n l ,., v . . W. v! :4 c . EM »‘ ‘ . ‘ f" ‘ .. ' r— .. v ‘ .- - a A.‘ o O 1 ,_._a:,‘ -. £. 4 .7-..“ r 731'. 9.-. ,- ': “ . . . _...,. a , 343 Kat-1;,- .. 4 .- ' .... .... ‘u 4 .Au‘ ”.4. u we {$7.11. {:2 .5.— . r.-. -.-' “- . #uo-Mg.‘ A__-. .g n ,. .... ... ,, _ It. 9.. "' '.‘.‘.. '11“ ”w, - .. -—~“....‘: ‘v _ A. ‘ n. .. . . .LA' .. w- A f" .. .._ .. 1—5 M O - 4.“ " "m¢~< o-‘ ‘ ....4 .1- :- . '33:: w; rm -:‘~:.'f.'.. - . . x. ' a ' A r- ' “of"- -- z - . . —¢4‘ .w’r » n2 . ":z. —- _ . . L *3: a .4..- a -4 . "-0 “"3": n. 4: .~. ,.. (w u. - .u-r-I 1—‘0‘ '. a.“ m tn r > > . ‘5“-.- " p...o f.....— ~ .fl.. - -'::':. w 47‘” ‘1...”3“ ‘ M .4 .0 ”~22... cs- ' ‘ .moL, 1..“ ‘.f:-.-o-- 4. «:00: "‘ . ~ . . a 2! ._J .—- .— . '- ,.. n ma- -. $2.:- _ - ."f 4 .. . u. "7:. «4 321:: ; if??? t unu— acn- . .‘ I . "w. , .w- .4"- I 3 L». 215 0‘0": 3 . ‘ l n—‘ -0..- .. V > '7. 'u.‘ 0—. '31: o .. o a n ‘ . 5"1': - f4 7“. ' wy—vv ”.J a I . ..~ .- x- ‘31::- '— JILL" .. - . r » ._o- .. . M a _- o—o-¢.w--" "f ’ -— z.‘ .u. . >- ‘.'.a~— - 3' ‘F-v-r. o.- . .- . 2:: ' .a- L-¢o‘-:‘ 'fi’. .—. , .....-... a .- p - _'. .. ... .'r~‘ ‘ v . r-> . .- ..,.y-— .. ; g n.. ..J:w .«u. ‘ ‘2‘ .uh gév.‘ :‘-l ' I s V ‘ I 3’3 ‘51". ‘1‘, PH ....--... .4 .._. . u-l -:-.‘ 7—1.“ I 0". '1‘. -... ..— . «e. p.“ "x - -. - .. M ..- .«u-. “-g‘ -.- - 1.5.. ‘33}; C» —. .r “a o- W .- Y’ 5'... ,. —-<- — o.) ".4 fi‘- - ’2... ...._‘ -a’ I?“ “"41.- a «’1' -..- m.” . “.0... ..-....... .0... anvm‘ . . .. ..— 71.. M . . .. o .o ..a.. ‘affl a! “an - .—-.. .¢:-.v < 4., ~.a .. _ Ma ;.’. g. '3: . . .. AP)?“ #3:}. :7. o 3 141.3 a .. .- .42“- ’0 J .1 A P. .L J ~. 1 1‘ W ‘ ."H"'*.‘ {3}!“ ._ ' ‘ ‘ ‘. C’. ! ‘ , 31* ' l .. ..... ”I «243" . .. m. ‘ 0,312.3» £3“ 0'- 2133‘ a .57; gs“ .1 I: _' 5 I .- ‘4 V ' "2'4"" .677“- ” ,... .. _ J. a“ .33..-- 4 Jo'- 433+." a ’ : .u ' o4 ' 4. «~45. .: w. Whit" 7..” -—_, ~ "21% '- .. ... r::,.g.§. J, J. as" r 4 “ . 4 u ‘ 1 .-” J . *1." t A n L. .... 1"" 1', ‘ ‘1. .. w r a tit: '5'? 3:1" I I. " 71'3““ iv ‘ was .i 2 '1 I! U! -‘ “'3! .._Q " .7." w: “' W5!“ 13"9' ‘5 5"- 11‘1 ‘ ' $11}. n J“ .E} in ‘31“ WE}??? “ ”n-..— .- 5k 1 I? ‘2‘,“91: v ‘ 333531;." , ‘ é 51.31“”: 5%" ~: 01:50 ~§ . . . 1 I , 2 “1&1? 5%“ *5 wt "' --\ J ‘ no I m “m ‘ SIS :- 3351‘- ‘ .gflw .... . .f ‘ ‘ may" «'5» P€EZF“ 3: (1", g;- ¢ "Aw I .33? w .." J a.“ 31’ < 5:. .1" r1}: 2‘ IO . . . THESlS /2\ | V / Illill'llll llllllilllilllllllllllllill 3 1293 01571 967 This is to certify that the dissertation entitled THE ROLE OF STROMAL MOLECULAR CHAPERONES IN CHLOROPLASTIC PROTEIN TRANSLOCATION presented by Erik E. Nielsen has been accepted towards fulfillment of the requirements for Ph . D. degree in Botany & Plant Pathology kafi Major profess?’ Date%‘:_/% 7/ it??? MS U it an Affirmative Action/Equal Opportunity Institution 0-12771 LIBRARY Mlchlgan State Unlversity PLACE II RETURN BOX to remove We checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE __] L___ii:_ E MSU le An motive Wipe! Opponunlty lnetltulon WWI [:j—T __l L__ E 1 1 THE ROLE OF STROMAL MOLECULAR CHAPERONES IN CHLOROPLASTIC PROTEIN TRANSLOCATION By Erik E. Nielsen A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1997 ABSTRACT THE ROLE OF STROMAL MOLECULAR CHAPERONES IN CHLOROPLASTIC PROTEIN TRANSLOCATION By Erik E. Nielsen Cytoplasmically synthesized precursors interact with translocation components in both the outer and inner envelope membranes during transport into chloroplasts. The roles of three stromal molecular chaperones, Hsp60, S78, and CIpC, in chloroplastic protein translocation were evaluated using coimmunoprecipitation techniques. Stable translocation complexes between precursor proteins and associated membrane translocation components were first identified in detergent-solubilized chloroplastic membrane fractions using antibodies specific to known translocation components. Antibodies specific to Toc75 and Toc34 (Iranslocon of the guter membrane of chloroplasts, 75 and 34 kDa proteins respectively), Tic110 (Iranslocon of the inner membrane of ghloroplasts, 110 kDa protein), and the stromal Hsp100, ClpC, specifically coimmunoprecipitated precursor proteins under limiting ATP conditions. A portion of these same translocation components were coimmunoprecipitated as a complex, even in the absence of added precursors, and could also be detected by cosedimentation through a sucrose density gradient. Hsp60 associated with newly-imported prSS and processed mSS in a manner consistent with a role in folding or assembly rather than protein translocation. 878 also coimmunoprecipitated with precursors, however this association was most likely an aggregation of insoluble protein complexes with residual membrane fragments containing protein translocation complexes. CIpC, however, associated with precursor-containing complexes in a manner consistent with a physiologically significant role in chloroplastic protein translocation. ClpC, was observed only in complexes with those precursors utilizing the general import apparatus, and its interaction with precursor-containing translocation complexes was destabilized by ATP. Despite the ATP-dependence of ClpC’s association with precursor-containing complexes, ClpC maintained a tight-association with envelope membranes regardless of whether ATP was present or not. In memory of my grandmother, Sarah C. Nielsen, who showed me how fascinating plants can be Acknowledgements I would like to thank all the people who played a part, directly or indirectly, in this thesis. My advisor, Ken Keegstra, provided me with the opportunity to work in his lab and l have learned a lot under his tutelage. Lee McIntosh, Natasha Raikhel, and Micheal Thomashow provided guidance as members of my graduate committee. Members of the lab, Mitsuru Akita, Jenny Davila-Aponte, Amy DeRocher, Karen Ford, Johann Frdhlich, Arun Goyal, Diane Jackson, Robyn Perrin, Sigrun Reumann, Pat Tranel, and Zhaohong Wang, all provided technical assistance and lively discussions. (Johann, John for the rest of us entertained everyone greatly with his whistling expertise, and Sigrun, came late, but made up for it by forcing us all to go to concerts and plays.) Kurt Stepnitz and Marlene Cameron helped with the preparation of the figures in this thesis. The PRL office staff (who always seemed to know what was going on when paperwork was involved). My family, who always listened when I wanted to complain, and Christiane Wobus who managed to put up with my absent-minded scientist act for the last five years. TABLE OF CONTENTS LIST OF FIGURES ............................................................................................... ix CHAPTER 1 Introduction .......................................................................................... 