFP is localized to punctate structures in FZL-GFP complemented le mutants. (a, b and e) FZL-GFP-labeled punctate structures are associated with the chloroplast. (a) Extended focus confocal image. (b) Single section of the confocal images from a. (c and d) Single sections of confocal images show FZL-GFP labeled punctate structures in the stroma of chloroplast. (e) Conventional fluorescence image. Arrows indicate FZL-GFP Iabled punctate structures. Image depth is 0.5 pm in b, c and d. Scale bar represents 5 pm in a—d. Scale bar represents 10 pm in e. Images in this figure are presented in color. 136 137 Figure 6 FZL-GFP is localized to vesicle-like structure in FZL-GFP complemented le mutant with immune gold labeling. (a) FZL-GFP is localized to vesicle-like structures underneath the chloroplast envelope. (b) FZL- GF P is localized to stroma and protrusion-like structure on chloroplast envelope. (c) FZL-GFP is localized to protrusion-like structure on chloroplast envelope. (d and e) FZL-GF P is localized to vesicle-like structures in cytosol. Black arrows indicate gold particles. White arrows indicate chloroplast envelope; adjacent dark areas are stroma. Triangles indicate cell wall. Scale bar represents 200 nm in a, c with the same magnification. Scale bar represents 200 nm in b, d and e with the same magnification. 138 ..5. > ‘ .{u— _‘ .,r," E‘V‘i‘hr . "Y .3," ~..-“"i‘.*9" a." “ g. ‘ is- -. -{rk-§u%v C ‘ ‘ 139 Increased levels of FZL result in more punctate and vesicle-like structures on the surface of chloroplasts To gain further insight into the function of F ZL, we analyzed the effect of F ZL- GFP expression level in the le mutant background. In the F ZL-GFP complemented mutant plants, probably because of positional effect and the number of the inserted transgenes, the level of FZL-GFP was variable. The relative levels of FZL-GFP were estimated by visual estimation of the strength of the GFP fluorescence signal or by immuno-blot detection (Figure 7d, g, j and k; Figure 8a, b and c). When FZL-GFP is expressed at a level to be able to fully complement the chloroplast division phenotype with normal thylakoid morphology, the GFP-labeled punctate structures are often seen associated with the chloroplast envelope or in the cytosol (Figure 7d-f; Figure 5 a, b and c). When F ZL-GFP is expressed at higher levels, the chloroplast division defect is still rescued but more punctate structures are found associated with the chloroplast (Figure 7g). Smaller GFP-labeled structures can also be seen in the stroma in these plants (Figure 7g). The number and size of GFP-labeled punctate structures inside the chloroplast increased with increasing FZL-GFP expression level (Figure 5c and (1; Figure 7 g). This probably means that, when these punctate structures are in excess, they accumulate and fuse together inside chloroplasts. Scanning electron microscopy (SEM) of isolated chloroplasts from plants expressing different levels of FZL-GFP showed that higher FZL- GFP levels are associated with an increased number of structures resembling vesicles protruding from the outer envelope (Figure 7b, e and h). TEM of leaf sections further indicated a positive correlation between F ZL-GFP levels and the number of vesicle-like 140 Figure 7 Correlation between number of punctate and vesicle-like structures and the level of FZL-GFP. (a, b and c) Chloroplasts of wild type plants. (d, e and f) Chloroplasts of le mutant fully complemented by FZL-GFP. (g, h and i) Chloroplasts of f2] mutant with higher FZL-GFP levels. (a, d and g) Confocal Laser Scanning Microscopy (CLSM) image. (b, e and h) Scanning Electron Microscopy (SEM) image. (c, f and i) Transmission Electron Microscopy (TEM) image. Scale bars, 5 pm in a, d and g, 1 pm in b, e and h, 500 nm in c, f and i. (j and k) Relative levels of FZL-GFP in different fzI transgenic lines expressing FZL-GFP. Shown inj is the immunoblot with GFP antibodies. The loading of the proteins was measured by Coomassie blue staining and shown in k. Lane 1 and 2, f2]; lane 3 and 4, f2] partially complemented by FZL-GFP; lane 5 and 6, f2! fully complemented by FZL-GFP; lane 7 and 8, f2] plants with higher FZL-GFP levels. Images in this figure are presented in color. 141 Wild type d e m e m b P m o C Overexpressed 6 “CM .... its A a... ‘4“... u. ‘5... 142 “as male WQQQQNQQ structures on the chloroplast surface (Figure 7c, f and i). Although infrequent, these structure were also observed in wild-type plants (Figure 7b) (Robertson et al., 1996), suggesting they are naturally occurring structures. These data further suggest that the GFP-labeled punctate structures correspond to the vesicle-like structures and that F ZL may be involved in their formation. The GTPase domain is important for FZL function To further investigate the role of FZL in the chloroplast, a mutant of F ZL, FZL (K362M)-GFP was made. In the gene product of this mutant, lysine 362 (Figure 1c) of F ZL was changed to a methionine. This lysine is conserved not only in the GTPase domains of dynamin family members, but also in the GTPase domains of many other proteins. Mutating the equivalent residue has been shown to abolish the function of F Z0 and other dynamin-related proteins (Hermann et al., 1998; Arimura and Tsutsumi, 2002; Orth et al., 2002). Mutation of residue 362 from lysine to methionine was predicted to reduce the GTPase activity of FZL without altering its structure. When the chloroplast division phenotype was partially complemented by F ZL- GFP, GFP-labeled punctate structures were rarely seen (Figure 8a). In fully complemented plants, GFP-labeled punctate-like structures were observed more frequently (Figure 8b). Inle mutant plants transformed by FZL(K362M)-GFP, the mutant phenotype was not rescued and GFP-labeled punctate structures were absent (Figure 8c). Nor were these structures found in wild type plants transformed with 143 Figure 8 Mutations in the GTPsae domain affect the localization and function of mutant FZL-GFP. (a and b) FZL-GFP in le plants (a) le mutant is partially complemented by low levels of FZL-GFP. (b)le mutant is complemented by normal levels of FZL-GFP. FZL-GFP is localized to vesicle-like structures. (c and d) K362M mutation blocks the formation of GFP labeled vesicle-like structures. (c) FZL(K362M)-GFP cannot complement the mutant phenotype of f2]. (d) FZL(K362M)—GFP in wild type plant. The GFP labeled vesicle-like structure is not seen as in b. Images are of same magnification in (a- d) and scale bar represents 5 pm. (e) lmmuno blot of FZL-GFP and FZL(K362M)—GF P. Approximately 2 mg of fresh weight was loaded in each lane. Lane 1 and (a), 1‘2] mutant partially complemented by low level of FZL-GFP; Lane 2 and (b), fzI mutant fully complemented by low level of FZL-GFP; Lanes 3-5, le mutant plants expressing FZL(K362M)—GFP; Lane 6-7, wild type plants expressing FZL(K362M)—GFP. Lane 3 and 6 are corresponding to (c) and (d) respectively. Images in this figure are presented in color. 144 11M anm Dwmfl I||ill||||||||||||||||lll||||ll|||||||||||||||||||||||l inn (: mum [MW e unfit nun GFRu rah? ts 1234567 mhl 145 F ZL(K362M)-GFP (Figure 8d). Irnmuno blots showed that the mutant proteins were expressed at levels comparable to those in mutant plants complemented by F ZL-GF P (Figure 8e). These data indicate that the GTPase activity of FZL is required for the localization of FZL in a punctate pattern, and also suggest that this punctate localization pattern is important for FZL function. Discussion: In this paper, we identified F ZL, an F ZO-like protein in plants, and studied its function. Mutation of FZL causes abnormal thylakoid morphology and a chloroplast division defect with heterogeneity in chloroplast size. FZL-GF P was shown to be present in both the inner envelope membrane and thylakoids of the chloroplast. F ZL-GFP was mainly localized to punctate or vesicle-like structures associated with chloroplasts and infrequently with the cytosol. These data indicate that FZL is not an orthorolog of F20 and mitofusins, mitochondrial fusion proteins in fungi and metazoa, but has a unique function in chloroplasts. FZL is very similar to two proteins found in Chloroflexus aurantiacus (Genebank accession number: ZP_00021046) and Deinococcus radiodurans R1 (Genebank accession number: NP_29495 9). Because the GC content of these two genes is very different from that of the flanking sequences and the whole genomes, the two genes seem to be the result of horizontal gene transfer (Makarova et al., 2001; Da Lage et al., 2004). The GTPase domains of FZL and FZO proteins