.i e . 39: 54...... $1..- .W‘Lbfin... ,1 |GANSTATE UISINVER IUTY IHIJIHIIWIIII llll Hllllilllililiihllii 31293 01688 5380 This is to certify that the dissertation entitled Functional Analysis and Intracellular Targeting of the Nuclear Localization Signal Receptor, Importin Alpha, in Arabidopsis thaliana presented by Harley M. S. Smith has been accepted towards fulfillment of the requirements for Ph.D. degree in GEUECiCS Mam flaw Major professor Date August 4, 1998 MSU is an Affirmative Action/Equal Opportunity Institution 0-12771 LIBRARY Michigan State i University PLACE iN RETURN BOX to remove this checkout from your record. TO AVOID FINE return on or before date due. MTE DUE ‘ I DATE DUE DATE DUE 2ng 65i99‘ 1m CJCIRCIDatoDIDpGS-p.“ FUNCTIONAL ANALYSIS AND INTRACELLULAR TARGETING OF THE NUCLEAR LOCALIZATION SIGNAL RECEPTOR, IMPORTIN a. BY Harley M. S. Smith A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Genetics Program 1998 ABSTRACT FUNCTIONAL ANALYSIS AND INTRACELLULAR TARGETING OF THE NUCLEAR LOCALIZATION SIGNAL RECEPTOR, IMPORTIN (1, IN PLANTS BY Harley M. S. Smith Macromolecular translocation into and out of the nucleus occurs through the nuclear pore complex (NPC) which is embedded in the nuclear envelope. A subset of proteins that are imported into the nucleus contain the classical nuclear localization signals (NLSs). The importin a/B heterodimer is required for NLS-protein import in vertebrates and yeast. Importin a is the NLS receptor that binds to the NLS-containing proteins in the cytoplasm and importin B interacts with the import machinery. To characterize the NLS—protein import pathway in plants, an importin a homologue (At—IMPa) was cloned from Arabidopsis thaliana. Antibodies generated against the recombinant expressed At—IMPa recognized a protein of the correct mass in Arabidopsis roots, stems, leaves and flowers as well as in tobacco suspension cells. In vitro binding studies demonstrated that At-IMPa specifically associated with three types of NLSs that function in plants. Immunolocalization of importin a in tobacco protoplasts demonstrated that this receptor was found in the cytoplasm and nucleus which is constant with its function as a nuclear shuttling NLS receptor. In contrast to yeast and vertebrate importin a subunits, At—IMPa mediated nuclear import in the absence of an importin B subunit in a vertebrate import system, suggesting that plants may possess nuclear import pathways exclusively mediated by importin G subunits. These studies combined strongly suggest that At-IMPd is a functional NLS receptor in plants. Prior to NLS-protein import, importin a/NLS-containing proteins complexes need to be somehow directed and transported to the NPC. Using antibodies to At-IMPa as a tool, we were interested in determining if the cytoskeleton could function in this transport process. Double—labeling immunofluorescence studies showed that most of the cytoplasmic importin a coaligned with microtubules and microfilaments in tobacco protoplasts. Treatment of tobacco protoplasts with microtubule or microfilament depolymerizing agents disrupted the strands of importin a in the cytoplasm, whereas a microtubule stabilizing agent had no effect. Subcellular localization studies indicated that a fraction of the cellular importin a cofractionated with the cytoskeleton and is extracted from this fraction under conditions similar to those that extract microtubule motor proteins. Lastly, importin a associated with microtubules and microfilaments in vitro in an NLS~ dependent manner. The interaction of importin a with the cytoskeleton could be an essential element of protein transport from the cytoplasm to the nucleus in vivo. FERTILE FIELDS Sometimes those simple things won’t turn the trick no more And our self-important dreams lie shattered on the floor Even the proletariat receives his royalty And as the battle rages on and on I wish it wasn’t me And it seem so cruel The last one breaking up Until the winter finds its worth As we glide upon the earth Now the trees are swept aside by wind and sheets of rain And the fertile fields once gilded have now withered and refrained She (mother earth) who longs for comfort feels instead a savage thrust And the ashen sky grows ever darker as dawn gives way to dust As we set our dogs upon the earth Feast on the dead until no life remains Forward towards a pointless end we squander never gain When What Will will I don died Cool Will What Will Will Will They Will What Will GOOD RIDDANCE I Get Old will it be like when I get old I hop on my bike and ride around town I still want to be someone and not just sit around ’t want to be like other adults cause they’ve already condescending, fossilized I be rich will I be poor will I still sleep on the floor will it be like when I get old I still kiss my girlfriend and try to grab her ass I still hate the cops and have no class all my grown up friends say they’ve seen it all before say hey act your age and I’m immature I do myself proud or only what’s allowed will it be like when I get old I sit around and talk about the old days sit around and watch T.V. I never want to go that way Never burn out not fade away As I What travel through time will I like what I find will it be like when I get old DESCENDENTS iv ACKNOWLEDGMENTS Thanks to Natasha (the boss) for support, enthusiasm, patience, and letting me be ME. Thanks to Glenn (Glennda) for showing me the ropes, support, and being a great friend. Thanks to the Raikhel laboratory (Past and Present) for their support and love, especially the lab wolfers, Jim, Louie, Alex, Ivan (sweet cheeks), and Cindy. Thanks to Joanne and Shirley at the Confocal Laboratory at MSU for their help with the confocal microscopy. Thanks to David Jans and his group at at the Nuclear Signaling Laboratory, Division for Biochemistry and Molecular Biology, John Curtin School of Medical Research, Canberra, Australia for their collaboration. Thanks to the friends I made at the PRL/genetics/Botany/CSS and thier support in graduate school (put your name here ). Thanks to everyone who supported me for IRONMAN (especially to the Natasha (for the plane ticket), Newman lab: Tom, Therese, Sue, Shari, and Barbara, the Office Staff: Karen, Jackie, Karen, Alice, Jan, Carol, Kate and Joe for setting up all the bake sales, lunch and dinner gatherings, and Ken Poff who helped make it possible for me to race in Kona). Thanks to Kurt and Marlene for their services, photography and graphics. Thanks to my committee for guidance: Pam Green, Ken Keegstra, and John Wang. Thanks to the people at UCSD: Maarten, Milo, Nigel, Ron, Sam, Jack, Bob, Andrew and the Harris lab. Special Thanks to the Triathlon/Running group who made life outside the lab fricken awesome: Jim (Man—o—pain), Bill, Steve, Vaughn (VC; 10:10 IM) and Erica (Swim Coach), Hal (BAMF), Danny, Judy, and Scott. Special Thanks to Family and Friends back home: Dad, Mom, Marie, Sue, Karyn, Brian, and especially Mark and Lisa for the hospitality, truck, surfboards and wetsuits, Katie (IHEARTU4EVER) and the Farley/Johnson family, Ben, Marker—TIO-Chordata and Jeannie, Pat, Bike, Dog, Eric, Dave and Darcy, Eli, Jim, Felix, Ted, Aaron, RJ, Dave A. and Tim, Mike, Jimmy, Marshall, Nicole, John Carothers, Carol, Janet and Lesile, MOCK, Good Riddance, Blasti, Lab, Enemies, Milo and the Descendents, NOFX, Rancid, Pennywise, Lagwagon, No Use, Ten Foot Pole, ALL, Coffee Mug, Bonus Cup, Offspring, Down by Law—Dag Nasty, Trader Joe’s, Minor Threat, DI, Adolescents, DOA, Black Flag, Power Bar, GU, Cytomax, Metabolol, Triathlon—Ironman. vi TABLE OF CONTENTS LIST OF TABLES ...................................... X LIST OF FIGURES ..................................... Xi CHAPTER 1 Introduction: Nuclear Localization Signal Protein Import Pathway ...................................... l l. The Nucleus ................................. 2 2. Nuclear Targeting Signals ................... 3 3. The NLS—Protein Import Pathway .............. 5 4. The Importin a Family ....................... 10 S. The Importin B Family ....................... l3 6. The Ran System .............................. 17 7. Mechanism of Translocation .................. 19 8. Regulatory Proteins ......................... 20 9. Regulation of the Import Apparatus .......... 22 10. Conclusion ................................. 23 ll. Thesis Scheme .............................. 23 References ..................................... 25 CHAPTER 2 Three Classes of Nuclear Import Signals Bind to Plant Nuclei ..................................... 36 Abstract ....................................... 37 vfi Introduction ................................... 38 Methods ........................................ 39 Results ............................ l ............ 41 Discussion ..................................... 45 References ..................................... 47 CHAPTER 3 Characterization of a Nuclear Localization Signal Receptor, Importin a, in Plants ..................... 48 Abstract ....................................... 49 Introduction ................................... 51 Methods ........................................ 55 Results ........................................ 64 Discussion ..................................... 92 References ..................................... 97 CHAPTER 4 Association of Importin a with the Cytoskeleton ........................................ 105 Abstract ....................................... 106 Introduction ................................... 107 Methods ........................................ 109 Results ........................................ 117 Discussion ..................................... 133 References ..................................... 140 fin CHAPTER 5 Conclusions and Future Directions ................... 145 References ..................................... 156 ix Table Table Table Table LIST OF TABLES Three classes of NLSs identified in plants ............................ 4 The importin B family of nuclear import/export receptors .............. 14 Comparison of amino acid identities for At-IMPa other importin a homologues ........................... 69 Kinetics of nuclear importin a reconstituted in vitro using purified subunits ............................. 9O Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure LIST OF FIGURES Schematic diagram of the NLS—import pathway .............................. 8 Specific binding of NLSs to purified tobacco nuclei ....................... 42 Specific binding of NLSs to purified maize nuclei ......................... 44 Amino acid sequence of At—IMPa and other protein homologues ............. 65 Purified antibodies to At-IMPa are specific in both Arabidopsis and tobacco .......................... 71 Immunoprecipitation of in vitro— translated At-IMPa ................... 73 At—IMPa recognizes three classes of NLSs .............................. 75 Specific interaction of At—IMPa with a functional NLS— substrate ............................ 78 Immunolocalization of importin a in fixed tobacco protoplasts ......... 8O Biochemical properties of nuclear importin a ........................... 82 Ability of At-IMPa to mediate nuclear protein import reconstituted in vitro using purified components in the absence of exogenously added importin B subunit ................... 84 Nuclear import reconstituted in vitro using purified components mediated by At—IMPa and m—IMPa in the absence and presence of exogenously added m—IMPB ......................... 87 Immunolocalization of importin a, tubulin, ELP and GUS in fixed tobacco protoplasts .................. 118 Importin a colocalized with microtubules in fixed tobacco protoplasts .......................... 120 Figure 4.3 The cytoplasmic strands of importin a colocalized with microfilaments in fixed tobacco protoplasts .......................... 123 Figure 4.4 Subcellular localization of importin a ........................... 125 Figure 4.5 The biochemical properties of the P27form importin a .................. 