1 Protein transport across the envelope membranes .............................. 3 Binding of precursors to chloroplastic envelope membranes .................. 4 Translocation of precursors across chloroplastic envelope membranes....5 Identification of protein translocation components ................................ 6 Protein transport in other systems ................................................... 10 Maintenance of translocation-competent conformation and delivery to the translocation channel ................................ 10 Precursor interaction with proteinaceous receptors on the target membrane ........................................ 14 Precursors traverse membranes in proteinaceous translocation channels .................................... 16 Generation of unidirectional movement through the translocation channel ......................................... 19 Molecular chaperones: pushing precursors into, and pulling precursors out of, protein translocation channels ...... 22 Molecular chaperones: Definition of activity ............................ 24 The chloroplast stroma contains homologues of several molecular chaperone families .......................................... 27 Do stromal chaperones play a role in protein import into chloroplasts?...30 Statement of problem and attribution ............................................... 31 References ................................................................................ 34 vi CHAPTER 2 Detergent solubilization of chloroplastic protein translocation complexes .............................................................. 46 Introduction ................................................................................ 47 Materials and Methods ................................................................. 49 Results ..................................................................................... 52 Translocation components of the outer membrane, inner membrane, and stroma form a stable complex with precursor under binding conditions .................................. 52 A translocation complex can form in the absence of added precursors ....................................... 66 Discussion ................................................................................. 70 References ................................................................................ 78 CHAPTER 3 Evaluation of the roles of three stromal molecular chaperones in chloroplastic protein import ................................................. 83 Introduction ................................................................................ 84 Material and Methods .................................................................. 88 Results .............................................................. . ...................... 92 Preparation of antibodies that specifically recognize and immunoprecipitate S78 .................................... 92 ClpC, S78, and Hsp60 each associate with precursors or newly imported proteins during an import timecourse ............. 99 ClpC and S78 interact with membrane-associated precursor, but Hsp60 interacts with soluble mSS ..................... 103 ClpC and S78 both interact with translocation complexes, but only the association with ClpC is stable in solubilized complexes ........................................ 107 S78 displays different solubilization characteristics than precursor and translocation components ........................ 111 Discussion ............................................................................... 1 19 References .............................................................................. 127 vii CHAPTER 4 ClpC interacts with the chloroplastic protein import machinery in a physiologically relevant manner ........................................ 131 lntrod uction .............................................................................. 1 32 Materials and Methods ............................................................... 134 Results ................................................................................... 1 39 A translocation complex containing ClpC is formed with several chloroplast-targeted precursors, and is destabilized by ATP ................................. 139 ClpC interacts specifically with chloroplastic envelope membranes in an ATP-independent manner .......................... 145 ClpC is tightly associated with chloroplastic membranes ........... 153 Discussion ............................................................................... 1 58 References .............................................................................. 171 CHAPTER 5 Conclusions ...................................................................................... 175 Effect of nucleotides on protein import complexes... .....182 Are chloroplastic protein translocation complexes permanent structures, or is their formation regulated by nucleotides? .......... 183 What role does GTP play in chloroplastic protein import? ........... 185 What effect does ATP have on the association of ClpC with translocation complexes? ........................................................... 186 Does ClpC interact directly with precursors during protein import? ....... 187 Identification of inner membrane proteins that interact with ClpC ........ 190 References .............................................................................. 