can be aligned with those of dynamin and 146 dynamin-related proteins, but they lack the other domains of the dynamin family and are only more similar to dynamin-related proteins when compared with other GTPases. The fact that F Z0 is involved in mitochondrial fusion and FZL is apparently involved in chloroplast function implies that they diverged very early. However, F ZL and FZO are similar in both sequence and domain arrangement and a phylogenetic analysis indicated that they are more related to each other than other dynamin related proteins, suggesting that FZL may be mechanistically similar to FZO, mediating membrane fusion through the interaction of its coiled-coil domains and GTP hydrolysis. F ZL may have membrane fusion activity as does F ZO. We speculate that F ZL is involved in a vesicle-trafficking process in the chloroplast and blocking of this process affects the properties of the thylakoid and chloroplast envelope membranes, and results in a change in thylakoid morphology and dysfunction of the chloroplast division machinery. This is based on the following evidence: First, F ZL has a chloroplast transit peptide and F ZL is a membrane protein found in both the thylakoid and the envelope fiaction of chloroplasts (Figure 4). Second, F ZL-GFP was localized to thylakoids, vesicle-like structures associated with the inner chloroplast envelope, membrane protrusions from the chloroplast and vesicle-like structures in the cytosol based on both fluorescence microscopy and immuno-gold labeling (Figure 5 and Figure 6), suggesting that F ZL and these structures undergo trafficking. Third, the number of GFP-labeled punctate structures and vesicle-like structures on the surface of chloroplasts was positively correlated with the level of FZL-GF P, further supporting the hypothesis that 147 the punctate structures correspond to vesicle-like structures and that F ZL is involved in the formation and (or) the trafficking of these structures. Fourth, in fizl mutant plants partially complemented with low levels of F ZL-GFP, the punctate pattern of GP P signal can rarely be seen; in 1221 mutant plants fully complemented with F ZL-GFP and with normal thylakoid morphology, many FZL-GFP-labeled punctate structures were visible on the cytosolic side of the chloroplast envelope; in plants with high levels of F ZL-GF P, some of the F ZL-GFP-labeled punctate structures were found in the stroma but were smaller in size (Figure 5c, (1). If the function of FZL is membrane fusion as is true for FZO in fungi and metazoa, it is likely that the vesicles are originally very small and then aggregate and fuse to form larger vesicles as they are transferred across the chloroplast envelope. Finally, a mutation, K362M, completely abolished the function of FZL-GFP and its punctate localization pattern. This further supports the notion that FZL mediates the fusion of small vesicles into larger vesicles in the stroma and their subsequent trafficking to the envelope and cytosol, and this dynamic vesicle-trafficking process is required for normal chloroplast thylakoid morphology. In most eukaryotic cells, the Golgi apparatus is an array of stacked membrane sacs. Vesicles traffic from one sac to another by fusion and regeneration (Mayer, 2002). The preper functioning of the Golgi apparatus requires many GTPases of the dynamin (Henley and McNiven, 1996; Jones et al., 1998) and Rab GTPase families and other proteins such as golgins with coiled-coil domains (Barr, 1999; Short et al., 2001; Barr and Short, 2003; Diao et al., 2003; Jackson, 2003; Satoh et al., 2003). GTPase activity and the interaction between the coiled-coil domains of these proteins are important for 148 the maintenance of the dynamic structure of the Golgi apparatus and for the fusion and trafficking of vesicles for secretion. In spite of their many differences, the structure of the Golgi apparatus and chloroplast thylakoid are somewhat similar as they are both made up of well-organized, stacked membrane sacs. Also similarly, a dynamin-like GTPase with two coiled-coil domains and two trans-membrane domains, F ZL, is involved in the maintenance of the structure of thylakoid stacks, suggesting that thylakoid and Golgi may share similarities in their biogenesis. Although it has been shown that the vesicle- transport system seen in the Golgi is related to Golgi structure (Short et al., 2001; Panic et al., 2003), no such relationship has been shown to exist for thylakoids and vesicle- transport. If, as proposed here, FZL is involved in both the structural maintenance of the thylakoids and chloroplast vesicle-trafficking, the Golgi apparatus and thylakoid may also have in common the feature of vesicle-trafficking as a means of maintaining structure. In the vesicle secretion system of yeast and animal cells, vesicles originate from the Golgi and are transported to and then fuse with the plasma membrane (Tooze et al., 2001; Mayer, 2002). If the FZL-GFP-labeled vesicles are transported out of chloroplasts, this process should involve transport across both the inner membrane and outer chloroplast envelope. This is in contrast to the single membrane involved in Golgi vesicles-trafficking and these two processes may have many mechanistic differences. However, as with the chloroplast vesicle fusion proposed here, mitochondrial fusion requires the fusion of double membranes (Mozdy and Shaw, 2003). Recent work has shown that three mitochondrial fusion proteins in S. cerevisiae, FZOlp, Mgrnlp and 149 Ugolp, form a complex spanning the outer and inner membranes of the mitochondria and that their interactions are important for mitochondrial fusion (McQuibban et al., 2003; Sesaki et al., 2003; Wong et al., 2003). Although the mechanism of fusion is thus far not very clear, a similar mechanism may be responsible for vesicle trafficking out of chloroplasts and include a protein complex spanning the inner and the outer membranes of the chloroplast. Vesicle-like structures on the outer envelope or in the stroma and close to the inner envelope have been observed either by chance or under specific experimental conditions (Robertson et al., 1996; Kroll et al., 2001; Westphal et al., 2001b; Westphal et al., 2001a; Chiba et al., 2003; Westphal et al., 2003). VlPPl is proposed to be a protein involved in thylakoid biogenesis and maintenance via the budding of vesicles from the inner envelope membrane (Kroll et al., 2001; Westphal et al., 2001b). The vesicle transport of FZL seems to be a distinct pathway for two reasons: first, for FZL, the direction is from stroma to cytosol, but for VIPPl, the direction is likely from inner envelope to thylakoid. Second, the thylakoid membrane system is degraded in vippl mutants but not in le mutants, suggesting that FZL influence the morphology of the thylakoids whereas VIPPl is involved in biogenesis of the thylakoids. Vesicles close to the inner envelope of the chloroplast were also observed to accumulate at 4°C and their formation and fusion were affected by specific inhibitors(Westphal et al., 2001a, 2003); however, the direction of the movement of these vesicles is unclear. Rubisco-eentaining vesicles with double membranes have been found in the cytoplasm in naturally senescing 150 leaves of wheat (Chiba etal., 2003). This supports the proposal that there is a vesicle- trafficking process from the chloroplast to the cytosol. Stromules are described as stroma-filled tubular structures extending from the plastid enve10pe membrane (Kohler et al., 1997b) and are clearly shown to be tubules dynamically protruding outward from chloroplasts and connected to each other (Kohler et al., 2000; Arimura et al., 2001). Vesicle-like structures filled with GFP have been observed either associated with stromules or detached from the plastid body (Kohler et al., 1997b; Pyke and Howells, 2002). It was proposed that the vesicle-like structures are cleaved fi'om the plastids and represent a plastid export mechanism (Pyke and Howells, 2002). We studied le mutant plants with a chloroplast-targeted GFP as a marker for the stroma and found no defects in stromule formation (data not shown). Additionally, GFP- filled vesicles were shown to be bead-like or located at the enlarged end of stromules (Kohler et al., 1997b). Therefore, FZL does not appear to be involved in stromule formation. However, stromules may provide tracts for the trafficking of FZL-GFP- labeled vesicles or some of the FZL-GFP-labeled vesicles may make up a portion of the stromules. Bacteria have been known to secrete vesicles from the outer