127 Figure 4.6 Association of importin a with microtubules was dependent on functional NLSs ...................... 129 Figure 4.7 Association of importin a with microfilaments was dependent on functional NLSs ...................... 131 Figure 4.8 A Schematic representation of NLS— transport from the cytoplasm to the NPC ........................... 138 xfi Chapter 1 Introduction Nuclear Localization Signal Protein Import Pathway 1. The Nucleus In eukaryotic cells, nuclear activities are compartmentalized from other biochemical processes by the double—membrane nuclear envelope. Macromolecular translocation into and out of the nucleus occurs through the nuclear pore complex (NPC), which is a 124 megaDalton proteinaceous complex that spans the nuclear envelope (for reviews see Davis, 1995; Pante and Aebi, 1996a). The NPC is a cylindrical structure with eight-fold symmetry that contains a cytoplasmic and nucleoplasmic ring. Both of the rings are 120 nm in diameter. These rings are connected to each other by 8 spoke-like structures that converge on a central gate where the pore is located. The pore has a diameter of 26-28 nm and contains a central plug, referred to as the transporter. It is hypothesized that the transporter is involved in the import and export processes (for reviews see Davis, 1995; Pante and Aebi, 1996a). Eight thin projections extend from the cytoplasmic ring, called “cytoplasmic fibrils,” and they are distinct from the cytoskeleton. Connected to the nucleoplasmic ring is a structure called the nuclear basket that extends into the nucleoplasm. The distance from the cytoplasmic fibrils to the nuclear basket is z75 nm. At least 25 NPC proteins, called nucleoporins, have been identified in yeast and mammals (for reviews see Davis, 1995; Pante and Aebi, 1996). Small molecules, ions and metabolites can passively move through the 9 nm aqueous channels found in NPC, while proteins and macromolecules larger than 40 kiloDaltons require targeting information for import into and export out of the nucleus (for reviews see Davis, 1995; Pante and Aebi, 1996a). 2. Nuclear Targeting Signals Nuclear localization signals (NLSs) are targeting signals found in many nuclear proteins that required for import into the nucleus (Dingwall and Laskey, 1991). These signals can be grouped into one of three classes (Table 1.1; for reviews see Boulikas, 1993, 1994; Hicks and Raikhel, 1995b). The SV40 large T—antigen—like and bipartite classes have been identified in animals, fungi and plants, whereas the Mat a2- like NLSs have been found only in fungi and plants (Hicks and Raikhel, 1995b). Interestingly, the NLS from the yeast protein Mat a2 does not function in mammalian cells in vivo (Chelsky et a1., 1989; Lanford et a1., 1990). In each of these classes, there is no consensus sequence for these signals, however regions of basic amino acids are a common feature (Table 1.1; Hicks and Raikhel, 1995b). Another group of targeting signals has been identified in mRNA binding proteins that shuttle into and out of the nucleus. The M9 signal, a 38 amino acid sequence found in the hnRNPAl protein (Michael et a1., 1995; Siomi and Dreyfuss, 1995; Weighardt et a1., 1995), and the K signal, a 24 amino acid sequence found in hnRNPK (Michael, et a1., 1997), function as nuclear shuttling signals and are distinct Table 1.1 Three Classes of NLSs lndentified in Plants Class NLS Sequence Example 1. Bipartite MESNRESAESBYBE 02 (NLS B) 2. SV40-Iike MSEBKEREKL R (NLS M) 3. Mat a2-like MISEALB_KAIG§_R_ R (NLS C) The 02 NLS (NLSB) was identified in a maize transcription factor Opaque-2 (Varagona et al., 1992). The NLSs M and C were identified a maize regulatory protein called R (Shieh et al., 1993). from NLSs (Michael et a1., 1995; Siomi and Dreyfuss, 1995; Weighardt et a1., 1995; Michael, et a1., 1997). In addition, nuclear export signals (NES) have also been characterized in several proteins (for review see Gerace, 1995). 3. The NLS-Protein Import Pathway Experimentally, NLS-protein import into the nucleus is divided into two distinct steps, docking and translocation. Docking is NLS dependent and occurs when nuclear proteins dock at the cytoplasmic side of the NPC in an energy independent fashion. Translocation through the NPC is an energy dependent process (Newmeyer and Forbes, 1988; Richardson et a1., 1988). In vitro import systems using permeabilized vertebrate cells suggested that some of the factors necessary for import are soluble, since they are depleted from these cells during permeabilization (Adam et a1., 1990; Moore and Blobel, 1993). Subsequently, three soluble factors were identified that can mediate import in vitro (for reviews see Hicks and Raikhel 1995b; Gorlich and Mattaj, 1996). The factors in vertebrates are known as importin a (Adam and Gerace, 1991, Enenkel et a1., 1995, Gorlich et a1., 1994, 1995a, b; Imamoto et al., 1995a,b; Moroianu et a1., 1995; Radu et al., 1995a; Weis et a1., 1995), importin B (Adam and Adam, 1994; Chi et a1., 1995: Gorlich et al., 1995a; Imamoto et al.,1995a; Iovine et a1., 1995; Radu et al., 1995b) and Ran/TC4 (Melchior et a1., 1993; Moore and Blobel, 1993). Mutations in homologous import factors in yeast block import in vivo (Loeb et a1., 1995; Schenstedt et a1., 1995). The following outline summarizes the roles of the NLS— import factors in the nuclear import process (shown schematically in Figure 1.1). Nuclear import occurs when a heterodimer of importins a and B bind to an NLS—containing protein in the cytoplasm via the NLS binding region of importin a (Adam 1995; Azuma et a1., 1995; Gorlich et al., 1995a; Imamato et al., 1995a; Moroianu et a1., 1995; Radu et al., 1995a,b; Weis et a1., 1995). In yeast and vertebrates, importin a requires importin B for high affinity interaction with NLSs (Gorlich et al., 1995a; Rexach and Blobel, 1996; Efthymiadis et a1., 1997; Hubner et a1., 1997). After importin a binds to the NLS-containing protein, importin B mediates the docking of the trimeric complex to the cytoplasmic side of the NPC (Gorlich et al., 1995a; Imamato et al., 1995a; Moroianu et a1., 1995; Pante and Aebi, 1996; Radu et al., 1995a,b). Translocation of the trimeric complex through the NPC requires free GTP (Gorlich et al., 1996c) and a small GTPase, Ran, (Melchior et a1., 1993; Moore and Blobel, 1993; Gorlich et al., 1996c), in the GDP bound form (Gorlich et al., 1996c; Weis et a1., 1996). Recently, it was shown that only the GDP bound form of Ran can bind to the NPC (Gorlich et al., 1996c), but the energy necessary for the translocation step requires GTP hydrolysis by Ran (Melchior et a1., 1993; Moore and Blobel, 1993; Richards et a1., 1997). Therefore, these results suggest that RanGDP must targeted to the NPC and converted to RanGTP by a nucleotide exchange factor during the import process. The identity of the RanGDP receptor and the nucleotide exchange factor at the NPC have yet to be determined. After translocation, the trimeric complex docks at the nuclear basket of the NPC where the termination step occurs (Kutay et a1., 1997a). In vitro binding studies indicate that the binding of RanGTP to importin B terminates import by releasing the importin a/NLS—protein complex into the nucleoplasm (Rexach and Blobel, 1995; Chi et a1., 1996; Gorlich et al., 1996a). Subsequently, importin a somehow dissociates from the NLS—containing protein and the importin a and B subunits are exported to the cytoplasm, where they can participate in another cycle of import (Gorlich et al., 1996a; Weis et a1., 1996). Importin B is probably exported to the cytoplasm in a complex with RanGTP (Bischoff and Gorlich, 1997; Floer et a1., 1997). This complex is dissociated in the cytoplasm through the action of importin a and a set of RanGTP- activation proteins that are exclusively found in this compartment (Hopper et a1., 1990; Melchior et al., 1993b; Bischoff et a1., 1994; Coutavas et a1., 1993; Beddow et a1., 1995; Bischoff et a1., 1995; Richards et al, 1996). Export of importin a is facilitated by a heterodimer consisting of an importin B homologue, called Cas, and RanGTP (Bischoff and Gorlich, 1997; Gorlich, 1997; Kutay et al., 1997b). Cas Figure 1.1 Schematic diagram of the NLS-import pathway CYTOPLA SM RanGAP1 RanBP1 NUCLEOPLASM g} binds to importin a in a RanGTP manner creating a trimeric complex that is exported to the cytoplasm (Kutay et al., 1997b) Complex dissociation occurs in the cytoplasm by the RanGTP activating proteins that are located in this compartment (Bischoff and Gorlich, 1997; Gorlich, 1997; Kutay et al., 1997b). Cas has a low binding affinity for importin a in the absence of RanGTP which allows the formation of the importin a/B heterodimer in the cytoplasm (Kutay et al., 1997b). 4. The Importin or Family In Saccharomyces cerevisiae, a unicellular organism, importin a is encoded by a single gene, called Srpl (Yano et a1., 1992). However, in multicellular organisms, such as vertebrates and insects, importin a is encoded by a small gene family that can be divided into three subgroups (Gorlich and Mattaj, 1996; Kohler et a1., 1997; Tsuji et a1., 1997). Amino acid comparison between subgroups shows that they are 45-50% identical to each other, while the isoforms within each subgroup are 80% or more identical to each other. The overall protein structure is conserved in all the importin a isoforms especially in the importin B binding domain (IBB) found in the N—terminus. These isoforms also contain eight armadillo repeat motifs in the central domain, where the order and the number of repeats is conserved in fungi, vertebrates and insects (Gorlich and Mattaj, 1996; Kohler et a1., 1997; Malik et a1., 1997; Tsuji et a1., 1997; Ryder et 10 a1., 1998). Interestingly, these armadillo repeats are found in many cytoskeleton associated proteins (Barth et a1., 1997). The C—terminal domain and a small region between the IBB domain and the first armadillo repeat are highly divergent between the different subgroups (Gorlich and Mattaj, 1996; Kohler et a1., 1997; Malik et a1., 1997; Tsuji et a1., 1997; Ryder et a1., 1998). These observations suggest that divergent regions allow the different isoforms to perform unique roles in nuclear import. The presence of multiple importin a genes in higher eukaryotes suggest that these genes may recognize specific types of NLSs and/or be involved in nuclear import in certain tissues. Multiple isoforms of importin 0 also suggests that they are functionally redundant. In Drosophila, a mutation in an importin a isoform causes larvae to develop malignant brain tumors, suggesting that this isoform is involved in brain development (Kussel and Frasch, 1995; Torok et a1., 1995). Northern and western blot analysis demonstrates that many of the importin a isoforms are differently expressed in mouse and humans (Prieve et a1., 1996; Kohler et a1., 1997; Tsuji et a1., 1997; Ryder et a1., 1998). Interestingly, up to 5 different isoforms are expressed in the testes of mouse suggesting that different NLS—protein import pathways may co- exist in the same tissue (Tsuji et a1., 1997). A recently characterized importin a isoform from humans is highly expressed in skeletal muscle and represents more than 1% of the total protein in this tissue (Ryder et a1., 1998). ll Initial studies in humans demonstrate that two isoforms of importin a, NPI-l and Rchl, are not only expressed in the same tissues (Gorlich and Mattaj, 1996), but they can import SV40 T-antigen and bipartite NLS substrates in permeabilized cells (Moroianu et a1., 1995). However, recent results suggest that these isoforms are not fully redundant. First, in vitro NLS binding assays demonstrate that they associate differently with NLSs identified in a variety of nuclear proteins (Nadler et a1., 1997). Second, only NPI-l can bind and facilitate nuclear import of Statl, a transcription factor involved in cytokine signaling. Furthermore, NPI—l can associate with Statl and a SV40 T-antigen NLS-substrate at the same time, implying that NPI—l can import different NLS-cargos at the same time (Sekimoto et a1., 1997). This study suggests that different isoforms may play roles in specific signal transduction pathways. Unlike NPI—l and Rchl, Qipl, a third importin a isoform characterized in humans, cannot recognize the SV40-like NLS peptides from helicase unless surrounding sequences are present (Miyamoto et a1., 1997). In summary, it appears that NLS-protein import can be controlled through NLS-recognition by different importin a isoforms. In addition, many of these isoforms may also be involved in NLS-protein import in specific tissues that would contribute to the development of multicellular organisms. 