1 92 viii Figure 1.1 Figure 2.1 Figure 2.2 Figure 2.3 Figure 2.4 Figure 2.5 Figure 3.1 Figure 3.2 Figure 3.3 Figure 3.4 Figure 3.5 Figure 3.6 Figure 3.7 Figure 4.1 LIST OF FIGURES Protein translocation systems ...................................... 12 Association of prSS with known translocation components ........................................... 56 Complexes immunoprecipitated by anti-Toc75 and anti-ClpC contain other translocation components ..... 59 Translocation components and prSS cosediment as a complex ........................................... 64 Translocation complexes containing ClpC and Toc75 form in the absence of added precursor ............... 68 A model for formation of translocation complexes during ATP-dependent docking of precursors to chloroplasts ............................. 73 The C-tenninal peptide-binding domain of S78 is not highly conserved ..................................... 95 Specificity of anti-S78 antiserum .................................. 97 Association of ClpC, S78, and Hsp60 with translocation complexes during import ......................... 102 ClpC and S78 interact with membrane- associated prSS, but Hsp60 interacts with mSS in the soluble fraction .................................. 106 Translocation complexes containing 678 are insoluble ..................................................... 1 10 S78 displays different solubilization characteristics than precursor and translocation components ............... 113 Some 878 is released from detergent-insoluble material by addition of ATP ....................................... 116 ClpC interacts with translocation complexes ix Figure 4.2 Figure 4.3 Figure 4.4 Figure 4.5 Figure 4.6 Figure 4.7 Figure 4.8 Figure 5.1 formed by other precursors that use the general translocation apparatus ................................. 141 ATP destabilizes the association of ClpC and translocation complexes ..................................... 144 ClpC is present in both chloroplast envelope and stromal compartments ........................................ 148 ClpC’s association with chloroplastic membranes is not ATP-dependent .............................. 151 ClpC is tightly associated with chloroplastic membranes .......................................... 155 Working model for the ATP-dependent dissociation of ClpC and precursor- containing translocation complexes ............................. 161 Structural features of Hsp100 and SecA proteins ........... 167 Conserved motifs similar to those found in Hsp1005 can be identified in corresponding regions of SecA proteins ........................................... 170 Working model of the general chloroplastic protein import apparatus ........................................... 178 Chapter 1 INTRODUCTION Chloroplasts contain their own genetic system which manufactures most of the chloroplastic RNAs and some chloroplastic proteins (Shinozaki and Sugiura, 1986). However, the majority of chloroplastic proteins are encoded by genes located in the nucleus. Proteins targeted to chloroplasts are synthesized on cytoplasmic ribosomes, and must be translocated across the chloroplastic envelope membranes to gain access to the organelle interior. Once inside, these proteins then must be further sorted to one of three distinct compartments which are separated by three different membrane systems (outer envelope membrane, inner envelope membrane, and thylakoid membrane). Newly-synthesized proteins destined for the chloroplast cannot traverse the chloroplastic envelope membranes unless they are equipped with specific information in the form of an amino-terminal extension, the transit peptide (Schreier et al., 1985; Van den Broeck et al., 1985). Protein import into chloroplasts occurs post-translationally, and therefore the transit peptide is both structurally and functionally distinct from signal sequences responsible for co- translational targeting of proteins to the endoplasmic reticulum (Ellis, 1981). Transit peptides are generally between 50-70 amino acid residues in length, and are characterized by the presence of numerous hydroxy amino acids such as serine and threonine and the lack of negatively charged amino acids or tyrosine (de Boer and Weisbeek, 1991). Following import, the transit peptide is cleaved off by a specific stromal protease (Oblong and Lamppa, 1992). These mature- sized proteins are then assembled into active holoenzymes, or mobilized by the presence of additional targeting sequences to other intra-chloroplastic compartments. PROTEIN TRANSPORT ACROSS THE ENVELOPE MEMBRANES Entry of cytoplasmically synthesized precursors into the chloroplastic interior is thought to occur via a single translocation apparatus. The basis for this hypothesis rests on two lines of evidence. First, transit peptides from proteins destined to different locations within chloroplasts are interchangeable. For example, the transit peptides from precursors of the stromally-localized RUBISCO small-subunit (prSS), inner membrane-localized Bt1 protein (prBt1), and thylakoid membrane-localized light-harvesting chlorophyll binding protein (prLHCP) can be swapped (Lamppa 1988; Li et al., 1992). Second, competition studies using either synthetic precursor peptides, or full-length precursor proteins expressed in Escherichia coli, can inhibit binding and import of other precursors into chloroplasts (Perry of al., 1991; Schnell et al., 1991; Oblong and Lamppa, 1992). However, it should be noted that many chloroplastic precursor proteins remain unidentified, and their import pathways uncharacterized. In light of this, at some point in the future other import pathways to the interior of chloroplasts may be discovered. BINDING OF PRECURSORS TO CHLOROPLASTIC ENVELOPE MEMBRANES At present, transport across the chloroplastic envelope membranes can be divided into two discrete steps. The first is the binding of precursor proteins to chloroplasts, and the second is the translocation of bound precursors across the two envelope membranes. Binding of precursors to chloroplasts requires protease-sensitive components on the outer envelope membrane (Cline et al., 1985), and occurs at approximately 1500-3500 binding sites on each chloroplast (Friedman and Keegstra, 1989). Bound precursors