membrane. Recently, it was shown that in Escherichia coli these vesicles have an important role in the secretion and delivery of bacterial protein toxins to mammalian cells (Beveridge, 1999; Miller et al., 2003; Wai et al., 2003). Since chloroplasts originated from cyanobacteria and many bacterial cellular processes are still preserved within the organelle, chloroplasts 151 may have a vesicle secretion system similar to that in bacteria. To date, the cargo of F ZL- GFP-labeled vesicles is unknown, but the fact that vesicle formation is involved in the maintenance of thylakoid structure and chloroplast division implies that blocking of the dynamic vesicle-trafficking process may affect the underlying composition of the thylakoid and inner chloroplast membranes and consequently the function of chloroplasts. Thin layer chromatograph (TLC) analysis of total lipids and gas chromatograph (GC) analysis of total fatty acids showed no difference between wild type and le plants (data not shown); however, the mutant phenotype may be due to abnormal distribution of lipids or (and) fatty acids. Alternatively, FZL may be involved in some unknown signaling pathway that controls the development and division of chloroplasts. Further dissection and discovery of more components involved in this chloroplast vesicle-trafficking process will provide more details of its role. If FZL is involved in the dynamic process of vesicle trafficking from the thylakoid to the cytosol, as proposed, loss of F ZL fimction may severely affect the properties of the inner membrane. Subsequently, the function and (or) the localization of some chloroplast division proteins may be affected, resulting in enlarged chloroplasts in le mutants. The lack of visible GFP-labeled vesicle-like structures and the lack of complementation in the F ZL(K362M)-GFP transformed le plants firrther indicates that the vesicles formed in the chloroplast and (or) their trafficking are important for chloroplast division. Alternatively, the abnormal thylakoids in le mutants may not be separated well during chloroplast division, affecting the constriction of chloroplasts and resulting in enlarged chloroplasts with heterogeneity. 152 Materials and Methods Plant material Arabidopsis thaliana ecotype Columbia (Col-0) was used for all experiments as wild type. T -DNA insertion lines of FZL in the Col-0 background were ordered from Arabidopsis Biological Resource Center (ABRC) (Alonso et al., 2003).Thele mutant ’7 was identified in the segregation group from Salk lines: Salk_033745 and Salk_009051 (Alonso et al., 2003). Plants were grown as described (Gao et al., 2003). 61.! A v Microscopy Phenotypes were analyzed as previously described (Gao et al., 2003), except that the images were recorded with a Coolpix 4500 digital camera (Nikon Corporation, Tokyo). For in vivo detection of GFP with conventional fluorescence microscope, fresh leaf tissue was mounted in water and viewed with an L5 filter set (excitation 460-500 nm, emission 512-542 nm) and 40X objective lenses of a Leica DMR A2 microscope (Leica, Wetzlar, Germany) equipped with epifluorescence illumination. Images were captured with a cooled charge-coupled device camera (Retiga 1350EX, Qimaging, Burnaby, British Columbia, Canada). Chlorophyll was detected by the same method but with the TX2 filter set (excitation 540-5 80 nm, emission 608-683 nm). Confocal laser scanning microscopy (CLSM) was done with a Zeiss Pascal LSM 5.0 (Carl Zeiss, Germany). A 488nm argon ion laser line was used for excitation of GFP and chlorophyll flourescence. A BP 505-515 nm emission filter was used for GFP and a LP 650 nm emission filter was 153 used for chlorophyll. Images were viewed with a 40X oil immersion objective and processed with the Zeiss Pascal software (version 3.2 SP2). F or scanning electron microscopy (SEM) of isolated chloroplasts, chloroplasts from 3-week-old plants were isolated as in (Fitzpatrick and Keegstra, 2001), fixed with 4% glutaraldehyde for 2 hours and dehydrated in a ethanol sereies (Klomparens, 1986).The samples were sputter coated with gold, and imaged using a JEOL J SM 6400V SEM (JEOL USA, Inc., Peabody, MA, USA) at 7-10 kV of accelerating voltage. For transmission electron microscopy (TEM) of chloroplasts, 3-week-old leaf tissue was cut and fixed in 2.5% glutaraldehyde and 2.0% paraforrnaldehyde in phosphate buffer 0.1 M, pH7 .4 for approximately 24 hours at 4 °C. Postfixation was done in 1% osmium tetroxide. Samples were embedded in silicone molds and the polymerization was done for 24 hours at 60 °C. Thin sections were stained with 2% uranyl acetate and lead citrate before viewing with a J EOL 100CX transmission electron microscope (J EOL Inc., Peabody, MA, USA). Immune-gold labeling was done as in (Vandenbosch, 1991). The 3-week-old leaf tissue was cut and fixed in 0.2% glutaraldehyde and 3.0% paraforrnaldehyde in phosphate buffer 0.1 M, pH7.4, for approximately 24 hours at 4 °C. The primary antibodies against GF P (Clontech, Palo Alto, CA, USA) were diluted 30 times and the secondary antibodies conjugated with 10m colloidal gold (Sigma, Saint Louis, MO, USA) were diluted 20 times. The samples were viewed with TEM as above. 154 All images were processed with PHOTOSHOP imaging software (Adobe Systems, San Jose, CA). Sequence analysis of FZL Primers used for RT-PCR amplification of F ZL cDNA were 5'- CACAGACGAAGGTATCTCACTCTC -3' and 5'- TGTCAAGAAGGGAAAACCCGAC -3'. Amplified cDNAs were sub-cloned into Bluescript KS+ (Stratagene, La J olla, CA, USA) before sequencing. The amino acid sequence of FZL was deduced from the cDNA sequence. TargetP (Emanuelsson et al., 2000) (http://www.cbs.dtu.dk/services/TargetP/) was used for predicting the subcellular location the FZL. The transmembrane domains of FZL were predicted with TMHMM program (http:/lbiowb.sdsc.edu/) and TMpred program (http://www.ch.embnet.org/software/TMPRED_form.html). The coiled-coil domains were predicted with the Paircoil program (Berger et al., 1995). The sequence alignment shown in Figure 1c was obtained using the program CLUSTALW (Thompson et al., 1994) and Boxshade at the Biology Workbench 3.2 website (http://biowb.sdsc.edu/). The GenBank accession number for FZO fi'om Drosophila melanogaster is AAF561 10. Protein sequences of the GTPase domains were used for the phylogenetic analysis in Figure lb and aligned with CLUSTALW (Thompson et al., 1994) using default settings. The alignment is available on request. The phylogenetic tree was drawn by using the 155 program DRAWTREE at the Biology Workbench 3.2 website (http://biowb.sdsc.edu/). Neighbor joining analysis was performed using the program PROTPARS at the same website. Bootstrap analysis was performed on the neighbor-joining tree with one thousand replications. GenBank accession numbers for proteins aligned with F ZL are as follows: human Dynamin] (NP_004399), human Dynamin H (NP_OO4936), yeast Dnmlp (NP_013100), AtDRP3B (NP_565362), AtDRPlA (NP_851120), Glycine phragrnoplastin (AABOS992), AtDRP2A (AAF22291), AtDRPZB (NP_176170), AtDRPSA (NP_175722), AtDRPSB (AY212885), Drosophila FZO (AAF56110). Test of the transit peptide of FZL A derivative of the transformation vector pCAMBIA-l302 (CAMBIA, Canberra, Australia), pCAMBIA-l302-bar (the hptII gene was replaced with the bar gene from pCAMBIA-3300), which has a 35S CaMV promoter-GFP-NOS terminator cassette, was used as a negative control (Figure2 d-f). The corresponding coding sequence of the predicted transit peptide was amplified by PCR using the primers 5'- GCTTCCATGGGAACTCTAATCTCTCACCGGC -3' and 5'- GCATAGATCTGGACGCTTGTAACCACCAG -3'. The PCR product was digested with Ncol and BgIII and ligated into pCAMBIA-l302-bar. The start codon is in the NcoI cutting site. This constructs were transferred to Agrobacterium tumefaciens GV3101 and introduced into Arabidopsis plants by floral dipping. Green fluorescence from GF P and red fluorescence from chlorophyll in the transgenic (F igure2 a-c) and wild type plants (Figure2 g-h) were detected by fluorescence microscopy. 