12 5. The Importin B Family Studies indicate that the importin B family of proteins are nuclear shuttle proteins that function as receptors for the import and export of most proteins and ribonucleoprotein (RNP) complexes through the NPC (Table 1.2). For example, importin B and transportin, specifically mediate nuclear import of NLS-containing proteins and M9—containing proteins respectively, while exportin and CAS specifically mediate the export of NBS—containing proteins and importin a, respectively (for reviews see Gorlich, 1997; Ullman et a1., 1997; Wozniak et a1., 1998). A conserved feature of the importin B-like proteins is the RanGTP binding domain located in the N-terminus which allow these receptors to bind RanGTP, not RanGDP (Gorlich et a1., 1997; Wozniak et a1., 1998). Mutations that block the interaction of importin B with RanGTP also prevent the release and disassembly of the importin a/B/NLS—cargo complex from the nuclear basket of the NPC (Kutay et al., 1997a). 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The nucleotide exchange factor for Ran is RCCl, a chromatin binding protein that generates RanGTP inside the nucleus (Ohtsubo et al., 19989; Bischoff and Postingl, 1991). The GTPase activating protein, RanGAP1 stimulates Ran GTPase activity (Hopper et a1., 1990; Melchior et al., 1993b; Bischoff et a1., 1994). RanGAP1 is found in the cytoplasm (Hopper et a1., 1990; Melchior et al., 1993b; Bischoff et a1., 1994) and the cytoplasmic face of the NPC (Matunis et a1., 1996; Mahajan, et a1., 1997). A Ran binding protein, RanBP1, is a cytoplasmic protein that induces GTP hydrolysis by Ran together with RanGAP1 (Coutavas et a1., 1993; Beddow et a1., 1995; Bischoff et a1., 1995; Richards et al, 1996). Interestingly, a nucleoporin, RanBP2, found on the cytoplasmic fibrils of the NPC contains two RanGTP binding domains and it is implicated in Ran activation (Wu et a1., 1995; Wilken et a1., 1995; Yokoyama et a1., 1995). Based on the compartmental localization of RCCl and the RanGTP activating proteins, it is hypothesized that there is 17 a RanGTP gradient across the nuclear envelope, where the concentration of RanGTP is high in the nucleus and low in the cytoplasm (Gorlich, 1997). This gradient is crucial for nuclear transport, because microinjection of RanGAP1 into the nuclei of xenopus oocytes, which should raise the RanGDP levels inside the nucleus, blocks NLS— and M9-protein import as well as the export of mRNA, U snRNA, tRNA, importin a, importin B, transportin, and NBS—containing proteins. Genetic studies also support this model in vivo. Mutations in the yeast Ran homologue, Gspl, cause NLS-proteins to accumulate in the cytoplasm.and poly'(A.)+ RNA to accumulate inside the nucleus (Schlenstedt et a1., 1995). Similar studies have also shown that Ran mutants block NLS—protein import in mammalian cells (Carey et a1., 1996; Palacios et a1., 1996). Mutations in the yeast RanGAP1 and RCC1, called Rnal and Prp20, respectively, block RNA processing and export (Hartwell et a1., 1967; Hopper et a1., 1978; Amberg et a1., 1990; Forrester et a1., 1992; Amberg et a1., 1993), and mutations in the yeast RanBP1, Yrbl, cause NLS-containing proteins to accumulate in the cytoplasm while poly (A)‘iflmA is retained in the nucleus (Schlenstedt et a1., 1995). In mammalian cells, mutations that cause mislocalization of RanBP1 to the nucleus block NLS—protein import (Richards et a1., 1996). Similarly, mutations in RCC1 also blocks mRNA processing and transport (Cheng et a1., 1995) as well as NLS— protein import in mammalian cells (Tachibana et a1., 1994). In summary, mutations in these proteins should lead to 18 disruption in the RanGTP gradient, which would result in a block in macromolecular transport into and out of the nucleus. Why is the RanGTP gradient so important for nuclear translocation? In vitro binding studies show that RanGTP regulates the interaction between import/export receptors and their cargo (Bischoff and Gorlich, 1997; Floer et a1., 1997; Gorlich 1997; Kutay et al., 1997a). This model predicts that import receptors, like importin B, interact with their cargo in the cytoplasm where the levels of RanGTP are low and release their cargo in the nucleus where RanGTP levels are high. On the other hand, export receptors bind and release their cargo in the opposite manner. Thus, the Ran gradient may provide a directionality for nuclear import/export so that cargo binding and release occurs in the appropriate cellular compartments (Gorlich, 1997; Nigg, 1997). 7. Mechanism of Translocation Proteins destined for the nucleus must move z75 nm from the cytoplasmic fibrils, through the central gate, and to the nuclear basket (for reviews see Davis, 1995; Pante and Aebi, 1996a). Recently, electron microscopy studies have mapped two distinct binding regions on the cytoplasmic side of the NPC (Pante and Aebi, 1996b). The initial binding site is found near the ends of the cytoplasmic fibrils 50 nm from central plain of the NPC. The second binding site is about 10 nm from the central plane of the NPC, adjacent to the 19 central gate. Immunoelectron micrograph pictures displayed some fibrils containing NLS—substrates bent inward toward the central gate where the second binding site is located. Therefore, it was postulated the NLS—substrates may be transferred from the initial binding site to the second binding site by the inward bending of cytoplasmic fibrils (Pante and Aebi, 1996b). A model based on in vitro binding studies predicts that the translocation of the NLS—protein import complex through the pore may occur by a series of binding steps mediated by importin B and release steps mediated RanGTP (Rexach and Blobel, 1995; Chi et a1., 1996; Gorlich et al., 1996a). This model is referred to as the guided diffusion model (Rexach and Blobel, 1995). However, the mechanism that “guides" the NLS—protein import complex is not known. Clearly, a more defined structure of the NPC and the identification of more nucleoporins is required for better understanding of import as well as export. In addition, the localization of intermediates during the translocation process will also provide more information about this poorly defined process. 8. Regulatory Proteins An import regulatory factor, p10, stimulates NLS-protein import in the presence of importin a, B, RanGDP, and free GTP in permeabilized cells (Moore and Blobel, 1994; Paschal and Gerace, 1995). Biochemical studies demonstrate that p10 can bind directly to RanGDP, importin B and to a subset of 20 nucleoporins suggesting that p10 may regulate the interaction of the import factors with the NPC transport machinery. (Nehrbass and Blobel, 1996; Paschal et a1., 1996). Genetic studies in yeast demonstrate that this protein is an essential gene involved in NLS-protein import in vivo (Corbett and Silver, 1996; Nehrbass and Blobel, 1996; Paschal et a1., 1996). A significant step in the human immunodeficiency virus type 1 (HIV-1) infection process requires nuclear import of the pre—integration complex (Popov et a1., 1998; Vodicka et a1., 1998). The pre—integration complex contains the HIV—1 genome as well as some accessory proteins that contain NLSs. Nuclear import of this complex requires an HIV—1 encoded protein, viral protein R (Vpr), which stimulates the import of the pre—integration complex by enhancing the interaction of the viral NLS-proteins with importin a (Popov et a1., 1998). In addition, Vpr mediates the import of importin a/NLS protein complex in the absence of importin B, suggesting that Vpr, which is not homologous to importin B, can interact with the import machinery during the import process (Vodicka et a1., 1998). Interestingly, at high concentrations, 40 nM, Vpr blocks nuclear import indicating that the cytopathic effect observed in late stages of HIV—1 infection may be due to high concentrations of Vpr in the cell (Popov et a1., 1998). Lastly, Hsp 70 is involved in the import of some proteins (Imamoto et a1., 1992; Shi and Thomas, 1992; Shulga et a1., 21 1996), however its role may be in exposing the NLSs to the import machinery. 9. Regulation of the Import Apparatus Nuclear import of numerous transcription factors is regulated by various stimuli (environmental, chemical, or cell cycle progression) which can lead to changes in cell fate or metabolism indicating that this process is an important component in gene expression (Jans and Huebner, 1996; Mishra and Parnaik, 1995; Vandromme et a1., 1996). For example, protein phosphorylation adjacent to NLSs can either stimulate or block the import of various transcription factors (Jans and Hubner, 1996). In addition, studies also indicate that the import apparatus itself is regulated by the cell (Mishra and Parnaik, 1995; Vandromme et a1., 1996). Proliferating and serum stimulated cells have a higher rate of NLS-protein import than quiescent and serum starved cells (Feldherr and Akin, 1990; 1993; Vriz et a1., 1992). Comparisons between the NPCs found in these cells revealed that the diameter of the pore is two times smaller in quiescent cells than in proliferating cells (Feldherr and Akin, 1990). In serum starved cells, NLS—protein import can be enhanced by treating the cells with mitogens to raise the CAMP levels (Roux et a1., 1990) or with protein kinase A (PKA) activators (Vandromme, et a1., 1994; 1996) suggesting that the import apparatus may be regulated by a signal transduction pathway that utilizes CAMP and PKA 22 phosphorylation (Vandromme, et a1., 1996). Interestingly, when permeabilized mammalian cells are treated with alkaline phosphatase to dephosphorylate proteins, NLS—protein import is blocked. However, import can be restored by incubating permeabilized cells with cytosol enriched with PKA or protein kinase C (PKC). Interestingly, two highly phosphorylated proteins in these cytosolic extracts are similar in mass as importin a (Mishra and Parnaik, 1995) suggesting that a PKA or PKC signal transduction pathway may regulate the function of the NLS—receptor. 10. Conclusion Understanding of the NLS-protein import process has blossomed since the development of the permeabilize cell system that was used to identify proteins involved in this process. However, despite the recent advances in nuclear import and export field, the translocation process still remains a complete mystery. More attention needs to be focused in this area of research to understand the mechanism of translocation and how the import/export factors interact translocation machinery. 11. Thesis Scheme Macromolecular traffic through the NPC, including NLS- protein import, is an essential process in all eukaryotes. When I started this project, the goal was to identify and characterize the NLS receptor in plants. At this time, the 23 NLS receptor had been purified in vertebrates (Adam and Gerace, 1991), but the gene had not been cloned and characterized. In plants, an NLS binding site at the NPC and nuclear envelope had been characterized (Hicks and Raikhel, 1993). The SV40 large T—antigen and bipartite NLSs specifically and reversibly bound to and competed for this low-affinity site (Hicks and Raikhel, 1993). To identify the NLS binding site biochemically, a crosslinking approach was used and at least four NLS binding proteins (NBPs) were identified that specifically associated the bipartite NLS from Opaque—2 (Hicks and Raikhel, 1995a). The binding affinity and biochemical properties of the NBPs correlated closely with the NLS binding site. This evidence (Hicks and Raikhel, 1993; Hicks and Raikhel 1995a) indicated that at least one component of NLS recognition was located at the NPC and nuclear envelope in plants. 