are true intermediates on the import pathway, as demonstrated by the ability to chase them into the chloroplast by presenting the chloroplast with conditions capable of allowing import (Cline et al., 1985). Binding can be detected only if subsequent import is prevented. This can be done by providing low levels of exogenous ATP, which presumably limits the ATP concentration in the chloroplast stroma (Olsen et al., 1989), or by performing import with high levels of ATP at low temperatures (Bauerle et al., 1991). Blockage of import by low temperatures probably also occurs by limiting stromal ATP concentration through the inhibition of the inner envelope membrane ATP/ADP translocator because import is restored at low temperatures if stromal ATP is produced by photophosphorylation (Leheny and Theg, 1994). Bound precursors are stably associated with envelope membranes and are no longer in equilibrium with free precursors (Olsen et al., 1989). These bound precursors remain accessible to exogenous protease, and are not processed by the stromal processing protease (Cline et al., 1985), and so are considered to be halted at an early stage of translocation. Interestingly, precursors halted at this stage localize to intermediate density envelope fractions containing both outer and inner envelope membranes rather than outer envelope fractions after chloroplast lysis and density fractionation (Perry and Keegstra, 1994; Ostrom and Keegstra, unpublished results). Also, when visualized by immunolocalization with electron microscopy precursors bound to chloroplasts were found to localize in “patches” coinciding with regions where both outer and inner envelope membranes were closely associated (Schnell and Blobel, 1993). Taken together these two observations suggest that at this stage of import either the translocation complexes contain members of both outer and inner envelope membranes, or that binding is restricted to special regions of the envelope membranes. TRANSLOCATION OF PRECURSORS ACROSS CHLOROPLASTIC ENVELOPE MEMBRANES Import of precursor proteins across chloroplastic envelope membranes requires ATP in the stromal compartment (Pain and Blobel, 1987; Theg et al., 1989). Unlike the mitochondrial system, in chloroplasts neither binding nor import of precursor proteins require a membrane potential (T heg et al., 1989; Neupert et al., 1990). While the exact mechanism by which transport of the precursor protein through the translocation apparatus is not yet understood, the precursor is thought to traverse the membranes in an unfolded or loosely folded conformation (Schnell and Blobel, 1993). This model is based on analogy to the mitochondrial protein translocation system (Eilers and Schatz, 1986; Rassow et al., 1990), and the observation in chloroplasts of a “late” import intermediate that is processed to the mature-size yet remains associated with the envelope membranes in a protease accessible conformation (Schnell and Blobel, 1993). IDENTIFICATION OF PROTEIN TRANSLOCATION COMPONENTS An important step towards understanding how precursor proteins are translocated into chloroplasts is the identification of members of the protein transport apparatus. At the time this thesis was initiated, a cross-linking strategy, utilizing a heterobifunctional, photo-activateable cross-linker which could be cleaved by reducing agents, had led to the identification of two outer envelope membrane proteins of 86 and 75 kDa (T oc86, and Toc75 respectively) in association with prSS under binding conditions (Peny and Keegstra, 1994). Prior to the current identification of Toc75 and Toc86, several unsuccessful efforts to identify chloroplastic protein translocation components had been attempted. The use of a heterobifunctional, photo-activated cross- Iinker created an 86 kDa adduct of prSS and an unidentified protein. This complex was shown to be envelope associated, however because the cross- linker was unable to be cleaved, no identification of the interacting protein could be determined (Comwell and Keegstra, 1987). In an alternative strategy, anti- idiotypic antibodies were raised against antibodies that had been raised against a synthetic transit peptide analog of prSS. Theoretically by mimicking the structure of the original transit peptide analog, these anti-idiotypic antibodies should interact with a chloroplastic transit peptide receptor protein (Pain of al., 1988). Using this strategy a 30 kDa protein of the inner envelope was identified, but upon cloning, this protein was determined to be identical to the phosphate translocator (Flthge et al., 1989; Schnell et al., 1990; Willey et al., 1990). The relevance of the anti-idiotypic antibody strategy for identifying receptor proteins has since been called into question in several systems (Davis et al., 1992). Therefore the significance of involvement of this protein in chloroplastic protein import remains equivocal (Flflgge et al., 1991). Interestingly, upon lysis and sucrose density fractionation, cross-linked complexes containing Toc75 and T0086 were observed primarily in fractions consistent with their localization to contact sites after addition of low levels of ATP (Perry and Keegstra, 1994). However, if no ATP was included during cross- linking, the efficiency of cross-linking to Toc75 was reduced, Toc86 became the primary cross-link, and these complexes were observed primarily in outer envelope fractions. This suggested that in the absence of ATP the initial interaction of precursors with Toc86 was not restricted to special regions of the outer envelope membranes. The subsequent association of precursors with T0075 and redistribution of these complexes to envelope fractions containing contact sites perhaps reflects the association of precursor and/or Toc75 or Toc86 with additional components located in the inner envelope membrane or stromal compartment of the chloroplast. During the course of this thesis Toc 75 and Toe 86 have been cloned and sequenced and several other translocation components of the outer and inner envelope membranes have been identified. Upon sequencing Toc75, it was determined that despite the