156 Complementation analysis and FZL-GFP localization The genomic fragment corresponding to F ZL, including ~740bp of the 5' flanking DNA, was amplified from genomic DNA by PCR using the primers 5'- GACACACATATTAGGATTGGTTGCAC -3' and 5'- TACCACTAGTTCTCATCTCGTC TCGTGATACATG -3'. The PCR product was digested with Spel and ligated into the transformation vector pCAMBIA-1302-bar, which was digested with Smal and SpeI. The construct was transferred to Agrobacterium tumefaciens GV3101 and introduced into le plants by the floral dipping method. The phenotypes of the T1 plants were determined by microscopy. In addition to fluorescence microscopy, FZL-GF P levels were measured by immunoblot. Monoclonal GF P antibodies were purchased from Clontech (Palo Alto, CA, USA). Protease protection assay, fractionation of chloroplasts and immunoblotting Chloroplast isolation, protease protection assays and immunoblotting were done as described (Bruce et al., 1994; McAndrew et al., 2001). Crude proteins representing approximately 4 ug chlorophyll content in each lane were analyzed by SDS-PAGE and immunoblotting in Figure 4a. Chlorophyll was quantified by adding 10 pl chloroplast suspensions to 990 pl 80% acetone and measuring the absorbance of the supernatant at 645 and 663 nm (Bruce et al., 1994). Fractionation of chloroplasts was done as in (Block et al., 2002). To fractionate chloroplasts into envelope and thylakoid, chloroplasts were broken in 10 mM MOPS pH 157 7.6, 4 mM MgC12 , 1 mM phenylrnethanesulfonyl fluoride and 1 mM caproic acid, and then separated on a step gradient of 0.93 M to 0.6 M sucrose in 10 mM MOPS pH 7.6, 1 mM Mng by centrifugation at 70 000 g for l h. The thylakoid and envelope fractions were precipitated by acetone and suspended in equal amounts of SDS loading buffer so that equal sample volumes represent an equivalent proportion of the chloroplast. Crude proteins of different fractions in each lane of Figure 4b and 4c were from chloroplasts with 4 pg chlorophyll content. For immunoblottings, GFP antibodies were the same as above with a 1:10,000 dilution. Ticl 10 and FtsZ1 antibodies antibodies were diluted as described (Jackson et al., 1998; Stokes etal., 2000). LHCb antibodies used at 1: 20,000 were kindly provided by Kenneth Cline. Mutagenesis of FZL To create FZLK362M mutation, the 5’ part of the genomic DNA of FZL was amplified by the PCR using the primers 5'- GACACACATATTAGGATTGGTTGCAC - 3' and 5'- Phos-TTCCAGAGTTAAATTCCCCCAC -3'. The 3’ end of the genomic DNA of FZL was amplified by PCR using the primers 5'- TGTCAACGGTTATCAATGCACTTCT -3' and 5'- TACCACTAGTTCTCATCTCGTCTCGTGATACATG -3' and digested with SpeI. The two PCR products were ligated into transformation vector pCAMBIA-1302-bar as above. The constructs were introduced into plants as above. 158 Literature cited Alonso, J.M., Stepaneva, A.N., Leisse, T.J., Kim, C.J., Chen, H., Shinn, P., Stevenson, D.K., Zimmerman, J., Barajas, P., Cheuk, R., Gadrinab, C., Heller, C., Jeske, A., Keesema, E., Meyers, C.C., Parker, H., Prednis, L., Ansari, Y., Choy, N., Deen, H., Geralt, M., Hazari, N., Hem, E., Karnes, M., Mulhelland, C., Ndubaku, R., Schmidt, 1., Guzman, P., Aguilar-Henenin, L., Schmid, M., Weigel, D., Carter, D.E., Marchand, T., Risseeuw, E., Brogden, D., Zeke, A., Crosby, W.L., Berry, C.C., and Ecker, J.R. (2003). Genome- Wide Insertional Mutagenesis of Arabidopsis thaliana. Science 301, 653-657. Anderssen, B., and Anderson, J .M. (1980). Lateral heterogeneity in the distribution of chlorophyll-protein complexes of the thylakoid membranes of spinach chloroplasts. Biochim Biophys Acta 593, 427-440. Arimura, S., and Tsutsumi, N. (2002). A dynamin-like protein (ADL2b), rather than FtsZ, is involved in Arabidopsis mitochondrial division. Proc Natl Acad Sci U S A 99, 5727-5731. Arimura, S., Hirai, A., and Tsutsumi, N. (2001). Numerous and highly developed tubular projections from plastids observed in Tobacco epidermal cells. Plant Sci 160, 449-454. Barr, EA. (1999). A novel Rab6-interacting domain defines a family of Golgi-targeted coiled-coil proteins. Curr Biol 9, 381-384. Barr, F.A., and Short, B. (2003). Golgins in the structure and dynamics of the Golgi apparatus. Curr Opin Cell Biol 15, 405-413. Berger, B., Wilson, D.B., Wolf, E., Tenchev, T., Milla, M., and Kim, P.S. (1995). Predicting coiled coils by use of pairwise residue correlations. Proc Natl Acad Sci U S A 92, 8259-8263. Beveridge, T.J. (1999). Structures of Gram-Negative Cell Walls and Their Derived Membrane Vesicles. J. Bacteriol. 181, 4725-4733. Block, M.A., Tewari, A.K., Albrieux, C., Marechal, E., and Joyard, J. (2002). The plant S-adenosyl-L-methioninezMg-protoporphyrin IX methyltransferase is located in both envelope and thylakoid chloroplast membranes. Eur J Biochem 269, 240-248. Borodich, A., Rejdestvenski, I., and Cottam, M. (2003). Lateral heterogeneity of photosystems in thylakoid membranes studied by Brownian dynamics simulations. Biophys J 85, 774-789. 159 Bruce, B.D., Perry, S., Froehlich, J., and and Keegstra, K. (1994). Plant Molecular Biology Manual. (Kluwer Academic Publishers, Boston, MA). Chiba, A., Ishida, H., Nishizawa, N.K., Makino, A., and Mac, T. (2003). Exclusion of ribulose-l,5-bisphosphate carboxylase/oxygenase from chloroplasts by specific bodies in naturally senescing leaves of wheat. Plant Cell Physiol 44, 914-921. Chow, W.S. (1984). The extent to which the spatial separation between photosystems I and 11 associated with granal formation limits noncyclie electron flow in isolated lettuce chloroplasts. Arch Biochem Biophys 232, 162-171. Clough, S.J., and Bent, A.F. (1998). Floral dip: a simplified method for Agrobacterium- mediated transformation of Arabidopsis thaliana. Plant J 16, 735-743. Colletti, K.S., Tattersall, E.A., Pyke, K.A., Froelich, J.E., Stokes, K.D., and h Osteryoung, K.W. (2000). A homologue of the bacterial cell division site- ' determining factor MinD mediates placement of the chloroplast division apparatus. Curr Biol 10, 507-516. Da Lage, J.L., Feller, G., and J anecek, S. (2004). Horizontal gene transfer from Eukarya to bacteria and domain shuffling: the alpha-amylase model. Cell Mol Life Sci 61, 97-109. Dai, K., and Lutkenhaus, J. (1992). The proper ratio of F tsZ to FtsA is required for cell division to occur in Escherichia coli. J Bacteriol 174, 6145-6151. de Beer, P.A., Crossley, R.E., and Rothfield, LL (1992). Roles of MinC and MinD in the site-specific septation block mediated by the MinCDE system of Escherichia coli. J Bacteriol 174, 63-70. Diao, A., Rahman, D., Pappin, D.J., Lucocq, J ., and Lowe, M. (2003). The coiled-coil membrane protein golgin-84 is a novel rab effector required for Golgi ribbon formation. J Cell Biol 160, 201-212. Emanuelssen, 0., Nielsen, H., Brunak, S., and ven Heijne, G. (2000). Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. J Mol Biol 300, 1005-1016. Fitzpatrick, L.M., and Keegstra, K. (2001). A method for isolating a high yield of Arabidopsis chloroplasts capable of efficient import of precursor proteins. Plant J 27, 59-65. Gae, H., Kadirjan-Kalbach, D., Froehlich, J.E., and Osteryoung, K.W. (2003). ARCS, a cytosolic dynamin-like protein from plants, is part of the chloroplast division machinery. Proc Natl Acad Sci U S A 100, 4328-4333. 160 Hales, K.G., and Fuller, M.T. (1997). Developmentally regulated mitochondrial fusion mediated by a conserved, novel, predicted GTPase. Cell 90, 121-129. Henley, J.R., and McNiven, M.A. (1996). Association of a dynamin-like protein with the Golgi apparatus in mammalian cells. J Cell Biol 133, 761-775. Hermann, C.J., Thatcher, J.W., Mills, J.P., Hales, K.G., Fuller, M.T., Nunnari, J., and Shaw, J.M. (1998). Mitochondrial firsion in yeast requires the transmembrane GTPase onlp. J Cell Biol 143, 359-373. Itoh, R., Fujiwara, M., Nagata, N., and Yoshida, S. (2001). A chloroplast protein homologous to the eubacterial topological specificity factor minE plays a role in chloroplast division. Plant Physiol 127, 1644-1655. Jackson, C.L. (2003). Membrane traffic: Arl GTPases get a GRIP on the Golgi. Curr Biol 13, R174-176. Jackson, D.T., Froehlich, J.E., and Keegstra, K. (1998). The hydrophilic domain of Ticl 10, an inner envelope membrane component of the chloroplastic protein translocation apparatus, faces the stromal compartment. J Biol Chem 273, 16583- 16588. Jenes, S.M., Howell, K.E., Henley, J.R., Cae, H., and McNiven, M.A. (1998). Role of dynamin in the formation of transport vesicles from the trans-Golgi network. Science 279, 573-577. Klemparens, K., Flegler, S. L. and Hooper, G.R. (1986). Procedures for Transmission and Scanning Electron Microcsopy for Biological and Medical Science, Burlington, VT: Ladd Research Industries. 146p. ( Kohler, R.H., Zipfel, W.R., Webb, W.W., and Hanson, M.R. (1997a). The green fluorescent protein as a marker to visualize plant mitochondria in vivo. Plant J 11, 613-621. Kohler, R.H., Schwille, P., Webb, W.W., and Hanson, MR. (2000). Active protein transport through plastid tubules: velocity quantified by fluorescence correlation spectroscopy. J Cell Sci 113 ( Pt 22), 3921-3930. Kohler, R.H., Cae, J., Zipfel, W.R., Webb, W.W., and Hanson, M.R. (1997b). Exchange of protein molecules through connections between higher plant plastids. Science 276, 2039-2042. Koshiba, T., Detmer, S.A., Kaiser, J.T., Chen, H., McCaffery, J.M., and Chan, D.C. (2004). Structural basis of mitochondrial tethering by mitofusin complexes. Science 305, 858-862. 