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(1997) Extracellular signal-dependent nuclear import of Statl is mediated by nuclear pore-targeting complex formation with NPI-l, but not Rchl. EMBO J. 16; 7067- 7077. Shi, Y. and Thomas, J. O. (1992) The transport of proteins into the nucleus requires the 70-kilodalton heat shock protein or its cytosolic cognate. Mbl. Cell. Biol. 12; 2186- 2192. Shieh, M., Wessler, S.R. and Raikhel, N.V. (1993) Nuclear targeting of the maize R protein requires two localization sequences. Plant Physiol. 101; 353-361. Shulga, N., Roberts, P., Zhenyu, G., Spitz, L., Tabb, M.M., Nomura, M. and Goldfarb, D.S. (1996) In vivo transport kinetics in Saccharomyces cerevisiae: A role for heat shock protein 70 during targeting and translocation. J. Cell Biol. 135; 329—339. 33 Siomi, H. and Dreyfuss G. (1995) A nuclear localization domain in the hnRNP A1 protein. J. Cell Biol. 129; 551-560. Tachibana, T., Imamoto, N., Seino, H., Nishimoto, T. and Yoshihiro, Y. (1994) Loss of RCC1 leads to suppression of nuclear protein import in living cells. J: Biol. Chem. 269; 24542—24545. Tiganis, T., Flint, A.J., Adam, A.S. and Tonks, N.R. (1997) Association of the T-cell protein tyrosine phosphatase with nuclear import factor p97. J. Biol. Chem. 272; 21548- 21557. Torok, 1., Strand, D., Schmitt, R., Tick, G., Torok, T., Kiss, 1., and Mechler, D.M. (1995) The overgrown hematopoietic organs-31 tumor suppressor gene of Drosophila encodes an importin-like protein accumulating in the nucleus at the onset of mitosis. J. Cell Biol. 129; 1473—1489. Tsuji, L., Takumi, T., Imamoto, N. and Yoneda, Y. (1997) Identification of novel homologues of mouse importin a, the a subunit of the nuclear pore-targeting complex, and their tissue-specific expression. FEBS Lett. 416; 30-34. Ullman, R., Powers, M. A., and Forbes, D. J. (1997) Nuclear export receptors from importin to exportin. Cell 90; 967—970. Vandromme, M., Gauthier-Rouviere, C., Lamb, N., and Fernandez, A. 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(1995) Localization of the RanGTP binding protein RanBP2 at the cytoplasmic side of the nuclear pore complex. Eur. J. Cell. Biol. 68; 211—219. Wozniak, R.W., Rout, M.P. and Aitchison, J.D. (1998) Karyopherins and kissing cousins. Trends Cell Biol. 8; 184- 188. Wu, J., Matunis, M.J., Rraemer, D., Blobel, G. and Coutavas, E. (1995) Nup358, a cytoplasmically exposed nucleoporin with peptide repeats, RanGTP binding sites, zinc fingers, a cyclophilin A homologous domain, and a leucine- rich region. J; Biol. Chem. 270; 14209-14213. Yano, R., Oakes, M.L., Yamaghishi, M., Dodd, J.A., and Ncmura, M. (1992) Cloning and characterization of SRPl, a suppressor of temperature—sensitive RNA polymerase I mutations, in Saccharomyces cerevisiae..Mol. Cell. Biol. 12; 5640-5651. Yokoyama, N., Hayashi, N., Seki, T., Pante, N., Ohba, T., Nishii, R., Ruma, R., Hyashida, T., Miyata, T., Aebi, U., Fukui, M. and Nishimoto, T. (1995) A giant nucleopore protein that binds Ran/TC4. Nature 376; 184-188. 35 Chapter 2 Three Classes of Nuclear Import Signals Bind to Plant Nuclei This chapter was published in: Hicks, G.R., Smith, H.M.S., Shieh, M. and Raikhel, N.V. (1995) Three classes of nuclear import signals bind to plant nuclei. Plant Physiol. 107, 1055-1058. 36 ABSTRACT Three nuclear localization signals (NLS), including an unusual Mat a2-like NLS from maize (Zea mays) R, were found to compete for binding to plant nuclei. Our results, indicate that plants possess a site at the nuclear pore complex that recognizes the three known classes of NLSs. 37 INTRODUCTION Since there are no strict consensus sequences for NLSs, a thorough study of each of the classes of signals is an essential step in identifying components of the import apparatus of plants. The initial event of import, binding at the NPC, has been examined with an in vitro nuclear binding assay using peptides to two classes of NLSs (Hicks and Raikhel, 1993). The bipartite NLS from Opaque-2 (02; Varagona, et a1., 1992) and the SV40 large T-antigen NLS (Raikhel, 1992) specifically bind to and compete for a single low affinity site that is firmly associated with the nuclear envelope and NPC (Hicks and Raikhel, 1993). However, mutant 02 (Varagona and Raikhel, 1994) and mutant SV40 large T- antigen NLSs (Raikhel, 1992) that are impaired in import in vivo do not compete for binding (Hicks and Raikhel, 1993). In addition, a peptide to the SV40 large T-antigen that was synthsized in reverse order is an inefficent competitor compared to the the wild type SV40 large T-antigen NLS peptide. To determine if the third known class of NLSs, the Mat a2-like NLSs, could compete for binding to the same nuclear site, we examined the association of NLS C from the R protein with purified tobacco (Ndcatiana tabacum) and maize (Zea mays) nuclei. 38 METHODS Materials The peptide to NLS C (CWT) was synthesized at the Peptide Synthesis Facility (Yale University, New Haven, CT). Synthetic peptides to the functional 02 bipartite NLS (OZWT), the functional SV40 large T—antigen NLS (SV4OWT), and the peptide unrelated to NLSs (non-NLS) were previously described (Hicks and Raikhel, 1993). All peptides contained a Cys residue at the amino or carboxy terminus to facilitate radiolabeling with carbon—14. The peptides were as follows: (CWT) CYMISEALRK AIGKR; (OZWT) MPTEERVRKR KESNRESARR SRYRKAAHLK C; (SV4OWT) CTPPKKKRKV; (non~NLS) CDGVFAGGG. Nuc lear binding assays For binding assays, nuclei were prepared from protoplasts of Nicotiana tabacum or maize (Zea mays) Black Mexican Sweet suspension-cultured cells abbreviated protocol previously described (Hicks and Raikhel, 1993), except that DTT was omitted from the nuclei isolation buffer. The NLS peptide, CWT, was radiolabeled with carbon—14 by carboxymethylation of cysteine residues in the prescence of 14C—iodoacetamide as described by Hicks and Raikehl, 1993. For NLS binding experiments to purified nuclei using the radiolabled [“C]CWT NLS were performed as described by Hicks and Raikhel, 1993 except that 50 mM Tris—HCl, pH 7.3, was replaced by 50 mM Hepes-KOH, pH 7.3, in binding buffer. Briefly, 1 X 106 tobacco or maize nuclei were diluted to 70 ul with binding 39 buffer, and 200,000 cpm of [”CJCWT (76 uCi/mmol; approximately 5 uM final concentration) in 30 ul of binding buffer was added. Binding was allowed to occur for 5 min on ice, after which time the nuclei were pelleted. The supernatant was removed and the cpm associated with the nuclei were quantitated by scintillation counting. For competitive displacement curves, unlabeled peptides were added from concentrated stocks made in binding buffer. Nonspecific backgrounds were estimated from the addition of 10 mM unlabeled CWT. Note: this assay measures relative binding of NLS peptides to purified nuclei. Assays points were the average of at least duplicate samples, and all experiments were done at least twice. 4O RESULTS Three classes of NLSs compete for the same NLS binding site in purified nuclei To determine whether NLS C could specifically bind to purified nuclei, CWT was synthesized that corresponded to the minimum region of R that functionally defined this signal in vivo (Sheih et a1., 1993). In addition, the previously described (Hicks and Raikhel, 1993) O2WT and SV40WT peptides, and a peptide unrelated to NLSs (non—NLS), were used. The latter peptide, which corresponds to a defective vacuolar import signal (Dombrowski et a1., 1993), served as a “non— NLS” control. To examine the relative binding of NLS C, the CWT peptide was carbon—14 labeled and allowed to bind to purified tobacco nuclei. Displacement curves resulting from the addition of the CWT peptide as a competitor indicated that [“C]CWT could specifically bind to plant nuclei at low affinity with an apparent dissociation constant of approximately 100 uM (Fig. 2.1 A). This is similar to the apparent dissociation constant for the binding of [”C]O2WT and [”C]SV40WT to tobacco nuclei (200 uM; Hicks and Raikhel, 1993). Displacement of [”C]CWT by unlabeled SV40WT and OZWT indicated that the three peptides competed for binding to the same nuclear site (Fig. 2.1, B and C). The non—NLS control peptide was a poor competitor compared to the functional NLSs (Fig. 2.1 C). We also examined the binding of [”CJCWT to nuclei from the distantly related monocot maize. Experiments 41 100 (A a ' i {\i E 1 8 :wr OS 50‘ \ e l {I *2 \ \l '5 0 M.,," 1-..... 10" 10'5 10“ 10'3 10'2 ;B §1°°‘—§—§\ g {\{ SV40WT 32 50- \ V \ e . \i E '5 0 - , - ---... . 10° 105 10" 103 102 100 'Q-I—i—‘i—§~§\} non-NLS =§ , i E O 0 3 5°' \ V I osz a) . s \ E o v-.." v v if" . rfiw {\r? rv.n ‘0 10“ 10‘5 10" 10'3 10°2 peptide (M) Figure 2.1 Specific binding of NLSs to purified tobacco nuclei. The [“C]CWT peptide was incubated with nuclei in the presence of CWT (CYMISEALRKAIGKR) (A), SV40WT (CTPPKKKRKV) (B), or 02WT (MPTEERVRKRKESNRESARRSRYRKAAHLKC) or non-NLS (CDGNFAGGG) (C) competitor peptides. Results are reported as a percent binding of control (:1: SE) versus concentration (M) of added competitor. Average total binding and nonspecific association were, respectively, 16,900 and 11,000 cpm. 42 using purified nuclei from maize indicated that monocots possess a similar binding site to which [“CJCWT could associate. Addition of CWT as a competitor demonstrated that binding was specific and of an affinity similar to that found with tobacco nuclei (Fig. 2.2 A). The SV40WT and O2WT peptides also competed effectively with [”C]CWT for binding to the nuclear site (Fig. 2.2, B and C), whereas the non—NLS control peptide displayed no ability to compete (Fig. 2.2 C). 43 100 J,_\ 9 a O a \- O o \ sso~ , 2 a: \ .E E §\§ E \i o .... 1 q vvv' m 10“ 10'5 10" 10'3 10'2 €100 ’ § \- E ’ \ swowr ° \ O E?“ 50‘ RE 0) \§ ,5 1 'U .5 D 0 ' ' "'"I ' ' """I ' T ""l V ' "'"I 10“ 10‘5 10“ 10'3 10'2 ' i_.. non-NLS 1 "' \ __ . _ A °° t-—-- —§§I I— i T? ‘5 i\ O 0 9‘: 5° ‘ \ 02WT 2 n B \{ .E Q o . ......., . . ...... . ......., . . ...... 10" 10'5 1o" 10" 10‘2 peptide (M) Figure 2.2 Specific binding of NLSs to purified maize nuclei. The [“0]er peptide was incubated with nuclei in the presence of CM (A), SV40WT (B), or 02WT or non- NLS (C) competitor peptides. Results are reported as percent binding of control (t SE) versus concentration (M) of added competitor. Average total binding and non-specific association were, respectively, 25,400 and 14,000 cpm. DISCUSSION A careful study of the binding of NLSs is an important step prior to identifying the corresponding NLS binding proteins. Although the binding of individual NLSs has been examined in animals and yeast (Forbes, 1992), a systematic approach using the three known classes of NLSs has not been reported. We previously demonstrated that higher plants possess a saturable low—affinity site at the NPC that can specifically bind to the SV40 large T—antigen NLS and the 02 bipartite NLS (Hicks and Raikhel, 1993). In this study, we examined the binding of the Mat a2—like NLSs, such as NLS C from the endogenous maize transcription factor R with purified nuclei isolated from tobacco and maize cell suspension cultures. In these experiments, we used 5 uM [“C]CWT in our competition studies which is below the dissociation constant, Kd, for this site. Under these conditions the NLS binding sites are not saturated. Therefore, we expected to displace approximately half of the (”CJCWT when the concentration of the competitor peptide, CWT, equals the dissociation constant. We found that this occurs at approximately 100 UN, which is similar to the apparent dissociation constant for the binding of [”C]02WT and [”C]SV40WT to tobacco nuclei (Hicks and Raikehl, 1993). The SV40WT and 02WT peptides were also found to compete with [”C]CWT for binding. Similar results were obtained using nuclei from maize, indicating that the binding site is probably a component that is common among divergent species. 