integral membrane nature of this protein, it did not contain typical membrane-spanning helixes (Schnell et al., 1994; Tranel et al., 1995). Toc75 has been tentatively identified as a part of an outer membrane protein-conducting channel, as secondary structure predictions suggest that this protein may contain extensive B-sheet structure similar to bacterial porins (Tranel et al., 1995). Toc86 has a cytoplasmically-exposed GTP-binding domain, and has been proposed as a potential receptor (Kessler et al., 1994; Hirsch et al., 1994). In addtion to Toc75 and Toc86, several other proteins of the outer envelope membrane have been identified as chloroplastic protein translocation components. In the outer membrane Toc34 has been identified and sequenced, and also contains a cytoplasmically-exposed GTP-binding domain (Kessler et al., 1994; Seedorf et al., 1995). The role of Toc34 in chloroplastic protein import remains unidentified. Two additional outer membrane translocation components are homologues of the Hsp70 molecular chaperone family. One of these proteins is peripherally associated with the cytoplasmic surface of the outer membrane and is closely related in primary structure to the major cytoplasmic eukaryotic Hsp703 (Kc et al., 1992; Wu et al., 1994). The other Hsp70 homologue, Hsp70-IAP, was identified in association with complexes containing Toc75, T0086, and Toc34, and precursor. This Hsp70 is tightly-associated with the outer membrane, and is thought to be primarily exposed to the inter- membrane space (Marshall at al., 1991; Schnell et al., 1994). These two Hsp70 homologues are proposed to play roles in the ATP-dependent association of precursors with the envelope membranes of chloroplasts (Kouranov and Schnell, 1996) Several candidates for inner envelope membrane components have been identified. Tic110 and Tic36 were identified as members of translocation complexes containing precursors that had spanned the inner envelope membrane (Schnell et al., 1994). As a result of the investigations presented in this thesis, and in collaboration with others, an inner envelope membrane protein, Tic110, has been cloned and identified as a chloroplastic protein translocation component (Lilbeck et al., 1996; Kessler and Blobel, 1996). Tic36 has not been further characterized. Two additional components, Tic21 and IAP25, have recently been cross-linked directly to the transit-peptides of precursors associated with chloroplasts in the presence of limiting ATP concentrations (Ma et al., 1996). The localization of IAP25 has not been confirmed, but Tic21 is an inner envelope membrane component (Ma et al., 1996). As these proteins interact directly with the transit-peptide of precursors they have been proposed as candidates for components that mediate the presentation of precursors to the inner membrane translocation machinery (Ma ef al., 1996). 10 PROTEIN TRANSPORT IN OTHER SYSTEMS The identification of new components of the chloroplastic import apparatus might benefit from comparison with the mechanisms by which other protein import systems accomplish transport of precursors through membranes. Identifying homologous proteins that perform similar tasks in different protein translocation systems might serve as a basis for investigation of similar proteins’ involvement in the chloroplastic protein translocation apparatus. Such transmembrane protein translocation occurs across the endoplasmic reticulum, the plasma membrane of bacteria, and the mitochondrial membranes (Figure 1.1). MAINTENANCE OF TRANSLOCATION-COMPETENT CONFORMATION AND DELIVERY TO THE TRANSLOCATION CHANNEL Most proteins can only be efficiently translocated when they are at least partially unfolded (Eilers and Schatz, 1986). Bacterial protein transport across the plasma membrane, and eukaryotic protein transport across the endoplasmic reticulum occur by both post- and co-translational mechanisms (Wickner et al., 1991; Hardy and Randall, 1991; Luirink et al., 1994; Miller et al., 1994). In bacteria, proteins delivered post-translationally to the translocation apparatus associate with the transport-specific chaperone SecB (Wickner et al., 1991), Hsp70 (Phillips and Silhavy, 1990) or Hsp60 (Kusukawa et al., 1989) 11 Figure 1.1. Protein translocation systems. The protein export systems of the bacteria and endoplasmic reticulum (ER) are functionally related. In these two export systems the translocation channels SecYEG, and Sec61p share sequence homology, and precursors can be transported through these systems either co- or post-translationally. Import of cytoplasmically synthesized precursors into the chloroplastic stroma and the mitochondrial matrix requires a coupled interaction of outer and inner membrane translocation components. In mitochondria, these translocation complexes function independently of one another. In chloroplasts, it is still unclear whether the two translocation systems are independent. Receptor proteins recognize precursors in several translocation systems. In the ER, precursors translocating co-translationally utilize the SRP-SR receptor complex, whereas precursors transported post-translationally use the Sec63p. In mitochondria, a heteromeric recognizes precursors. In chloroplasts, the identity of the receptor protein remains unknown, but the outer membrane protein, Toc86, is a likely candidate. All protein transport systems utilize chaperone, or chaperone-like, proteins to generate unidirectional movement of precursors through translocation channels. In bacteria, SecA “pushes” precursors across the plasma membrane. In both mitochondria and the ER, Hsp70s (m-Hsp70 and BiP, respectively) “pull" precursors through the translocation channel. It is unclear whether stromal molecular chaperones are members of the chloroplastic protein translocation apparatus. 