161 Kroll, D., Meierhoff, K., Bechteld, N., Kinoshita, M., Westphal, S., Vothknecht, U.C., Sell, J., and Westhoff, P. (2001). VIPPl, a nuclear gene of Arabidopsis thaliana essential for thylakoid membrane formation. Proc Natl Acad Sci U S A 98, 423 8-4242. Legres, F., Lembes, A., Frachon, P., and Rojo, M. (2002). Mitochondrial fusion in human cells is efficient, requires the inner membrane potential, and is mediated by mitofusins. Mol Biol Cell 13, 4343-4354. Makarova, K.S., Aravind, L., Wolf, Y.I., Tatusov, R.L., Minten, K.W., Keonin, E.V., and Daly, M.J. (2001). Genome of the extremely radiation-resistant bacterium Deinococcus radiodurans viewed from the perspective of comparative genomics. Microbiol Mol Biol Rev 65, 44-79. Mayer, A. (2002). Membrane fusion in eukaryotic cells. Annu Rev Cell Dev Biol 18, 289-314. McAndrew, R.S., Froehlich, J.E., Vitha, S., Stokes, K.D., and Osteryoung, K.W. (2001). Colocalization of plastid division proteins in the chloroplast stromal compartment establishes a new functional relationship between FtsZ1 and F tsZZ in higher plants. Plant Physiol 127, 1656-1666. McQuibban, C.A., Saurya, S., and Freeman, M. (2003). Mitochondrial membrane remodelling regulated by a conserved rhomboid protease. Nature 423, 537-541. Meeusen, S., McCaffery, J.M., and N unnari, J. (2004). Mitochondrial Fusion Intermediates Revealed in Vitro. Science 305, 1747-1752. Miller, 8.1., Bader, M., and Guina, T. (2003). Bacterial vesicle formation as a mechanism of protein transfer to animals. Cell 115, 2-3. Mozdy, A.D., and Shaw, J.M. (2003). A fuzzy mitochondrial fusion apparatus comes into focus. Nat Rev Mol Cell Biol 4, 468-478. Neuhaus, H.E., and Wagner, R. (2000). Solute pores, ion channels, and metabolite transporters in the outer and inner envelope membranes of higher plant plastids. Biochim Biophys Acta 1465, 307-323. Orth, J.D., Krueger, E.W., Cae, H., and McNiven, M.A. (2002). The large GTPase dynamin regulates actin comet formation and movement in living cells. Proc Natl Acad Sci U S A 99, 167-172. Osteryoung, K.W., Stokes, K.D., Rutherford, S.M., Percival, A.L., and Lee, W.Y. (1998). Chloroplast division in higher plants requires members of two 162 functionally divergent gene families with homology to bacterial ftsZ. Plant Cell 10, 1991-2004. Panic, B., Perisic, 0., Veprintsev, D.B., Williams, R.L., and Munro, S. (2003). Structural basis for Arll-dependent targeting of homodirneric GRIP domains to the Golgi apparatus. Mol Cell 12, 863-874. Pyke, K.A., and Howells, C.A. (2002). Plastid and stromule morphogenesis in tomato. Ann Bot (Lend) 90, 559-566. Rapaport, D., Brunner, M., Neupert, W., and Westermann, B. (1998). onlp is a mitochondrial outer membrane protein essential for the biogenesis of firnctional mitochondria in Saccharomyces cerevisiae. J Biol Chem 273, 20150-20155. Robertson, E.J., Rutherford, S.M., and Leech, RM. (1996). Characterization of chloroplast division using the Arabidopsis mutant arc5. Plant Physiol 112, 149- 1 59. Rojo, M., Legros, F., Chateau, D., and Lembes, A. (2002). Membrane topology and mitochondrial targeting of mitofusins, ubiquitous mammalian homologs of the transmembrane GTPase on. J Cell Sci 115, 1663-1674. Santel, A., and Fuller, M.T. (2001). Control of mitochondrial morphology by a human mitofirsin. J Cell Sci 114, 867-874. Santel, A., Frank, S., Gaume, B., Herrler, M., Yeule, R.J., and Fuller, M.T. (2003). Mitofusin-l protein is a generally expressed mediator of mitochondrial fusion in mammalian cells. J Cell Sci 116, 2763-2774. Satoh, A., Wang, Y., Malsam, J., Beard, M.B., and Warren, G. (2003). Golgin-84 is a rabl binding partner involved in Golgi structure. Traffic 4, 153-161. Schleiff, E., and Sell, J. (2000). Travelling of proteins through membranes: translocation into chloroplasts. Planta 211, 449-456. Sesaki, H., and Jensen, RE. (2001). UGOl encodes an outer membrane protein required for mitochondrial fusion. J Cell Biol 152, 1123-1134. Sesaki, H., and Jensen, RE. (2004). Ugolp links the onlp and Mgrnlp GTPases for mitochondrial fusion. J Biol Chem 279, 28298-28303. Sesaki, H., Southard, S.M., Yaffe, M.P., and Jensen, RE. (2003). Mgrnlp, a dynamin- related GTPase, is essential for fusion of the mitochondrial outer membrane. Mol Biol Cell 14, 2342-2356. 163 Short, B., Preisinger, C., Korner, R., Kopajtich, R., Byron, 0., and Barr, EA. (2001). A GRASPSS-rabZ effector complex linking Golgi structure to membrane traffic. J Cell Biol 155, 877-883. Stokes, K.D., McAndrew, R.S., Figueroa, R., Vitha, S., and Osteryoung, K.W. (2000). Chloroplast division and morphology are differentially affected by overexpression of FtsZ1 and FtsZ2 genes in Arabidopsis. Plant Physiol 124, 1668-1677. Thompson, J.D., Higgins, D.G., and Gibson, T.J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673-4680. Teoze, S.A., Martens, G.J., and Huttner, W.B. (2001). Secretory granule biogenesis: . rafting to the SNARE. Trends Cell Biol 11, 116-122. L Trissl, H.W., and Wilhelm, C. (1993). Why do thylakoid membranes from higher plants form grana stacks? Trends Biochem Sci 18, 415-419. Vandenbosch, K. (1991). Irnmunogold labelling. In Hall J L, Hawes C, eds. Electron microscopy of plant cells (Academic Press Limited), pp. 183—218. Vitha, S., Froehlich, J.E., Koksharova, 0., Pyke, K.A., van Erp, H., and Osteryoung, K.W. (2003). ARC6 is a J-domain plastid division protein and an evolutionary descendant of the cyanobacterial cell division protein F m2. Plant Cell 15, 1918- 1933. Wai, S.N., Lindmark, B., Sederblem, T., Takade, A., Westermark, M., Oscarssen, J., J ass, J., Richter-Dahlfers, A., Mizunoe, Y., and Uhlin, B.E. (2003). Vesicle-mediated export and assembly of pore-forming oligomers of the enterobacterial ClyA cytotoxin. Cell 115, 25-35. Westermann, B. (2003). Mitochondrial membrane fusion. Biochim Biophys Acta 1641, 195-202. Westphal, S., Sell, J., and Vothknecht, U.C. (2001a). A vesicle transport system inside chloroplasts. F EBS Lett 506, 257-261. Westphal, 8., Sell, J ., and Vothknecht, U.C. (2003). Evolution of chloroplast vesicle transport. Plant Cell Physiol 44, 217-222. Westphal, S., Heins, L., Sell, J., and Vothknecht, U.C. (2001b). Vippl deletion mutant of Synechocystis: a connection between bacterial phage shock and thylakoid biogenesis? Proc Natl Acad Sci U S A 98, 4243-4248. 164 Wong, E.D., Wagner, J.A., Gorsich, S.W., McCaffery, J .M., Shaw, J .M., and Nunnari, J. (2000). The dynamin-related GTPase, Mgrnlp, is an intermembrane space protein required for maintenance of fusion competent mitochondria. J Cell Biol 151, 341-352. Wong, E.D., Wagner, J.A., Scott, S.V., Okreglak, V., Holewinske, T.J., Cassidy- Stone, A., and Nunnari, J. (2003). The intramitochondrial dynamin-related GTPase, Mgrnlp, is a component of a protein complex that mediates mitochondrial fusion. J Cell Biol 160, 303-311. 165 Chapter 5 5— Conclusion and Future Directions ‘14:}... .42." 1 ._vv Fm. 166 The genomes of many bacteria and eukaryotes, including several species of cyanobacteria and the model plant Arabidopsis thaliana, were sequenced in the last decade. This makes it much easier to identify the genes involved in many biological processes, including chloroplast division. Bacterial cell division is best studied in E. coli and many of the cell division proteins in E. coli are well conserved in cyanobacteria (Margolin, 2000; Errington et al., 2003). Several chloroplast division proteins, such as FtsZ1, FtsZZ, MinD, MinE, Ftn2 and SulA, were identified by searching for homologs of cyanobacterial cell division proteins in the Arabidopsis genome (Osteryoung and Vierling, 1995; Osteryoung et al., 1998; Strepp et al., 1998; Colletti et al., 2000; Stokes et al., 2000; Itoh et al., 2001; Vitha et al., 2003; Maple et al., 2004; Raynaud et al., 2004). This indicated that chloroplasts inherited some of the important components of the cyanobacterial cell division machinery during endosymbiosis. However, not all the choloroplast division genes are derived from cyanobacteria; ARC5, a eukaryotic-derived chloroplast division gene, was identified by positional cloning in Arabidopsis (Gao et al., 2003). Phylogenetic analysis indicated that ARC5 is related to a group of dynamin-related proteins unique to