45 It is generally accepted that animals require soluble factors for NLS recognition (Forbes, 1992; Moore and Blobel, 1994), and this may also be the case in yeast (Schenstedt et a1., 1993). Our results indicate that at least some components of NLS recognition are located at the NPC of plants. We have recently extended our in vitro studies to include the addition of cross-linked reagents during NLS binding. Using this approach, several NLS binding proteins have been specifically radiolabeled with the peptides to the functional 02 bipartite NLS (Hicks and Raikhel, 1995). Future work is aimed at identifying and characterizing NLS- binding proteins to understand NLS—protein import proteins in plants. 46 References Dombrowski, J.E., Schroeder, N.R., Bednarek, S.Y. and Raikhel, N;V. (1993) A mutational analysis of the carboxy— terminal propeptide of barley lectin: determining functional elements necessary for proper sorting to vacuoles of tobacco. Plant Cell 5; 587—596. Forbes, D.J. (1992) Structure and function of the nuclear pore complex. Annu. Rev. Cell Biol. 8; 495—527. Hicks, G.R. and Raikhel, N.V. (1995) Nuclear localization signal binding proteins in higher plant nuclei. Proc. Natl. Acad. Sci. USA 92; 734-738. Hicks, G.R. and Raikhel, N.V. (1993) Specific binding of nuclear localization sequences to plant nuclei. Plant Cell 5; 983—994. Moore, M.S. and Blobel, G. (1994) A G—protein involved in nucleocytoplasmic transport: the role of Ran. Trends in Cell Biol. 19; 211—216. Raikhel, N.V. (1992) Nuclear targeting in plants. Plant Physiol. 100; 1627—1632. Schlenstadt, G., Hurt, 3., Doye, V. and Silver, P.A. (1993) Reconstitution of nuclear protein transport with semi- intact yeast cells. J. Cell Biol. 123; 785—798. Shieh, M., Wessler, S.R. and Raikhel, N.V. (1993) Nuclear targeting of the maize R protein requires two localization sequences. Plant Physiol. 101; 353—361. Varagona, M.J. and Raikhel, N;V. (1994) The basic domain in the bZIP regulatory protein Opaque—2 serves two independent functions: DNA binding and nuclear localization. Plant J. 2; 207-214. Varagona, M.J., Schmidt, R.J. and Raikhel, N.V. (1992) Nuclear localization signal(s) required for nuclear targeting of the maize regulatory protein Opaque-2. Plant Cell 4; 1213- 1227. 47 Chapter 3 Characterization of a Nuclear Localization Signal Receptor, Importin a, in Plants This chapter was constructed from two published papers: 1. Hicks, G.R., Smith, H.M.S., Lobreaux, S. and Raikhel, N.V. (1996) Nuclear import in permeabilized protoplasts from higher plants has unique features. Plant Cell 8, 1337-1352. 2. Smith, H.M.S., Hicks, G.R., and Raikhel, N.V. (1997) Importin a from Arabidopsis thaliana is a nuclear import receptor that recognizes three classes of import signals. Plant Physiol. 114; 411—417. In addition, the functional analysis of the Arabidopsis importin a protein in the mammalian in vitro import system was done in collaboration with Dr. David A. Jans’s group at the Nuclear Signaling Laboratory, Division for Biochemistry and Molecular Biology, John Curtin School of Medical Research, Canberra, Australia. 48 ABSTRACT Protein import into the nucleus is a two-step process. In vitro import systems from vertebrate cell extracts have shown that several soluble factors are required. One of these factors is the receptor, importin a, which binds to nuclear localization signals (NLSs) in vitro. In this study, we cloned an importin a homologue (At—IMPa) from Arabidopsis thaliana and it was 36-48% identical to other importin a homologues identified in other organisms. Antibodies generated against the recombinant expressed At—IMPa specifically recognized the endogenous protein in Arabidopsis and tobacco. To determine if At-IMPa was a NLS receptor, we used an in vitro NLS binding assay. We found that NLS binding by At—IMPa was specific, and the receptor was able to recognize three classes of NLSs identified in plants. Purified antibodies to At—IMPa were used to determine the in vivo location of importin a in tobacco protoplasts. Importin a was found in the cytoplasm and nucleus, and it was highly concentrated at the nuclear envelope. The biochemical properties of nuclear importin a and localization studies using purified nuclei demonstrate that importin a was tightly associated with the plant nucleus. Moreover, these results suggest that a fraction of nuclear importin a interacts with the nuclear pore complex. Using purified subunits in a vertebrate in vitro import system, we showed that in the absence of exogenously added importin B subunit but in the 49 presence of RanGDP and NTF2, At-IMPa was able to mediate nuclear import to levels comparable to those mediated by the mouse importin a/B heterodimer. Neither mouse importin a nor B was able to mediate nuclear import in the absence of the other importin subunit. Therefore, At-IMPa's has unique properties that can fulfill the role of both mammalian importin subunits. In summary, our results taken together strongly suggest that At—IMPa is a functional NLS receptor in Arabidopsis. 50 INTRODUCTION Protein import into the nucleus occurs through the nuclear pore complex (NPC), which is a 124 megadalton proteinaceous complex embedded in the nuclear envelope which acts as a gateway for protein traffic in and out of the nucleus (for reviews see Davis, 1995). For many nuclear localized proteins, this process is receptor mediated and dependent upon targeting signals called nuclear localization signals (NLSs; Dingwall and Laskey, 1991). Although there is no consensus sequence for these signals, NLSs can be grouped into three classes (for reviews see Boulikas, 1993, 1994; Hicks and Raikhel, 1995b). A NLS within the SV40 large T- antigen (Kalderon et a1., 1984; Lanford and Butel, 1984) defines a class of signals, the SV40—like NLSs, that are composed of a single peptide region containing basic residues. The bipartite NLSs (Dingwall et a1., 1988) are composed of two peptide regions containing basic residues separated by a spacer of variable length. Finally, Mat a2- like NLSs share similarity with the amino—terminal NLS of the yeast protein Mat a2 (Hall et a1., 1984) and contain hydrophobic and basic residues. Experimentally, NLS-protein import into the nucleus is divided into two distinct steps, docking and translocation. Docking is NLS dependent and occurs when nuclear proteins dock at the cytoplasmic side of the NPC in an energy independent fashion. Translocation through the NPC is an energy driven process (Newmeyer and Forbes, 1988; Richardson 51 et a1., 1988). In vitro import systems using permeabilized vertebrate cells suggested that some of the factors necessary for import are soluble (Adam et a1., 1990; Moore and Blobel, 1993). Subsequently, four soluble factors were identified that can mediate import in vitro (for reviews see Hicks and Raikhel 1995b; Gorlich and Mattaj, 1996). The factors in vertebrates are known as importin a (Adam and Gerace, 1991, Enenkel et a1., 1995, Gorlich et a1., 1994, 1995a, b; Imamoto et al., 1995a,b; Moroianu et a1., 1995; Radu et al., 1995a; Weis et a1., 1995), importin B (Adam and Adam, 1994; Chi et a1., 1995: Gorlich et al., 1995a; Imamoto et al.,1995a; Iovine et a., 1995; Radu et al., 1995b), and Ran/TC4 (Melchior et a1., 1993; Moore and Blobel, 1993). Mutations in some homologous import factors in yeast also block import in vivo (Loeb et a1., 1995; Schenstedt et a1., 1995). NLS-protein import occurs when a heterodimer of importins a and B binds to an NLS-containing protein in the cytoplasm via the NLS binding region of importin 0 (Adam 1995; Azuma et a1., 1995; Gorlich et al., 1995a; Imamato et al., 1995a; Moroianu et a1., 1995; Radu et al., 1995a,b; Weis et a1., 1995). Importin a requires importin B for high affinity interaction with NLSs in vertebrates and yeast (Gorlich et al., 1995a; Rexach and Blobel, 1996; Efthymiadis et a1., 1997; Hubner et a1., 1997). Importin B mediates the docking of the trimeric complex to the cytoplasmic side of the NPC (Gorlich et al., 1995a; Imamato et al., 1995a; Moroianu et a1., 1995; Pante and Aebi, 1996; Radu et al., 1995a,b). 52 Translocation of the trimeric complex through the NPC requires free GTP (Gorlich et al., 1996c), and a small GTPase, Ran (Melchior et a1., 1993; Moore and Blobel, 1993; Gorlich et al., 1996c). In vitro studies in vertebrates and yeast suggest that importin B interacts with a subset of nucleoporins during the translocation process (Iovine et a1., 1995; Pascal and Gerace, 1995; Gorlich et al., 1996c; Nehrbass and Blobel, 1996; Rexach and Blobel, 1996). After translocation, dissociation of importin a with the NLS- containing protein leads to export of importin a into the cytoplasm, where it can participate in another cycle of import (Gorlich et al., 1996a; Weis et a1., 1996). In addition, importin B functions as a NLS—receptor for a subset of proteins in the absence of importin a (Palacios, et a1., 1997; Tiganis et a1., 1997; Chan et a1., 1998). Hsp 70 is involved in the import of some proteins (Imamoto et a1., 1992; Shi and Thomas, 1992; Shulga et a1., 1996), however its role may be in exposing the NLSs to the import machinery. The protein import process can be regulated by phosphorylation (Jans and Huebner, 1996; Mishra and Parnaik, 1995; Vandromme et a1., 1996). An NLS binding site at the NPC and nuclear envelope has been well characterized in plants (Hicks and Raikhel, 1993). Three classes of NLSs specifically and reversibly bind to and compete for this low-affinity site (Hicks and Raikhel, 1993; Hicks et a1., 1995). To identify the NLS binding site biochemically, a crosslinking approach was used and at least 53 four NLS binding proteins (NBPs) were identified using the bipartite NLS from a maize transcription factor, Opaque-2 (Hicks and Raikhel, 1995a). The binding affinity and biochemical properties of the NBPs correlate closely with the NLS binding site. This and other evidence (Hicks and Raikhel, 1993; Hicks and Raikhel 1995a) indicate that at least one component of NLS recognition is located at the NPC and nuclear envelope in plants. To get a better understanding of the NLS—protein import pathway in plants, we identified and characterized an Arabidopsis importin a homologue, At-IMPa. 54 METHODS Materials All chemicals were obtained from Sigma Chemical Co. unless otherwise noted. All NLS peptides were synthesized at the Peptide Synthesis Facility (Yale University, New Haven, CT). Cloning and sequencing of At-IMPa A 900-bp partial cDNA exhibiting significant homology with yeast SRPl and animal importin a homologues was obtained from the Michigan State University-Department of Energy Plant Research Laboratory Arabidopsis Sequencing Project (Newman et a1., 1994). The cDNA was used as a probe to screen the PRL2 Arabidopsis cDNA library (Newman et a1., 1994) in lZiplox (Gibco BRL, Gaithersburg, MD). The library was made from mRNA of etiolated seedlings, roots, leaves, and flowers. The probe was synthesized with a—32-dATP (3000 Ci/mmol; NEN Research Products, Boston, MA) by Klenow (Boehringer Mannheim) and random hexanucleotide primers. Approximately 2 )(11? plaques were screened, and 12 additional cDNAs were purified by standard methods (Sambrook et a1., 1989). The cDNAs were excised from lZiplox for sequencing, as described by Newman et al. (1994). A full length cDNA of 2.2 kb was obtained that hybridized to an mRNA of similar length (H.M.S. Smith and N.V. Raikhel, unpublished data), and 3' and 5’ deletions were made (Henikoff, 1987) and sequenced using Sequenase 2.0 (United States Biochemical Corp., Cleveland, 55 OH). Deduced amino acid sequences were aligned by MegaAlign, DNASTAR Inc. (Nov. 