12 Bacteria 13 chaperones. In eukaryotes, cytosolic members of the Hsp70 moleculuar chaperone family have been shown to be important in the post-translational delivery of precursors to the endoplasmic reticulum (099 at al., 1992; Walter and Johnson,1992) In both bacteria and eukaryotes the co-translational targeting pathways are mediated by cytosolic ribonucleoprotein complexes called signal recognition particles (SRPs; Luirink et al., 1994; Walter and Johnson, 1994). In both cases, the SRP interacts with the nascent polypeptide as it exits the ribosome. Efficient delivery of the nascent-chain-ribosome-SRP complex to their respective translocation complexes is a result of several different GTPases, whose interactions with one another stimulate a cascade of GTP hydrolysis and/or GTP/GDP exchanges, acting together to drive targeting of these complexes to completion (Gilmore, 1993; Crowley et al., 1993). Targeting of precursors to mitochondria occurs primarily post- translationally (Reid and Schatz, 1982; Wienhues et al., 1991). Cytosolic Hsp70$ also help to target some proteins to mitochondria (Deshaies et al., 1988). In addition, mitochondria also utilize a specific ATP-driven cytosolic chaperone (MSF) to target precursors to mitochondrial receptors (Hachiya et al., 1993). 14 PRECURSOR INTERACTION WITH PROTEINACEOUS RECEPTORS ON THE TARGET MEMBRANE In prokaryotes, cytoplasmically-synthesized precursors generally can only be targeted to the plasma membrane, and therefore receptor proteins are functional members of the translocation apparatus, SecA for post-translational targeting (Cunningham and Wickner, 1989; Lill et al., 1990) and SecY for co- tranlsational targeting (Osbourne and Silhavy, 1993). In eukaryotes, precursors synthesized in the cytoplasm have a multitude of different membranes with which they could associate. To aid in the targeting of these precursors to the correct organelle the translocation machinery in these membranes interact with receptor proteins that specifically recognize either the precursors targeting signals or associated chaperones. Receptors are generally not permanently associated with translocation components but rather interact dynamically (Walter and Johnson, 1994; Kiebler et al., 1993). The dynamic interaction of receptors and translocation complexes might help increase the efficiency of targeting and import of precursors across the membrane by releasing receptors to interact with additional precursors while the translocation channel is occupied by translocating the current precursor. Endoplasmic reticulum targeted precursors are recognized by different receptors depending on whether they are co-translationally targeted or post- translationally targeted (Rapoport, 1992). Nascent chain-SRP-ribosome complexes are recognized by a SRP-specific receptor called the signal- recognition-particle-receptor (SR, or docking protein; Connolly and Gilmore, 15 1989). The SR is a heterodimer consisting of two GTPases, SRa, a peripherally associated membrane protein, and SR8, an integral membrane protein. Upon binding of SRP and SR complexes, a GTPase cascade is initiated, leading eventually to interaction of the signal sequence of the precursor with the translocation channel (Gilmore, 1993). In addition to recognition of the SRP by SR, the main ribosome receptor of the endoplasmic reticulum is the translocation channel protein, SecG1 (Kalies et al., 1994). Precursors targeted to the endoplasmic reticulum post-translationally are not recognized by the SRP-receptor (SR; Perara et al., 1986; Schlenstedt et al., 1990). Membrane receptors for the post-translationally targeted precursors have not been identified yet, but candidates for this receptor are members of the Sec62-Sec63 complex (Caplan et al., 1993). This complex consists of the integral membrane proteins Sec62, 8e063, and Sec71, as well as Sec72, a peripheral membrane protein exposed to the cytosol (Feldheim and Schekman, 1994). In mitochondria, precursors are initially recognized by, and bind to, receptor proteins on the outer membrane. Genetic and biochemical studies have identified four outer membrane proteins that are thought to make up these receptors, with two of these, TOM20 and TOM22 (Harkness et al., 1994; Kiebler et al., 1990) making up one heteromeric complex, and the other heteromeric complex consisting of TOM37 and TOM70 (Gratzer et al, 1995; Hines et al., 1990). Interestingly, these receptor proteins appear to interact differently with different precursors. Of the four identified receptor proteins, TOM22 is the only 16 essential receptor (Baker et al., 1990; Lithgow et al., 1994), and can partially substitute for mutations in the other members when over-expressed. PRECURSORS TRAVERSE MEMBRANES IN PROTEINACEOUS TRANSLOCATION CHANNELS Two disparate models were originally proposed for the translocation of precursor proteins across lipid bilayers, one involving the spontaneous insertion of precursors across the membrane, and the second involving the assembly of a proteinaceous channel through which precursor proteins were translocated to the other side of the membrane. Overwhelming experimental evidence now suggests that the latter model of a proteinaceous transport channel is the correct one (For reviews see; Rapoport, 1992; Neupert et al., 1990; Wickner et al., 1991; Schnell, 1995). The best evidence for this was obtained using biophysical techniques to measure the ability of ions of various sizes to quench fluorescently-Iabeled precursors halted in the translocation channel of reticulum- derived vesicles (Crowley et al., 1993). In bacteria, reconstitution of translocation activity in proteo-liposomes indicates that the minimal protein translocation channel consists of a trimeric complex of integral membrane proteins, SecY, SecE, and SecG (Brundage et al., 1990). This trimeric complex interacting with the peripheral-membrane associated SecA ATPase are capable of supporting ATP, and Alum-dependent pre-protein translocation activity comparable to that observed with native plasma 17 membranes (Hartl et al., 1990; Bassilana and Wickner, 1993). In addition to these essential components, additional components SecD, SecF, yajC, and other components have been identified, but their roles in protein translocation through this channel remain unknown. Using genetic selection to obtain yeast mutants that were defective in translocation, Schekman and colleagues isolated conditional mutations in