plants (Gao et al., 2003; Miyagishima et al., 2003). Dynamin and its relatives are large GTPases that participate in a variety of organellar fission and fusion events in eukaryotes, including the budding of endocytotic and Golgi-derived vesicles, mitochondrial fission, mitochondrial fusion, and plant cell plate formation, etc. (Chen et al., 1991; van der Bliek and Meyerowitz, 1991; Wilsbach and Payne, 1993; Gu and Verma, 1996; Pelloquin et al., 1998; Bleazard et al., 1999; Sesaki and Jensen, 1999; Hinshaw, 2000; Danino and Hinshaw, 2001). Dynamin 167 was shown to constrict lipid tubes in vitro (Sweitzer and Hinshaw, 1998; Danino et al., 2004) and this family of proteins is proposed to be mechanochemical enzymes (Danino and Hinshaw, 2001). ARC5 was localized to the chloroplast division furrow on the cytosolic surface (Gao et al., 2003). The phenotype of the large dumbbell-shaped chloroplasts in the arcS mutant suggests that ARC5 may generate force during the constriction of the division firrrow (Pyke and Leech, 1994; Robertson et al., 1996; Gao et al., 2003). Thus, the chloroplast division apparatus is of mixed evolutionary origin and shares structural and mechanistic similarities with both the cell division machinery of bacteria and the dynamin-mediated organellar fission machineries of eukaryotes. The phenotype of the arc5 mutant also suggests that chloroplast division is not completely blocked (Pyke and Leech, 1994; Robertson et al., 1996; Gao et al., 2003). Since arc5 is a null allele, ARCS is either not essential for chloroplast division or is redundant to some other gene. A homolog of ARC5 , ARC5H, was found in the Arabidopsis genome, suggesting it might be redundant to ARC5. However, an arc5/arc5h double mutant showed the same phenotype as arc5 and a 35S-ARC5H transgene showed no effect on chloroplast division in either arc5 or wild type plants. Moreover, ARC5H- GFP was localized to the nucleus and not chloroplasts. These data strongly suggest that ARC5H does not function in chloroplast division. Therefore, although ARC5 is important for chloroplast division, it is not an essential chloroplast division protein. Cyanobacteria are Gram-negative bacteria with a cell wall between the inner and the outer cell membranes. F tsI, a transpeptidase conserved in many bacteria, is involved 168 in the synthesis of the new cell wall during cell division (Nakamura et al., 1983; Weiss et al., 1997; Wissel and Weiss, 2004). As there is no cell wall between the inner and outer membranes of chloroplasts and no homolog of Ftsl is found in the sequenced genomes of Arabidopsis and rice, the inner and outer membranes of chloroplasts may be connected by some protein complex, such as the chloroplast import machinery. In the arc5 mutant, although the constriction of the chloroplast outer membrane is defective, components of the chloroplast division machinery that are derived from cyanobacteria may be sufficient for the first several rounds of chloroplast division in the cells of very young tissues. The total volume of chloroplasts in one cell relative to the volume of the cell in leaf tissue is believed to be kept at a certain ratio (Pyke, 1999). When leaf cells start to quickly expand but the chloroplasts cannot divide efficiently, chloroplast size will increase. With the increase of chloroplast size, the chloroplast division machinery may work even less efficiently. When chloroplast envelopes were changed during evolution, some chloroplast division proteins may be lost and chloroplasts may divide inefficiently. However, there are may be some disadvantages to have less chloroplasts with relatively larger size (J eong et al., 2002). The chloroplast division machinery must be further modified to overcome this problem. Probably in this kind of situation, ARC5 evolved from the host cell to facilitate the division of chloroplasts from the cytosolic side. A similar scenario may have occurred in the evolution of Dnm1 for mitochondria division. However, mitochondria are more ancient than chloroplasts and mite-FtsZ, which was found only in some lower 169 eukaryotes (Beech et al., 2000; Gilson et al., 2003; Kiefel et al., 2004), may be the last relic of the bacterial cell division machinery in mitochondria. ARC5 is relatively divergent from other dynamin-related proteins (Gao et al., 2003). The GTPase and middle domains of ARC5 can be aligned with those in other dynamin-related proteins better than can the PH and GED domains (Gao et al., 2003). The PH domains of dynamin family proteins determine the specificity of membrane binding (Salim et al., 1996; Zheng et al., 1996). The lower conservation in the PH domains between ARC5 and other members of dynamin family may underlay their different locations in the cell. ARC5 may generate force to facilitate chloroplast division like dynamin. However, ARC5 fimctions at a different site and forms a ring with a much lager diameter than do other dynamin-related proteins. FZO is an even more diverged dynamin-related protein involved in the firsion of the mitochondrial outer membrane in metazoa and fungi (Hales and Fuller, 1997; Sesaki and Jensen, 1999). It has a GTPase domain, two transmembrane domains and two coiled- coil domains (Shaw and Nunnari, 2002). I found an F ZO-like protein in plants, FZL, with a similar domain arrangement. However, FZL was shown to be targeted to the chloroplast. The fizl knockout mutant has disorganized stacking of thylakoid grana and a chloroplast division defect with heterogeneity in chloroplast size. Overexpression of FZL-GFP resulted in very long thylakoid membrane sacs with considerably less stacking than in wild type. FZL-GFP is a membrane protein localized to both the thylakoid and the inner chloroplast envelope membrane. 170 Thylakoids in cyanobacteria are simple parallel membrane sacs without stacking or visible differentiation in the membrane structure (Vothknecht and Westhoff, 2001; Westphal et al., 2003). In contrast, thylakoids in plants are organized stacks of membrane sacs (grana) connected by fewer unstacked sacs (lamellae) (Dekker and Boekema, 2005). Grana and lamellae have different roles in photosynthesis and their differentiation makes photosynthesis more efficient (Trissl and Wilhehn, 1993). F ZL is in the same plylegenetic branch as FZO in the dynamin family and has a domain arrangement similar to that of FZO, suggesting that they may share similarity in the mechanisms of their functions. It is interesting that FZL evolved to regulate the morphology of thylakoid. Although it is not clear how the thylakoids are separated during chloroplast division and cyanobacterial cell division, analysis of FZL does indicate that the morphogenesis of thylakoids affects chloroplast division. ARC5 is the first cytosolic component of the chloroplast division machinery to be identified. FZL is the first known protein that dramatically affects both thylakoid morphology and chloroplast division. In budding yeast, Dnm1 is a dynamin-related protein localized to the division site of mitochondria. It interacts with other proteins, such as Mdvl, Net2 and Fisl (Cerveny et al., 2001; Tieu et al., 2002; Cerveny and Jensen, 2003), on the cytosolic side to control mitochondria division. In fungi and metazoa, F ZO, Ugol and Mgrnl are in a protein complex involved in the fusion of mitochondrial outer membranes (Wong et al., 2003). So, ARC5 and FZL may interact with other proteins during chloroplast division and thyalkoid morphogenesis. In E. coli, there are about ten 171 proteins localized to the cell division site and interacting with each other. In chloroplasts, it is possible that there are additional proteins that interact with F tsZ and ARC6 at the division site either on the stromal side or in the intermembrane space. Therefore, more genes need to be identified for a better understanding of the mechanism of chloroplast division. Homologs of cyanobacterial cell division proteins in Arabidopsis were shown to be involved in chloroplast division (Osteryoung and Vierling, 1995; Osteryoung et al., 1998; Colletti et al., 2000; Stokes et al., 2000; Itoh et al., 2001; Vitha et al., 2003; Maple F et al., 2004; Raynaud et al., 2004). Most of the cyanobacterial cell division proteins were found based on homology to known cell division proteins in E. coli. However, some cell division proteins in E. coli have no homologs in cyanobacteria. In contrast, F m2 and F tn6 are cell division proteins unique to cyanobacteria (Koksharova and Wolk, 2002). Since F tn2 and F tn6 were identified by transposon mutagenesis in a non-saturating