1997 version). Expression and purification of recombinant At-IMPa The At—IMPa cDNA was cloned into pGEX5—2 (Pharmacia, Piscataway, NJ) by using EcoRl and Notl sites to produce an N-terminal glutathione—S—transferase (GST) fusion protein. The plasmid was transformed into DHSa and the production of fusion protein was induced overnight at 37WC, according to the manufacture’s procedures. For protein purification, cells were chilled on ice for 20 min before centrifugation at 7500g and suspended in 20 ml of ice-cold PBS containing 1 mM PMSF. Cells were lysed by two treatments at 1100 p.s.i. in a French Press. Triton X—100 was added to 1%, and centrifuged at 1200g. Then, 40 ml of the supernatant was mixed with 2 ml of glutathione—agarose (Pharmacia) for 3 hr at 4W2. The beads were washed 10 times with 40 ml of ice-cold PBS containing 1 mM PMSF. The fusion protein was eluted with 5 ml of elution buffer (50 mM Tris-HCl, pH 8.0, 10 mM reduced glutathione) and concentrated using a Centricon—lO device (Amicon). The purified protein had a concentration of 2.5 mg/ml. For antibody production, the fusion protein was purified further by 10% SDS-PAGE and prepared as follows: gel strips containing a total of ~6 mg of fusion protein were equilibrated in extraction buffer (50 mM ammonium bicarbonate, pH 7.8 0.05% SDS) for 5 min at 233:, ground with mortal and pestle, and mixed overnight as a suspension in 56 extraction buffer at 37%1 The suspension was centrifuged twice at 1300g, filtered through a 45-um cellulose acetate filter, and washed by repeated dilution with 50 mM ammonium bicarbonate. pH 7.8, in a Centricon-30 device (Amicon). The sample was lyophilized twice and dissolved in 3.0 ml of PBS at a concentration of 0.9 mg/ml. For expression as a His-tagged fusion protein, At—IMPa was cloned into pET14b (Novagen, Madison, WI) by using Ndel and BamHl sites. The plasmid was transformed into BL21 (DE3) and then induced and purified by affinity chromatography, according to the manufacturer’s protocol. The final protein concentration was 1.6 mg/ml. Antibody production and purification Preimmune serum was collected; then one rabbit was immunized with 0.5 mg of purified GST—At-IMPa fusion protein in the presence of an adjuvant (TiterMax; Vaxel, Norcross, GA) for each of three injections over a 6-week period and a final injection of 1.0 mg. For affinity purification of At-IMPa antibodies, His-tagged At—IMPa was coupled to Affigel-lO (Bio—Rad Laboratories). Purified protein (3.5 mg) was mixed with 1 ml of Affigel—10 in 50 mM Hepes-KOH, pH 7.2 in a 3.5- ml column and rocked for 4 hr at 4%L The column was washed with three column volumes of 50 mM Tris-HCl, pH 7.2, three volumes of 100 mM glycine pH 2.5 and finally equilibrated in 50 mM Hepes—KOH, pH 7.2, and filtered through a 45-uM cellulose acetate filter; specific antibodies were bound to 57 the At—IMPa affinity column by passage through the column three times. The column was then washed sequentially with 10 mM Tris—HCl, pH 7.2, 10 mM Tris-HCl, pH 7.2, plus 0.5 M NaCl, and 50 mM Hepes—KOH, pH 7.2. Specific At—IMPa antibodies were eluted with 100 mM glycine, pH 2.5, and 1-ml fractions were collected and neutralized with 0.6 ml of 1 M Hepes-KOH, pH 7.5, 2 mM magnesium acetate, 50 mM potassium acetate, and 0.225 M mannitiol. Immunoblots Ten percent of SDS-PAGE and blotting to nitrocellulose were performed by standard methods (Sambrook et a1., 1989). Blots were blocked with nonfat dry milk and incubated overnight at 23%2Mdth.purified At—IMPa antibodies or preimmune sera (both stocks at 90 ug/ml) at a 1:3000 dilution. Blots were developed by using a 1:5000 dilution of goat anti-rabbit alkaline phosphatase-conjugated IgG (Kirkegaard and Perry Laboratories). Proteins were extracted from Arabidopsis roots, leaves, stems, and flowers by grinding in liquid nitrogen and suspended in extraction buffer (50 mM Tris-HCl, pH 6.8, 100 mM DTT, 2 % SDS) for SDS-PAGE (50 ug per lane). Tobacco nuclei were purified by the abbreviated procedure described by Hicks and Raikhel (1993). Nuclei (11” per gel lane) were diluted to 50 ul in 50 mM Tris-HCl, pH 7.3, 25 mM KCl, 2.5 mM MgC12, 3 mM CaClz, 20% glycerol and were incubated with 400 units per ml Dnase 1 (Boehringer Mannheim) at 23%? for 20 min. After centrifugation at 12,000g for 2 min, the 58 nuclear pellet was suspended in 25 ul of SDS sample buffer. Where indicated, 40 ug (0.53 x 1051cell equivalents per gel lane) of cytosol from evacuolated tobacco cells and 75 ng of His-tagged At—IMPa or mammalian importin a were loaded per gel lane. In 'vitro Ibinding/co-immunoprecipitation For in vitro transcription and translation, full length At- IMPa was cloned into Bluescript (SK-; Strategene, Inc., La Jolla, CA) with EcoRl (Boehringer and Mannheim, Indianapolis, IN) and BamHl (Boehringer and Mannheim, Indianapolis, IN). The vector was linearized at the 3' end of the gene with BamHl and transcribed with T7 RNA polymerase (Promega Biotech, Madison, WI). Two ug of mRNA was incubated in wheat germ extract (minus Met; Promega Biotech, Madison, WI) in the presence of 50 uCi [358] Met (New England Nuclear, Boston MA). Translation mix was diluted in Binding Buffer (50 mM Hepes pH 7.8, 25 mM KCl, 2.5 mM MgC12, 3 mM CaC12, 20% glycerol) and 250 ug of purified At—IMPa antibodies were added and mixed for 1 to 2 h at 40C. Next, Protein A Sepharose was added to a final concentration of 0.05 %. Precipitated proteins were washed 4 times in Binding Buffer to remove any contaminants and suspended in SDS sample buffer (50 mM Tris pH 6.8, 100mM DTT, 2.0% SDS, 20% glycerol). To determine if the antibodies could also recognize the recombinant expressed At-IMPa, immunoprecipitation was 59 checked by adding 500 ng of His—tagged At—IMPa to the immunoprecipitation reaction. Samples were separated by 10% SDS-PAGE and the gels were developed 24 h by autoradiography by standard methods (Sambrook et a1., 1989). For NLS binding substrates, 4 mg of Human Serum Albumin (HSA) was dissolved in PBS. The chemical crosslinker, Maleimidobenzoyl N-hydroxysuccinimide ester (MBS; Pierce, Rockford, IL), was added to a final concentration of 10 mM and incubated for 30 min at room temperature. Unbound crosslinker was removed by Gel filtration with a 2 ml G—25 column. NLS peptide was added to a final concentration of 1.2 uM to the flow through and incubated for 3 h at room temperature. Next, free peptide was removed by gel filtration through a 2 ml G-25 column and substrates were washed extensively in PBS. NLS substrates were concentrated in a Centricon—lO microfiltration device (Amicon, Beverly, MA), and they were aliquoted and stored at -80°C. For co— immunoprecipitation, 2 ug of NLS—HSA substrate was mixed with 500 ng of recombinant At-IMPa in Binding Buffer for 2 to 4 h at 40C. Then, 400 ng of purified At-IMPa antibodies were added and incubated for 1 to 2 h at 40C. Protein A Sepharose (Pharmacia Biotech, Piscataway, NJ) was added to 0.05% and mixed at 40C for 1 h. Samples were precipitated and washed 4 times in Binding Buffer to remove unbound substrates then suspended in SDS sample buffer. For competition experiments, NLS binding was performed as described above except the 60 Binding Buffer contained either 1 mM 02WT or 1 mM 02mut peptides. Separation of proteins by 10% SDS-PAGE and blotting were performed by standard methods (Sambrook et a1., 1989). Blots were rinsed with TBST (TBS, 0.05% Tween 20) then incubated overnight at room temperature with monoclonal antibodies raised against HSA (Sigma Chemicals Co., St. Louis, M0) at 1:2000 dilution in TBST. Blots were developed by using a 1:5000 dilution of goat anti-mouse alkaline phosphatase—conjugated IgG (Kirkegaard and Perry Laboratories, Guithersburg, MD). Immunolocalization .Nictotiana tabacum suspension—culture cells were maintained and protoplasts were prepared as described by Hicks and Raikhel, (1993). Protoplasts or purified nuclei were spun onto poly-lysine coated slides using a Cytospin 3 (Shandon Lipshaw, Pittsburg, PA), and immediatley fixed in Fix Buffer (3% paraformaldehyde in 50 mM Potassium Phosphate, pH 7.2) for 30 min at room temperature. Fixed cells were dried at room temperature and stored at 4%: for 24 h. Next, cells were dehydrated in cold methanol for 10 min, then washed in PBST (PBS, 0.5 % Tween 20). Affinity purified At-IMPa antibodies (100 ng/ul) were diluted 1:300 in PBST containing 60 ug of HSA and incubated on the cells at room temperature for 1 h in a moist chamber. After washing the cells in PBST, CY3- labeled goat anti—rabbit (Molecular Probes Inc. Eugene, OR) was diluted 1:50 in PBST and incubated on the cells for 1 h 61 in a moist dark chamber. After washing in PBST, cells were mounted in MOWIOL (Calbiochem, San Diego, CA) and optically sectioned (0.5—1.0 um sections) using Confocal Laser Scanning Microscopy (Model 10 Carl Zeiss) equipped with 514-nm argon laser. Micrographs were produced with Kodacolor Gold 100 film (Kodak). Extraction of importin or from purified nuclei Nuclei were prepared as described by Hicks and Raikhel, (1993). One million nuclei were diluted to 50 ul in Binding Buffer with 400 units of DNaseI (Boehringer Mannheim, Indianapolis, IN) at room temperature for 20 min. Nuclei were centrifuged at 12,000g for 2 min and suspended in 25 ul of cold Binding Buffer containing either 1.0 % Triton X-100, 0.25 M NaCl, 1.0% Triton X—100 and 0.25 M NaCl or 6M urea. The samples were incubated for 15 min at 40C, then centrifuged at 12,000g for 2 min. Ten ul of SDS sample buffer was added to the supernatant, and the pellet was resuspened in 25 ul of SDS sample buffer. Each sample was incubated at 650C for 5 min before separation by 10% SDS—PAGE and blotted to nitrocellulose by standard protocols (Sambrock et a1., 1989). Purified At—IMPa antibodies were diluted 1:2000 in TBST and blots were developed by using a 1:5000 dilution of goat anti-rabbit alkaline phosphatase—conjugated IgG (Kirkegaard and Perry Laboratories, Guithersburg, MD). 62 In Vitro Nuclear Transport These experiments were done in collaboration with Dr. David A. Jans group at the Nuclear Signaling Laboratory, Division for Biochemistry and Molecular Biology, John Curtin School of Medical Research, Canberra, Australia Analysis of the ability of At—IMPa to support nuclear import in vitro was performed by quantifying nuclear import kinetics at the single cell level using mechanically perforated HTC rat hepatoma cells in conjunction with confocal laser scanning microscopy (Jans et a1., 1991; Xiao et a1., 1997; Efthymiadis et a1., 1997, 1998). Experiments were performed for 40 min at room temperature in a 5 ml volume containing 30 mg/ml BSA, 2 mM GTP, and an ATP regenerating system (0.125 mg/ml creatine kinase, 30 mM creatine-phosphate, 2 mM ATP), transport substrate (0.2 mg/ml IAF—labelled fusion protein) or a control to assess nuclear integrity (70 kDa FITC-labelled dextran; Sigma Chem. Co.). Where indicated, 4 mM RanGDP, 0.15 mM NTF2, 1 mM mouse— importin B (m—IMPB), 1 mM mouse-importin a (m—IMPa) or 0.6 mM At-IMPa were added. Image analysis and curve—fitting was performed as described (Xiao et a1., 1997; Efthymiadis et a1., 1997, 1998); the level of accumulation at the nuclear envelope, relative to medium fluorescence, was measured using NIH Image 1.60 in line plot mode as previously (Piller et a1., 1998). Experiments were repeated three times. 