three yeast genes (se061, sec62, and sec63) that cause cytoplasmic accumulation of precursors (Rothblatt et al., 1989). Sec61, Sec62, and Sec63 are integral membrane proteins, and can be found in association with one another as part of a multisubunit complex in the endoplasmic reticulum (Deshaies et al., 1991). Interestingly, both Sec61 and SecY share high amounts of similarity (Hartmann et al., 1994). Because Sec61 is the major cross-linked product at both early and late stages of protein translocation (Sanders et al., 1992; High et al., 1991) into the endoplasmic reticulum this protein has been identified as the putative channel forming protein (Rapoport, 1992). Sec62 and Sec63 also associate to form a protein complex (Deshaies et al., 1991) and can be cross-linked to Sec61 only if cross-linking is performed prior to detergent solubilization, indicating that these proteins probably interact transiently (Sanders and Schekman, 1992). Both Sec62 and Sec63 contain large domains exposed to the cytosol, and thus are though to be involved in early stages of protein import into the endoplasmic reticulum. Support for this model comes from genetic studies in which mutations in either Sec62 or Sec63 inhibit the association of precursor with Sec61 protein (Sanders et al., 1992). 18 In mitochondria, the outer membrane and inner membrane translocation complexes act independently of one another (Segui-Real et al., 1993; Horst et al., 1995). The ability to selectively remove the outer membrane from mitochondria, either by osmotic shock (Eilers and Schatz, 1988) or selective detergent solubilization (Hwang et al., 1989), has enabled the creation of mitoplasts. Import into these organelles, which only have an inner membrane, demonstrate that the inner membrane translocation complex is capable of interacting with precursors and importing them in the absence of outer membrane receptors. In addition, binding and translocation of precursors can be reconstituted with purified outer envelope vesicles (Mayer et al., 1993). Passage of the precursor protein through the inner membrane requires both a membrane potential (AW) across the inner membrane, and ATP hydrolysis in the mitochondrial matrix (Eilers et al., 1987). This translocation event involves a proteinaceous translocation complex which includes several integral inner membrane proteins, TIM23 (Dekker et al., 1993), TIM17 (Kilbrich et al., 1994), and a peripheral membrane protein facing the matrix side of the inner membrane TIM44 (Blom et al., 1993; Horst et al., 1993). In addition to these membrane associated components, the matrix Hsp70 molecular chaperone is involved and is thought to be the ATP-requiring component of the import apparatus. 19 GENERATION OF UNIDIRECTIONAL MOVEMENT THROUGH THE TRANSLOCATION CHANNEL In both the mitochondrial and the endoplasmic reticulum protein transport systems, molecular chaperones of the Hsp70 family appear to play critical roles in the generation of unidirectional movement through the protein translocation channel (Kang et al., 1990; Brodsky and Schekman, 1993). Interestingly, in both these sytems these Hsp70 homologues associate with the translocation apparatus through specific interactions with members of the membrane translocation channels (Sanders et al., 1992; Schneider et al., 1994). In the endoplasmic reticulum, the translocation channel component Sec63 interacts with the endoplasmic reticulum localized Hsp70 (BiP, or Kar2p). In addition to a large cytoplasmic domain, SE063 contains a lumenal segment which shows significant homology to a portion of DnaJ, the Escherichia coli Hsp40 (Feldheim et al., 1992). This so-called “J-domain”'has been implicated as the site of interaction between members of the Hsp70 and Hsp40 molecular chaperones (Cyr and Douglas, 1994). It was therefore predicted that SEC63 would interact with BiP, a eukaryotic Hsp70 chaperone, located in the lumen of the endoplasmic reticulum. Evidence for this association comes from genetic experiments, in which double mutants of BiP and SEC63 were synthetically lethal (Sanders et al., 1992), and isolation of complexes containing both proteins (Brodsky and Schekman, 1993). What role does BiP play in protein Import into the endoplasmic reticulum? At the time of beginning this thesis, this question had not been answered. 20 Because temperature-sensitive mutations of BiP resulted in rapid appearance of translocation defects at non-pennissive temperatures (Vogel et al., 1990), and because this interruption of protein import could be reproduced in vitro as well as in vivo (Sanders et al., 1992), it was thought that BiP may play a direct role in protein import by interacting with the nascent chain of the precursor as it extended into the lumen of the endoplasmic reticulum. However, BiP was not required for protein translocation in a reconstituted translocation system utilizing detergent-solubilized rough-endoplasmic reticulum proteins (Gbrlich and Rapoport, 1993). However, two factors might explain the apparent lack of need for BiP in these import conditions. First, the reconstituted import was co- translational not post-translational and perhaps the components required for post-translational import are different than that for co-translational import. Second, the rates of import were much lower in the reconstituted system than for comparable import experiments utilizing endoplasmic reticulum microsomes, and additional stimulatory factors may be necessary for in vivo import. The matrix-H3p70 is essential for import of precursors into mitochondria (Kang et al., 1990; Gambill et al., 1993; Voos et al., 1993). While passage of the targeting presequence across the inner membrane is the first committed step of translocation across this membrane, it has become apparent that even at this early stage in transport the matrix-Hsp70 plays an important role. When matrix ATP is reduced to low levels but A‘P is maintained across the inner membrane, not only full translocation of the precursor but also passage of the transit peptide and its subsequent processing by the matrix-processing-peptidase is inhibited 21 (Cyr et al., 