screen, it is possible that there are still some cyanobacteria-specific cell division proteins have not been identified. Further genetic screening of cyanobacterial cell division mutants by transposon and chemical mutagenesis with high saturation may identify new cyanobacteria-specific cell division proteins and help to identify new chloroplast division proteins. Indeed, some additional cyanobacterial cell division mutants have recently been obtained by transposon mutagenesis (Miyagishima et al., 2005). Genetic screening by Leech’s group and others has identified 12 are loci in Arabidopsis (Pyke and Leech, 1994; Marrison et al., 1999). ARC6, ARC10, ARCll and 172 ARC 12 correspond to homologs of the cyanobacteria cell division proteins Ftn2, FtsZ1, MinD and MinE, respectively (Colletti et al., 2000; Vitha et al., 2003; Fujiwara et al., 2004) (and Miyagishima, personal communication). The N-terminal half of ARC3 has some similarity to the GTPase domain of FtsZ and the C-terminal half of ARC3 has a membrane-occupation-and-recognition-nexus (MORN) repeat motif (Shimada et al., 2004). ARC5 is a plant-specific dynamin-related protein of eukaryotic origin (Gao et al., 2003). Thus, mutant screening is another very useful approach for identifying chloroplast division genes. However, the genetic screening of the chloroplast division mutants is far from saturation. A further mutant screening in Arabidopsis and the subsequent gene cloning may identify new chloroplast division genes, especially genes of eukaryotic origin. Most of the arc mutants with an apparent chloroplast division phenotype have a non-sense mutation in the corresponding genes. It is not clear that whether mutations in the unknown chloroplast division genes cause gametophyte lethality or not. EMS causes alkylation of guanine and almost all the mutations are G/C-to-A/T transitions in EMS mutagenesis (Greene et al., 2003). Since the Arabidopsis genome has a ~40% GC content in the genie regions (Yu et al., 2002), there may be some limitations to use of EMS mutagenesis in Arabidopsis, although it has been successfully used. Other chemical mutagens can be applied as a complement to EMS in mutant screening. In Arabidopsis, there are many gene families (Arabidopsis_Genome_Initiative, 2000) and different members in the same gene family may be redundant. Mutations and 173 even the loss of function of these genes may not cause any phenotype. Therefore, other approaches are needed to overcome the shortcomings of mutant screening. Techniques to detect protein-protein interaction, such as yeast-two-hybrid (Y2H) screening and co- immunoprecipitation, are powerful and routine approaches for identifying new components in protein complexes and may be used for identifying unknown components in the chloroplast division machinery. Y2H screening has been used in many cases either for finding new proteins or for a better understanding of the relationship between interacting proteins in a biological process. A genome-wide Y2H screen identified 957 putative protein interactions in yeast (U etz et al., 2000). Y2H screening can be easily carried out. So, all the known proteins involved in chloroplast division can be used as baits for identifying the proteins with which they interact. This will not only identify new components of the chloroplast division machinery but also reveal the protein interactions in this complex. Another approach, co-immunoprecipitation (co-IP) is also very useful in the analysis of protein complexes. For example, the chloroplast protein import machineries are membrane protein complexes and they can be isolated in protein extracts by co-IP (Schnell et al., 1994; Akita et al., 1997). New components were found by analyzing all the proteins in the complex. Another example is the COP9 signalosome, a regulatory protein complex conserved fiom plants to humans (Wei and Deng, 2003). Some of the components in this complex were initially identified by genetic methods. Later on, all the components in this protein complex were identified by co-IP. This method may also be 174 applied to the chloroplast division machinery for discovering new chloroplast division proteins. Finally, the mechanism of chloroplast division at the biochemical and biophysical level is still largely unknown. Cyanobacteria have only one FtsZ, while plants have Ftle, FtsZZ and ARC3 (Osteryoung et al., 1998; Shimada et al., 2004). These three types of FtsZ proteins have distinct functions in chloroplast division (Osteryoung et al., 1998; Shimada et al., 2004). Why are three types of FtsZ proteins needed in plants for chloroplast division and how do they interact with each other? MinD and MinE are also present in the chloroplast (Colletti et al., 2000; Itoh et al., 2001), but MinC is missing. How are the MinD and MinE in plants different from those in cyanobacteria? What is the role of ARC6 in the chloroplast division? How does it regulate the activity of FtsZ at the chloroplast division site? How is ARC5 involved in chloroplast division? Is it also a mechanoenzyme like dynamin? Study of the crystal structures of the individual proteins or the proteins interacting with each other in the chloroplast division machinery will help us to answer the above questions and understand the molecular mechanism of chloroplast division. 175 Literature cited: Akita, M., Nielsen, E., and Keegstra, K. (1997). Identification of protein transport complexes in the chloroplastic envelope membranes via chemical cross-linking. J Cell Biol 136, 983-994. Arabidopsis_Geneme_Initiative. (2000). Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408, 796-815. Beech, P.L., Nheu, T., Schultz, T., Herbert, S., Lithgow, T., Gilson, P.R., and McFadden, G]. (2000). Mitochondrial F tsZ in a chromophyte alga. Science 287, 1276-1279. Bleazard, W., McCaffery, J .M., King, E.J., Bale, S., Mozdy, A., Tieu, Q., Nunnari, ; J., and Shaw, J.M. (1999). The dynamin-related GTPase Dnm1 regulates mitochondrial fission in yeast. Nat Cell Biol 1, 298-304. L Cerveny, KL, and Jensen, RE. (2003). The WD-repeats of Net2p interact with Dnmlp and F islp to regulate division of mitochondria. Mol Biol Cell 14, 4126- 4139. Cerveny, K.L., McCaffery, J.M., and Jensen, B.E. (2001). Division of mitochondria requires a novel DMN 1 -interacting protein, Net2p. Mol Biol Cell 12, 309-321. Chen, M.S., Obar, R.A., Schroeder, C.C., Austin, T.W., Poodry, C.A., Wadsworth, S.C., and Vallee, RB. (1991). Multiple forms of dynamin are encoded by shibire, a Drosophila gene involved in endocytosis. Nature 351, 583-586. Colletti, K.S., Tattersall, E.A., Pyke, K.A., Froelich, J.E., Stokes, K.D., and Osteryoung, K.W. (2000). A homologue of the bacterial cell division site- determining factor MinD mediates placement of the chloroplast division apparatus. Curr Biol 10, 507-516. Danino, D., and Hinshaw, J.E. (2001). Dynamin family of mechanoenzymes. Current Opinion in Cell Biology 13, 454-460. Danino, D., Moon, K.H., and Hinshaw, J.E. (2004). Rapid constriction of lipid bilayers by the mechanochemical enzyme dynamin. J Struct Biol 147, 259-267. Dekker, J.P., and Boekema, E.J. (2005). Suprarnoleeular organization of thylakoid membrane proteins in green plants. Biochim Biophys Acta 1706, 12-39. Errington, J., Daniel, R.A., and Scheffers, DJ. (2003). Cytokinesis in bacteria. Microbiol Mol Biol Rev 67, 52-65, table of contents. 176 Fujiwara, M.T., Nakamura, A., Itoh, R., Shimada, Y., Yoshida, S., and Meller, S.G. (2004). Chloroplast division site placement requires dimerization of the ARCl l/AtMinDl protein in Arabidopsis. J Cell Sci 117, 2399-2410. Gae, H., Kadirjan-Kalbach, D., Froehlich, J.E., and Osteryoung, K.W. (2003). ARC5, a cytosolic dynamin-like protein from plants, is part of the chloroplast division machinery. Proc Natl Acad Sci U S A 100, 4328-4333. Gilson, P.R., Yu, X.C., Hereld, D., Barth, C., Savage, A., Kiefel, B.R., Lay, S., Fisher, P.R., Margolin, W., and Beech, P.L. (2003). Two Dictyostelium orthologs of the prokaryotic cell division protein F tsZ localize to mitochondria and are required for the maintenance of normal mitochondrial morphology. Eukaryot Cell 2, 1315-1326. Greene, E.A., Cedeme, C.A., Taylor, N.E., Henikoff, J.G., Till, BJ., Reynolds, S.H., Enns, L.C., Burtner, C., Johnson, J.E., Odden, A.R., Comai, L., and Henikoff, S. (2003). Spectrum of chemically induced mutations from a large- scale reverse-genetic screen in Arabidopsis. Genetics 164, 731-740. Gu, X., and Verma, D. (1996). Phragrnoplastin, a dynamin-like protein associated with cell plate formation in plants. EMBO J. 15, 695-704. Hales, K.G., and Fuller, M.T. (1997). Developmentally regulated mitochondrial fusion mediated by a conserved, novel, predicted GTPase. Cell 90, 121-129. Hinshaw, J.E. (2000). Dynamin and its role in membrane fission. Annu Rev Cell Dev Biol 16, 483-519. Itoh, R., Fujiwara, M., Nagata, N., and Yoshida, S. (2001). A chloroplast protein homologous to the eubacterial topological specificity factor minE plays a role in chloroplast division. Plant Physiol 127, 1644-1655. J eong, W.J., Park, Y.I., Suh, K., Raven, J.A., Yoe, O.J., and Liu, J.R. (2002). A large population of small chloroplasts in tobacco leaf cells allows more effective chloroplast movement than a few enlarged chloroplasts. Plant Physiol 129, 112- 121. Kiefel, B.R., Gilson, P.R., and Beech, P.L. (2004). Diverse eukaryotes have retained mitochondrial homologues of the bacterial division protein FtsZ. Protist 155, 105- 115. Koksharova, O.A., and Welk, C.P. (2002). A novel gene that bears a DnaJ motif influences cyanobacterial cell division. J Bacteriol 184, 5524-5528. 