63 RESULTS Cloning of the Arabidopsis importin 0t homologue A partial 900 bp clone which had high homology to yeast and vertebrate importin a homologues was obtained from the MSU— DOE Plant Research Laboratory Arabidopsis Genome Sequencing Project (Newman et a1., 1994). This clone was used as a probe to screen the Arabidopsis PRL2 cDNA library (Newman et a1., 1994), and a full length clone (2.2 Kb) was identified that was similar in size to mRNA detected on Arabidopsis Northern blots (data not shown). The longest open reading frame encoded a polypeptide of 532 amino acids with an approximate molecular weight of 59 kD (Figure 3.1). This protein contains eight armadillo tandem repeats that are found in many proteins like armadillo, B-catenin, plakaglobin, a GTP exchange factor for small a Ras—GTPase (smgGDS), adenomatous polyposis coli tumor suppressor protein (APC) as well as importin a homologues (Figure 3.1). These repeats are 42 amino acids in length, highly hydrophobic and are believed to be involved in protein—protein interactions (Peifer et a1., 1994). Interestingly, most of the basic residues in the N-terminus of importin a homologues are highly conserved and may function as an NLS (Figure 3.1). At the amino acid level, the Arabidopsis importin a homologue (At—IMPa) is 36—49% identical with other homologues found in Saccharomyces cervisiae, Schizosaccharomyces pombe, HOmo sapiens,.MUs musculus, xenpus laevis, Caenorhabditis elegans, and Drosophila melanogaster (Table 3.1). Interestingly, the 64 Figure 3.1 Amino acid sequence of At-IMPa and other protein homologues. The sequences of At-IMPa was aligned with the other importin 0: sequences found in Sacchammyces cervisiae (Sc-IMP; GenBank accession number 002821), Schizosaccharomyces pombe (Sp-IMP; Z98887), Homo sapiens (Hs-IMP; P52294), Mus musculus (Mm-IMP; 060960), Xenpus Iaevis (XI-IMP; P52170), Caenorhabditis elegans (Ce-IMP; AFO40995), Drosophila melanogaster (Dm- IMP; AS7319), and Selaginella Iedpidophylla (SI-IMP; U96718). 65 Z¢mefl>mflmummmQO<>ZOA<3>DDmmmmqqo>hHm>¢OOMH>>dEmOm mUAZ>H>BB>ZmAm0HmHmmZHmOQAmOAHOAMAUWQmmmmUQDHHZUA43>mOmU>ZQZOUmQQOQmAm>¢O¢Z>>¢OBOMm mUQZmEQB>H2mAmmAmEmZZHdeHO0H>meH>HQmfidéwawfiHZOA<3>¢OM¢AZZmMmOQQ<>mmm>flzmmH>UmBODm mUQZmAEZBHZMAWOQmBOmZ>A¢AQmmH>ZUZHAfiflmwdeQU¢OmmHmdmmmmHQmHmdmH>mMBOQm mUQZmQ¢3>>QmUSBmQU¢OmO>DmmmmmQQMHhHm>¢0¢OH>ZMBOAm mUAZmA¢3>¢ZmBSBAmZOMmquAAmmqHZUQQ>>QmUZBmQU¢HZOA¢3>OmmmmmaqumHm>¢m¢OH>HmBOQm mUSZmQBBBmzmQSmAmmmeHHZQAmMQ>OUOQ>EQmUSQmflwfiHZOQ¢3>¢Omm>Dommmmqqm>h>m>@2¢m>>>mBOZD mUQZmQBBBdfimHQm.mMZmZmQDAHmMS¢ZUOQ>MQmNQBm00¢>204¢3HN>mUBWAQOHmam>¢0¢0>>>¥90¢fl mumZmAEBE¢ZMQZmAMdEMZQOZQQmAAdUUOA>Amm0mmm00¢>ZOA¢3>DQmmm4440>hHm>flzmQH>>MBmOm eommHzeqoemoqmo>qmm>>o.<«H>mmHmmmmmHmqzmmmoeemmqo¢4momms>om>z¢mam..mqmmm somaHzeqmzaammOAmeemquoo>q>quo.moHgooHmmzmoeqummmoo>¢eqom¢mommo¢¢<>e..qmqqzaomm somsHzeqazammmoqzmzzezoammHon>Ho.monqummzmmmmgammamozoqmommoommzzma>Hmo>mqo>mmoo sommHzeq<3<quHq>.mmHHozqmmzmmmmqzxmmHmmqmqmm>o>m zommHzeq>3mmmmoqeozmmmmqmm>mm¢>>omezH>moHmmmexmqqumsoemmqoommmmeHzoezomes>oom3mm¢>>omemH>monm2mmxmqqmmmmoemmqoommmmmmHzmHzomeH>oomzeqmmm>>o.4oH>MoHmmzemMmqqqmq...oqmqm eommHzeq<3mquoqzmmozmmzmm>qmm>>o.«mo>>onmmmmmmAHommx>emmm>>w.flmH>mmHmmmmMHmqammMOBBmMQOqfimQDm3>D¢>EGMAm..QAMMQ >QUmU...h.Q>EZDmmmmmB..BMVHmmeZ ..... Bmflommqmz UmmBBmBmm ......... DDMHOBZHzmMMmHdmmmMQmmHm>.>MMZMMWMOHOM.Mdzzxww40m ....... Z ..... mmz ZAWMAmmBA ......... QMQZ...HZMMMMSOQMMMmmmAmHfi>mmmm2meOBZ.HmZ¢Mwmwom ....... mZmD ¢Mm2 OZdzmmmmq ......... HammeU>ZMMMqHGammm¢MMAm>m>m>mmmem¢EQ.XUMZMWMMZM ....... MQ¢WZBBQE SZZHOfimmmwwam2>mmm8mmm49¢>ZMMMmAOmmmMOMMAOAUmmmmmmmSMDmZQ.mMZMmedm....mzmxwmfim...2 22mHOmMWBWMMZmMMmQOmmmmomquAUmmmmmmmZWQmZJ.mmzmmedm....mZMMUmBB...S QQZWMDOWAmHBmm¢mWO>D....>QZ¢MMZdmemmommHmH009mmmmmqHQdOmBUMUMVZOmmmmmHmmmmfim ..... 2 m>m000¢m>mmm0mmflmfldwflfimmHmZMMMOOBQmmmmdm04mmmom2¢m29mm>mm>hMmBmmOBUZQS >m<¢m¢mmmwoaddzwm ......... mMMMSAmmmmMmmmHm>ZZDmmmmm0mm¢Q>¢..>M>mzmm>m8....EMdmeQmS mtham mthmo mthso mthax mthsz mzHumm mzHuom mzHiom mzHlu4 mzHlam mzHumo mzHuso mzHuax mthez mzH-mm mzHlom mzHlum mzHuu< mzHIHm mthmo mzHueo mzHlfix mzHlez mzHlmm mzHuom mzHlum mzHuua 66 dewummHmmB...>Om<¢..m...OfiEMQHZZHOQA>>OHHOmQBUqufiummmfl>wd ........ 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HommOAOOmm< mqwmdflflHAO<>>ZEDOBZUQZMm¢MU>MQHZMAOMQU>BHHmemU>AAQUQmMHUOOW>A>MHUOOM>ANMHODmm00mB¢ ZmH<3moo>ooozom3>mmmszmM.smaqqomzmmu>omoq>qoeomoeoe ze>¢s¢emqoomHomoH>¢oHomozomsH2m>93¢¢mM>H2mx.zmoqqqoqmmqoom>oeoooeoe ze>qmmqqooeHzooHommmo<¢H2mHmsammMoHmam.ommqqomq>mq>oeoHooeoHoooe zeH<3<eoHoamzoosoHoooe ZmHmso>oH0mmzooommm.mqqmqqquaoozHH>oeo>oooe ZmHmzommxmem>m¢>mqqx>qmmqu¢QH>¢oHomezoomzH>>oeoqozoe 2mHmsaammxHommmzoqqm>qmmqu¢m>>eoHooMzomeH2mH93oooeoHoooe >Hzo>mmq<¢eo>x>o>moqqm>qme>>oamH>onmmzoooeqwmq¢3>equHzeoomquqmq>¢mmmHzo>mmqHHmmmozoqqx>qmm>4omo>>>mqoHo0mqqgomq>mdqmm>oommmmmzmz >Hzo>emqmme>Hmummmwsquamo>HoeM>>>oHmQZmooeqwmzasoeomqHomoommzqoeq>mqHoq>¢m2mmwmzmz >Hzo>4mq>mwozmzqqm>qmmo>oaoH>¢oHxo2moomnewq<3omooeom>mqqsmu>zqomm>M4mmmmmmmo >Hzo>>xwozmzqqm>qmmo>ooeom>mqqsmq>zqomm>MHzo>mmq¢meo>o¢mmmzqqm>qmmmHoq>omoqe>qumq>mH>0mHmzzomomzmo >Hzo>qemmmmqqm>qmmmHm>oH>mH<3o¢o>qemeozm>Hamaqemqaom>>mzomomxmo >Hzo>emq¢mHq>mmmmmqqqm>qmm>>oeoH>eoHmozeoomqwmq<3ommoommHammu¢mq¢mm>oommmom.mo mzHuam mzHlmo mzHlso mzHlax mzHusz mthmm mzHuom mthom mzHlu< mzHlmo mthso mzHuax mzHlsz mzHumm mzHuom mzHlum mzHlua mzHuam mzHumo mthso mzHuax mzHuez mthmm mzHuom mthom mzHuu< 67 momzzoomeom>mm>ooomzosmoommm. mewoommamm.oeazmmqaoz>mommqmomomew«omozmmmq«Hoo¢mmo mm aomom..2.memooomHSOOquo>omaHmmomomeom aomom..2.mamooomeooozqo>omMWmeOOABm4M .VMmHA¢Q¢43>>EEZQmBOAemmmqmm..Hmommom. ZmZmO....OZ>ZmOhDhBZOfiZOmdzEMD>¢QmWMOm. mmmHHz>mommooemoommmqswemqHm>mxmemzzommoqzmHm mzHlam mzHumo mzHuso mzHiax mzHlsz mzHlmm mzHlom mzHlum mzHuu< 68 Percent Identity Protein 2 3 4 5 6 7 8 9 At-IMP 48.1 43.3 48.5 48.1 37.7 36.4 37.0 72.2 SC-IMP - 54.1 46.3 46.5 39.1 35.8 37.5 47.0 Sp—IMP - - 48.0 47.8 39.7 38.5 38.7 39.9 Hs-IMP - - - 97.2 38.3 37.0 39.7 38.4 Ms-IMP - - - - 38.5 39.7 39.5 38.9 Xl-IMP - - - - - 41.4 47.5 41.9 Ce-IMP - - - - - - 42.2 39.9 Dm-IMP - - - - - - - 39.9 Sl-IMP - - - - - - - - Table 3.1 Comparison of amino acid identities of At-IMPa and other importin a homologues. For accession numbers, see Figure 3.1. 69 At-IMPa is 72% identical to an importin a homologue found in lower plants, Selaginella lepidophylla (Table 3.1). Because importin a is highly conserved throughout the different kingdoms, we propose that At-IMPa has a similar role in nuclear import of proteins in plants. Characterization of At-IMPa antibodies Antibodies were raised against a GST—At-IMPa fusion protein that was expressed in E. coli and purified from glutathione agarose beads. The fusion protein was further purified from SDS polyacrilaminde gels before injection. As a means to purify At-IMPa antibodies, His-tagged Arabidopsis importin a was expressed in E coli, purified from Ni—agarose beads, and cross—linked to Affigel 10 beads. Immune sera from the injected rabbit was passed through the Affigel His—tagged At- IMPa column, and the specificity of the purified antibodies were checked by immunoblot analysis. Arabidopsis protein isolated from roots, leaves, stems and flowers were separated by 10% SDS—PAGE, blotted to nitrocellulose and probed with At-IMPa antibodies. A 56 kD protein was detected in all tissues (Figure 3.2, right panel). Nuclei and cytosol were isolated from tobacco protoplasts for immunoblot analysis. At-IMPa antibodies also detected specific bands in isolated nuclei and cytosol fractions, that were similar in molecular weight as the recombinant His-tagged At-IMPa (Figure 3.2, right panel). Interestingly, a 57 kD band was detected in both nuclei and cytosol, however the cytosol contained a band 70 kD12341234 97- 66- 31- Figure 3.2 Purified antibodies to At-IMPa are specific in both Arabidopsis and tobacco. (A) Total protein (50 ug/gel lane) from Arabidopsis flowers (lanes 1), stems (lanes 2), leaves (lanes 3), or roots (lanes 4) was immunoblotted. Purified At-IMPa antibodies detected a protein of 56 kD in all tissues (left panel). The preimmune control blot is also shown (right panel). (B) Protein from tobacco cytosol (lanes 1) and nuclei (lanes 2) as well as purified His-tagged At-lMPa (lanes 3) was immunoblotted. Purified At-IMPa antibodies detected protein doublets of 56 kD in cytosol and nuclear extracts (left panel). The preimmune control blot is also shown (right panel) 71 at 56 kD whereas the nuclei contained a lower molecular weight band at 55 kD (Figure 3.2). The fact that importin a doublets were found in Arabidopsis and tobacco was not surprising since similar doublets have been found in bovine erthrocytes (Adam and Adam 1994), xenopus cytosol (Gorlich et a1., 1995; Moroianu et a1., 1995), yeast (Belanger et a1., 1994; Loeb et a1., 1995), and Drosophila (Torok et a1., 1995; Kussel and Frasch 1995). Furthermore, the importin a doublet in Drosophila has been shown to be the result of protein phosphorylation (Torok et a1., 1995). We concluded that At- IMPa antibodies are specific in both Arabidopsis and tobacco plants. At-IMPa binds to three classes of M88 To address the function of At-IMPa's, we used an in vitro binding/co-immunoprecipitation approach (Weis et al,. 1995). Affinity purified antibodies to At-IMPa were characterized to determine if in vitro translated [3SS]-Met At—IMPa could be specifically immunoprecipitated. To demonstrate that At-IMPa was translated, a sample of the translation mix was separated by SDS—PAGE, and analyzed by autoradiography (Figure 3.3, lane 1). Next, purified At—IMPa antibodies were added to the translation mix followed by protein A Sepharose for immunoprecipitation. When immunoprecipitates were separated by SDS—PAGE and visualized by autoradiography, [3SS]—Met At- IMPa was apparent (Figure 3.3, lane 2). The addition of 500 72 Figure 3.3 Immunoprecipitation of in vitro-translated At-IMPa. At-IMPa was in vitro translated with [35$]Met (lane 1) and immunoprecipitated with purified At-IMPa antibodies (lane 2). Immunoprecipitation was also examined in the presence of 500 ng of recombinant At-lMPoc (lane 3). The samples were analyzed by autoradiography after SDS-PAGE. 73 ng recombinant At—IMPa competed with the in vitro translated At—IMPa for immunoprecipitation (Figure 3.3, lane 3). Preimmune sera did not immunoprecipitate in vitro translated At-IMPa (data not shown). We next investigated whether At—IMPa could bind to NLSs in vitro. Representative NLS peptides from each of the three classes of NLSs were selected. The first NLS synthesized was the bipartite NLS, 02WT, identified in the maize transcription factor, Opaque—2 (Figure 3.4 A, Varagona et a1., 1992). The SV40 T—antigen NLS, identified from the simian virus 40 large T-antigen (Kalderon et a1., 1984; Lanford and Butel, 1984), was also synthesized (Figure 3.4 A). Lastly, a Mat a2-like NLS, NLSC, was synthesized which corresponds to one of the NLSs identified in the maize transcription factor, R (Figure 3.4 A, Shieh et a1., 1993). These NLSs are functional in vivo (Varagona et a1., 1992; Shieh et a1., 1993), and they bind specifically to the NLS binding site in tobacco nuclei (Hicks and Raikhel, 1993; Hicks et a1., 1995). A fourth peptide, 02mut (Figure 3.4 A), was also synthesized which corresponds to a mutant form of 02WT which does not function in vivo (Varagona and Raikhel, 1994) or compete with 02WT for binding to the site in tobacco nuclei (Hicks and Raikhel, 1993). The peptides corresponding to the functional NLSs were chemically coupled to human serum albumin (HSA) and allowed to interact with recombinant At- IMPa. Then, At-IMPa antibodies were added to the binding assay, and the protein complexes were immunoprecipitated with 74 Figure 3.4 At-lMPa recognizes three classes of NLSs. (A) Amino acid sequences of peptides corresponding to the wild-type bipartite (02WT) and mutant (02mut) 02 NLS from Opaque-2 and the SV40 large T-antigen NLS (SV40) and the Mat aZ-like NLS (NLSC) from the R protein are shown in a single-letter code. (B) HSA does not coimmunoprecipitate with At-lMPa (lane 1). Functional NLS substrates such as 02WT-HSA (lane 2), SV40 T-antigen-HSA (lane 3), or NLSC-HSA (lane 4) coimmunoprecipitate with At-IMPa. The secondary goat anti-mouse antibodies cross-react with the IgG heavy chain (50—kD protein band) of the rabbit importin a antibodies. HSA (lane 5), 02WT-HSA (lane 6), SV40 T-antigen-HSA (lane 7), and NLSC-HSA (lane 8) were immunoblotted directly to nitrocellulose to determine the migration pattern of these cross- linked