177 Maple, J., Fujiwara, M.T., Kitahata, N., Lawson, T., Baker, N.R., Yoshida, S., and Meller, S.G. (2004). GIANT CHLOROPLAST l is essential for correct plastid division in Arabidopsis. Curr Biol 14, 776-781. Margolin, W. (2000). Themes and variations in prokaryotic cell division. FEMS Microbiol Rev 24, 531-548. Marrison, J.L., Rutherford, S.M., Robertson, E.J., Lister, C., Dean, C., and Leech, RM. (1999). The distinctive roles of five different ARC genes in the chloroplast division process in Arabidopsis. Plant J 18, 651-662. A Miyagishima, S.Y., Welk, C.P., and Osteryoung, K.W. (2005). Identification of cyanobacterial cell division genes by comparative and mutational analyses. Mol Microbiol 56, 126-143. Miyagishima, S.Y., Nishida, K., Mori, T., Matsuzaki, M., Higashiyama, T., Kuroiwa, H., and Kuroiwa, T. (2003). A plant-specific dynamin-related protein forms a ring at the chloroplast division site. Plant Cell 15, 655-665. Nakamura, M., Maruyama, I.N., Soma, M., Kate, J., Suzuki, H., and Herota, Y. (1983). On the process of cellular division in Escherichia coli: nucleotide sequence of the gene for penicillin-binding protein 3. Mol Gen Genet 191, 1-9. Osteryoung, K.W., and Vierling, E. (1995). Conserved cell and organelle division. Nature 376, 473-474. Osteryoung, K.W., Stokes, K.D., Rutherford, S.M., Percival, A.L., and Lee, W.Y. (1998). Chloroplast division in higher plants requires members of two functionally divergent gene families with homology to bacterial ftsZ. Plant Cell 10, 1991-2004. Pelloquin, L., Belenguer, P., Menon, Y., and Ducommun, B. (1998). Identification of a fission yeast dynamin-related protein involved in mitochondrial DNA maintenance. Biochem Biophys Res Commun 251, 720-726. Pyke, KA. (1999). Plastid division and development. Plant Cell 11, 549-556. Pyke, K.A., and Leech, RM. (1994). A Genetic Analysis of Chloroplast Division and Expansion in Arabidopsis thaliana. Plant Physiol 104, 201-207. Raynaud, C., Cassier-Chauvat, C., Perennes, C., and Bergounieux, C. (2004). An Arabidopsis homolog of the bacterial cell division inhibitor SulA is involved in plastid division. Plant Cell 16, 1801-1811. 178 Robertson, E.J., Rutherford, S.M., and Leech, RM. (1996). Characterization of chloroplast division using the Arabidopsis mutant arc5. Plant Physiol 1 12, 149- 159. Salim, K., Bottomley, M.J., Querfurth, E., Zvelebil, M.J., Gout, I., Scaife, R., Margolis, R.L., Gigg, R., Smith, C.I., Driscoll, P.C., Waterfield, M.D., and Panayotou, G. (1996). Distinct specificity in the recognition of phosphoinositides by the pleckstrin homology domains of dynamin and Bruton's tyrosine kinase. Embo J 15, 6241-6250. Schnell, D.J., Kessler, F., and Blobel, G. (1994). Isolation of components of the chloroplast protein import machinery. Science 266, 1007-1012. Sesaki, H., and Jensen, RE. (1999). Division versus fusion: Dnmlp and onlp antagonistically regulate mitochondrial shape. J Cell Biol 147, 699-706. Shaw, J .M., and Nunnari, J. (2002). Mitochondrial dynamics and division in budding yeast. Trends Cell Biol 12, 178-184. Shimada, H., Koizumi, M., Kuroki, K., Mochizuki, M., Fujimoto, H., Ohta, H., Masuda, T., and Takamiya, K. (2004). ARC3, a chloroplast division factor, is a chimera of prokaryotic F tsZ and part of eukaryotic phosphatidylinositol-4- phosphate 5-kinase. Plant Cell Physiol 45, 960—967. Stokes, K.D., McAndrew, R.S., Figueroa, R., Vitha, S., and Osteryoung, K.W. (2000). Chloroplast division and morphology are differentially affected by overexpression of FtsZ1 and F tsZZ genes in Arabidopsis. Plant Physiol 124, 1668-1677. Strepp, R., Scholz, S., Kruse, S., Speth, V., and Reski, R. (1998). Plant nuclear gene knockout reveals a role in plastid division for the homolog of the bacterial cell division protein FtsZ, an ancestral tubulin. Proc Natl Acad Sci U S A 95, 4368- 4373. Sweitzer, S.M., and Hinshaw, J.E. (1998). Dynamin undergoes a GTP-dependent conformational change causing vesiculation. Cell 93, 1021-1029. Tieu, Q., Okreglak, V., N aylor, K., and N unnari, J. (2002). The WD repeat protein, Mdvlp, functions as a molecular adaptor by interacting with Dnmlp and Fislp during mitochondrial fission. J Cell Biol 158, 445-452. Trissl, H.W., and Wilhelm, C. (1993). Why do thylakoid membranes from higher plants form grana stacks? Trends Biochem Sci 18, 415-419. Uetz, P., Giet, L., Cagney, G., Mansfield, T.A., Judson, R.S., Knight, J.R., Leckshen, D., N arayan, V., Srinivasan, M., Pechart, P., Qureshi-Emili, A., 179 Li, Y., Godwin, B., Cenever, D., Kalbfleisch, T., Vijayadamodar, G., Yang, M., Johnston, M., Fields, S., and Rethberg, J.M. (2000). A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae. Nature 403, 623-627. van der Bliek, A.M., and Meyerowitz, E.M. (1991). Dynamin-like protein encoded by the Drosophila shibire gene associated with vesicular traffic. Nature 351, 411- 414. Vitha, S., Froehlich, J.E., Koksharova, O., Pyke, K.A., van Erp, H., and Osteryoung, K.W. (2003). ARC6 Is a J-Domain Plastid Division Protein and an Evolutionary Descendant of the Cyanobacteria] Cell Division Protein Ftn2. Plant Cell 15, 1918- 1933. Vothknecht, U.C., and Westhoff, P. (2001). Biogenesis and origin of thylakoid membranes. Biochim Biophys Acta 1541, 91-101. Wei, N., and Deng, X.W. (2003). The COP9 signalosome. Annu Rev Cell Dev Biol 19, 261-286. Weiss, D.S., Pogliano, K., Carson, M., Guzman, L.M., Fraipont, C., N guyen- Disteche, M., Losick, R., and Beckwith, J. (1997). Localization of the Escherichia coli cell division protein Ftsl (PBP3) to the division site and cell pole. Mol Microbiol 25, 671-681. Westphal, S., Sell, J., and Vothknecht, U.C. (2003). Evolution of chloroplast vesicle transport. Plant Cell Physiol 44, 217-222. Wilsbach, K., and Payne, G.S. (1993). Vpslp, a member of the dynamin GTPase family, is necessary for Golgi membrane protein retention in Saccharomyces cerevisiae. Embo J 12, 3049-3059. Wissel, M.C., and Weiss, D.S. (2004). Genetic analysis of the cell division protein Ftsl (PBP3): amino acid substitutions that impair septal localization of F tsI and recruitment of FtsN. J Bacteriol 186, 490-502. Wong, E.D., Wagner, J.A., Scott, S.V., Okreglak, V., Holewinske, T.J., Cassidy- Stone, A., and Nunnari, J. (2003). The intramitochondrial dynamin-related GTPase, Mgrnlp, is a component of a protein complex that mediates mitochondrial fusion. J Cell Biol 160, 303-311. Yu, J ., Hu, S., Wang, J., Wong, G.K., Li, S., Lin, B., Deng, Y., Dai, L., Zhou, Y., Zhang, X., Cae, M., Liu, J., Sun, J., Tang, J., Chen, Y., Huang, X., Lin, W., Ye, C., Tong, W., Cong, L., Geng, J., Han, Y., Li, L., Li, W., Hu, G., Li, J., Liu, Z., Qi, Q., Li, T., Wang, X., Lu, H., Wu, T., Zhu, M., Ni, P., Han, H., Dong, W., Ren, X., Feng, X., Cui, P., Li, X., Wang, H., Xu, X., Zhai, W., Xu, 180 Z., Zhang, J., He, S., Xu, J., Zhang, K., Zheng, X., Deng, J., Zeng, W., Tao, L., Ye, J., Tan, J., Chen, X., He, J., Liu, D., Tian, W., Tian, C., Xia, H., Bao, Q., Li, G., Gae, H., Cae, T., Zhao, W., Li, P., Chen, W., Zhang, Y., Hu, J., Liu, S., Yang, J., Zhang, G., Xiong, Y., Li, Z., Mao, L., Zhou, C., Zhu, Z., Chen, R., Hao, B., Zheng, W., Chen, S., Guo, W., Tao, M., Zhu, L., Yuan, L., and Yang, H. (2002). A draft sequence of the rice genome (Oryza sativa L. ssp. indica). Science 296, 79-92. Zheng, J., Cahill, S.M., Lemmon, M.A., Fushman, D., Schlessinger, J., and Cewburn, D. (1996). Identification of the binding site for acidic phospholipids on the pH domain of dynamin: implications for stimulation of GTPase activity. J Mol Biol 255, 14-21. 181