substrates after SDS-PAGE. 75 02WT 02mut SV40 NLSC MPTEERVRKR KESNRESARRS FiYRKAAHLK C MPTEERVRTN KESNRESARRS NYRKAAHLK C CTPPKKKRKV CYMISEALRK AIGKR 76 protein A Sepharose. The immunoprecipitated proteins were separated by SDS—PAGE and the NLS-HSA substrate that co- immunoprecipitated with At-IMPa was detected by Western blot analysis using monoclonal antibodies against HSA. Figure 3.4 B, lane 1 demonstrates that HSA alone was not co— immunoprecipitated with At—IMPa. However, co- immunoprecipitation occurred when HSA was coupled with peptides to the 02WT (Figure 3.4 B, lane 2), SV40 T-antigen (Figure 3.4 B, lane 3), or NLSC (Figure 3.4 B, lane 4). Figure 2B shows the mass of HSA (Figure 3.4 B, lane 8), 02WT— HSA (Figure 3.4 B, lane 7), SV40 T—antigen-HSA (Figure 3.4 B, lane 6), and NLSC-HSA (Figure 3.4 B, lane 5) when they are directly blotted to nitrocellulose after separation by SDS- PAGE. Note that NLS-HSA substrates do not migrate as distinct bands due to variations in the number of peptides coupled per HSA molecule. At-IMPa binding is specific for functional NLS: To test the specificity of whether At-IMPa binding for functional NLSs, competition studies were set up using the 02WT and 02mut peptides (Figure 3.4 A). The functional substrate, 02WT—HSA, was co—immunoprecipitated with recombinant At—IMPa (Figure 3.5, lane 1). In the presence of 1 mM of 02WT peptide 02WT-HSA binding was greatly reduced (Figure 3.5, lane 2), whereas 1 mM 02mut peptide did not compete with 02WT-HSA for At-IMPa binding (Figure 3.5, lane 3). In addition, 02WT-HSA binding to At-IMPa can be competed 77 Figure 3.5 Specific interaction of At-IMPa with a functional NLS substrate; 02WT-HSA is coimmunoprecipitated with At-IMPa (lane 1). Specificity of At-IMPa binding was determined by coimmunoprecipitation of 02WT-HSA in the prescence of 02WT (lane 2) or 02mut (lane 3) peptides. The secondary goat anti-mouse antibodies cross-react with the IgG heavy chain (50 kD protein band) of the rabbit importin (1 antibodies. 78 with 200 um of 02WT peptide (H.M.S. Smith and N.V. Raikhel, unpublished). Specific NLS binding indicates that At—IMPa is likely to be a functional NLS—receptor in plants. Furthermore, this single receptor recognizes the three classes of NLSs found in plants. Localization of importin or in tobacco protoplasts and nuclei To investigate the intracellular location of importin a, tobacco protoplasts were fixed and incubated with affinity purified antibodies to At—IMPa followed by CY3-labeled secondary antibodies. Immunofluorescence was detected in optical sections by Confocal Laser Scanning Microscopy. CY3 fluorescence from a 0.5 um optical section through the protoplasts indicated that importin a was located in the nucleus and cytoplasm (Figure 3.6, A). This also supports previous cell fractionation studies indicating that importin a is found in cytoplasmic and nuclear fractions from tobacco protoplasts (Hicks et a1., 1996). It also appears that importin a is excluded from the nucleolus in protoplasts and isolated nuclei (Figure 3.6; unstained region in the nucleus). Intense immunofluorescence at the nuclear envelope demonstrated that importin a was highly concentrated at the nuclear envelope (Figure 3.6, A; yellow region). A 1.0 um optical section displays similar fluorescence at the nuclear envelope when using tobacco nuclei purified in the presence 79 Figure 3.6 Immunolocalization of importin a in fixed tobacco protoplasts (A) or purified nuclei (B and C) were visualized by confocal laser scanning microscopy using affinity-purified At-lMPoz antibodies followed by CY3-labeled secondary antibodies. Bar = 10 um. 80 of 0.6% Triton X—100 (Figure 3.6, B and C), indicating that importin a is tightly associated with the nuclear envelope. Biochemical properties of nuclear importin or In vertebrates, importin a is soluble (Adam and Gerace, 1991), however in yeast it is associated with the NPC (Yano et a1., 1992; Belanger et a1., 1994, Aitchison et a1., 1996). In plants, previous studies demonstrate that importin a is strongly associated with cellular structures in tobacco permeabilized cells, even in the presence of 0.1% Triton X- 100 (Hicks et a1., 1996). In order to characterize the association of importin a with the nuclear envelope, we examined the biochemical properties of importin a in purified nuclei. Nuclei were purified from tobacco protoplasts and treated on ice for 15 minutes with 1% Triton X-100, 0.25 M NaCl, 1% Triton X-100 plus 0.25 M NaCl, or 6M urea. Samples were centrifuged and the nuclear pellet (P) and supernatant (S) were examined by immunoblot analysis using At—IMPa antibodies. Most of importin a was resistant to extraction by 1% Trition X—100 (Figure 3.7, lanes 1), 0.25 M NaCl (Figure 3.7, lanes 2) and 1% Triton X-100 plus 0.25 M NaCl (Figure 3.7, lanes 3); however, treatment of nuclei with 6 M Urea extracted some of the importin a (Figure 3.7, lanes 4). Importin a is also partially extracted with 0.5 M NaCl (H.M.S. Smith and N.V. Raikhel, unpublished). These biochemical properties indicate that importin a is tightly associated with the plant nucleus. In addition, the 81 Figure 3.7 Biochemical properties of nuclear importin a. Tobacco nuclei were treated with 1% Triton X-100 (lanes1), 0.25 M NaCl (lanes 2), 1% Triton X-100 plus 0.25 M NaCl (lanes 3), or 6 M urea (lanes 4). After treatment the samples were centrifuged and the supernatant (S) and nuclear pellet (P) were extracted with 2% SDS-PAGE sample buffer. Untreated nuclei were extracted with 2% SDS- PAGE sample buffer (C). The samples were separated by 10% SDS-PAGE, and importin a was detected with affinity-purified At-IMPa antibodies. 82 biochemical properties of nuclear importin a and NPC proteins (Heese-Peck et a1., 1995) correlate closely, indicating that a fraction of nuclear importin a is probably associated with the NPC. At-IMPa can mediate nuclear protein import independent of importin B These experiments were done in collaboration with Dr. David A. Jans’s group at the Nuclear Signaling Laboratory, Division for Biochemistry and Molecular Biology, John Curtin School of Medical Research, Canberra, Australia. Recently, an in vitro import system was developed in plants using evacuolated permeabilized tobacco protoplasts (Hicks et a1., 1996). However, importin a is not depleted from these permeabilized cells and its addition does not stimulate import (Hicks et a1., 1996; H.M.S. Smith, G.R. Hicks and N.V. Raikhel, unpublished data). Therefore, we analyzed the function of At-IMPa in permeabilized vertebrate cells. We compared the ability of At-IMPa to that of the mouse importins (m-IMP) to mediate nuclear import in a reconstituted in vitro system. In the presence of RanGDP and p10, m—IMPa or B alone could not mediate nuclear import of SV40 T—antigen NLS substrate (data not shown; Table 3.2), levels of nuclear accumulation being similar to those in the absence of importin subunits (data not shown). This was in contrast to the combination of m—IMPa/B where maximal nuclear accumulation relative to that in the cytoplasm (Fn/c was m... l 83 Figure 3.8 Ability of At-lMPa to mediate nuclear protein import reconstituted in vitro using purified components in the absence of exogenously added importin [3 subunit. Nuclear import was reconstituted in mechanically perforated rat HTC cells in the presence of an ATP-regenerating system containing GTP/GDP and using p10 and GDP-loaded Ran as described in the Methods. Quantitative results for nuclear import kinetics of the fusion proteins SV40WT-B-Galactosidase and SV40mut-B-Galactosidase mediated by mouse importin subunits (A) or At-IMPa (B) in the presence of Ran and p10. Results shown are from a single typical experiment where each data point represents at least 5 separate measurements of nuclear fluorescence (Fn), cytoplasmic fluorescence (Fc) and background fluorescence (see methods). Data was fitted for the function Fnlc = Fnlc max (1-e'k‘), where Fnlc is the nuclear/cytoplasmic ratio, k is the rate constant, and t is the time in minutes. Pooled data are presented in Table 3.2. 84 E5 we: . are as: an ac en an e— e an — P - bib I n I - b e .’ ts o¢>m 51:.— 32 9.5 ass 52 o¢>m ._.>> ov>m ._.>> o¢>m e95 oz... :_tan_-~< 3:895 omaos. m < 85 over S—fold (Figure 3.8 A; Table 3.2; half-maximal within 2.6 min). Significantly, At—IMPa, in the absence of exogenously added importin B subunit, was able to mediate nuclear import to comparable levels (Figure 3.8 B and 3.9 A; Fn/cmax of 3.7 - half-maximal within 8 min). The specificity of transport in all cases was demonstrated by the fact that the mutant SV40 T—antigen NLS substrate did not accumulate in the nuclei to any significant extent (Figure 3.8 A and B; Table 4). The results thus demonstrated that At-IMPa could mediate nuclear import of an NLS—containing transport substrate independent of an importin B subunit. Nuclear import mediated by At—IMPa was enhanced by p10, especially in terms of the rate of import. Nuclear import did not absolutely appear to require p10, since maximal import levels of 3.2 were observed in its absence (Figure 3.9; Table 3.2). In vitro binding studies showed that At—IMPa can bind to yeast and mouse importin B (Hubner et al., submitted). Nuclear import mediated by At-IMPa was tested in the presence of the m—IMPB subunit (Figure 3.9 B). Interestingly, nuclear import was reduced by more than 50%, compared to in the absence of m—IMPB, in either the absence or presence of p10 (Figure 3.9 B; Table 3.2). This inhibition of At—IMPa— mediated transport by m-IMPB is unlikely to be due to its ability to bind At—IMPa since At-IMPa's affinity for SV40 T— antigen NLS is over two times higher than that for m—IMPB (Hubner et al., submitted). Several studies (eg. Fagotto et 86 Figure 3.9 Nuclear import reconstituted in vitro using purified components mediated by At- lMPa and m-IMPa in the absence (A) and presence (B) of exogenously added m-IMPB. (A) Nuclear import kinetics of the fusion protein SV40WT-B- Galactosidase mediated (1) At-IMPa plus Ran plus p10, (2) At-lMPa plus Ran, (3) m-lMPa plus Ran plus p10, or (4) Ran plus p10. (B) Nuclear import kinetics of the fusion protein SV40WT-B-Galactosidase mediated (1) At-IMPa plus Ran, (2) At-IMPa plus Ran plus p10, (3) At-IMPa, or (4) Ran plus p10. Import assays were measured in the presence of an ATP-regenerating system containing GTP/GDP. Experiments for transport were performed as described in the legend to Figure 3.8. Results shown are from a single typical experiment where each data point represents at least 4 separate measurements of Fn, Fc, and background fluorescence. Pooled data are presented in Table 3.2. 87 ea: 2:: . of lm.v are as: — p n n n n n b p L e s m . J..— O O O In N . . 2:”. F . . rm; 32.5. 88 a1., 1998) have shown that importin B can inhibit nuclear import through effects not directly related to substrate recognition, but rather to binding to NPC components, implying that the effect observed in Figure 3.9 B may be similarly mediated. Nuclear import kinetic measurements were also performed in vitro for the first time using the bipartite NLS identified in the xenopus histone binding protein NIN2 (Kleinschmidt and Seiter, 1988). Peptides to this NLS were fused to B— galactosidase (N1N2-B—Gal) and produced results similar to those for SV40 T—antigen NLS substrates when they were added to the in vitro import system (Table 3.2 and data not shown). Maximal levels of At-IMPa—mediated import (Fn/cmax of about 3.4; half-maximal within 15.5 min) were obtained in the presence of RanGDP; the rate of import was almost twice as fast in the presence of p10 (t1/2 of c. 9 min; Table 3.2). In the presence of m—IMPB, accumulation was reduced by c. 40 and 60% in the presence and absence of p10 respectively. 89 496.0 u.» x538 2 3:28: .38: 883253 5 <28 cmic panama acacia... Oonaaoam 2:062 .260: umqm3m~o4m+ ”6822 mcomcdsm mm: 3 0 mice? cm “3.3 2..an micsaeba + + mauwloam elm: H 5 m~._§_u&3-_z=uu ‘ m