1993). To be chased into the matrix, these precursors required not only matrix ATP, but also A‘P across the inner membrane. Presequence translocation across the inner membrane appears to require A‘P, however in the absence of matrix ATP is a reversible process. While translocation of the presequence of the precursor requires a ALP across the inner membrane of the mitochondria, completion of translocation after this event has occurred requires only ATP (Stuart et al., 1994). This ATP- requirement is thought to reflect the ATP-dependent action of the matrix-Hsp70. This molecule is proposed to be the “motor” that drives unidirectional import across the mitochondrial membranes (Stuart et al., 1994). Several studies have shown that the matrix-Hsp70 binds to TIM44 in an ATP—dependent manner (Schneider et al., 1994; Kronidou et al., 1994; Rassow et al., 1995). It is thought that by associating with the membrane-associated TIM44 the matrix-Hsp70 is present to interact immediately with the precursor as it emerges from the translocation channel in the inner membrane (Schneider et al., 1994). In bacteria, translocation of precursors into the periplasmic space is accomplished by generation of a pushing force by the molecule SecA rather than a pulling force as seen in both the endoplasmic reticulum and mitochondria. Presumably, this difference reflects the need to maintain metabolic energy in the cytoplasmic compartment of the bacteria. Again, both membrane potential, in this case ApH+ and ATP hydrolysis are necessary for different steps in the translocation process. ApH-I- is required for insertion of the signal sequence into 22 the translocation channel and subsequent translocation is supported by ATP- hydrolysis by SecA (Wickner et al., 1991). SecA is a peripheral subunit of the bacterial translocase, and can be found in both the cytoplasm and tightly associated with the plasma membrane. The cytosolic form of SecA functions as a repressor of its own synthesis (Rollo at al., 1988), and may also function as a molecular chaperone (McFarland at al., 1993). The SecA protein binds with low affinity to lipids (Lill et al., 1990), but demonstrates a high affinity binding for the SecYEG complex when acidic phospholipids are present (Douville et al., 1995). Interestingly, while SecA does not contain traditional transmembrane domains, the binding of precursor and ATP drives a portion of this protein across the plasma membrane (Brundage et al., 1990; Kim et al., 1994). Associated precursor protein accompanies the SecA domain as it is driven across the membrane resulting in the stepwise translocation of roughly 20-25 amino acid residues per ATP hydrolysis (Hendrick and WIckner, 1991; Joly and Wickner, 1993). MOLECULAR CHAPERONES: PUSHING PRECURSORS INTO, AND PULLING PRECURSORS OUT OF, PROTEIN TRANSLOCATION CHANNELS While it is clear from the above discussion that the protein translocation systems of the bacterial plasma membrane, the endoplasmic reticulum, and the mitochondria display enormous functional diversity, utilizing a diverse array of proteins and energy sources to accomplish protein transport, several overlying 23 themes emerge from this complexity. First, secretion in both the bacterial and eukaryotic systems appear to have conserved co-translational elements, such as the members of the signal-recognition particle, as well as members of the translocation channel, Sec61/SecY. The second overlying theme is the ubiquitous presence of molecular chaperones in these protein translocation systems. These molecular chaperones are involved in multiple aspects in each of these translocation systems. In both bacterial and eukaryotic secretion systems, as well as the mitochondrial protein import system, these proteins are involved in the targeting of precursors to the translocation apparatus. Additionally, members of chaperone families localized to the interior of both the endoplasmic reticulum and mitochondria play essential roles in the generation of unidirectional transport of precursors into these organelles. This occurs despite the lack of obvious conservation between these respective protein translocation systems. The obvious question raised by this observation is, do molecular chaperones then also play a role in the import of proteins into the chloroplast? Before discussing this however, it is relevant to discuss the nature and activities of this class of proteins. 24 MOLECULAR CHAPERONES: DEFINITION OF ACTIVITY At a very early point in the evolution of living organisms, a series of structurally unrelated proteins were developed to regulate the interactions of the various proteins within the cytoplasm of the cell. These abundant proteins, which regulate many different activities in the cell, have been named molecular chaperones (Ellis, 1989). Essentially, molecular chaperone activity is based on a selective affinity for non-native protein conformations. This is generally characterized by recognition of exposed hydrophobic patches on target proteins by the molecular chaperone. As many of the molecular chaperones were initially identified by their induction in cells exposed to heat stress they are named heat shock proteins and the families are distinguished by their relative molecular weights, i.e. heat-shock protein 60 (Hsp60), heat-shock protein 70 (Hsp70), or heat-shock protein 100 (Hsp100; protein families of 60, 70, or 100 kDas respectively). However, in addition to being induced in various stress conditions most molecular chaperone families are also required in normal growth conditions, and perform essential functions for the maintenance of cellular viability (Gething and Sambrook, 1992). The ability of molecular chaperones to interact transiently with many different proteins has allowed them to fulfill many diverse functions in the cell. These activities run the full range of protein homeostasis, from association with nascent polypeptides as they extend from the translating ribosome (Nelson et al., 1992) and subsequent folding of these newly synthesized proteins to the 25 regulated association and disassociation with protein complexes in protein translocation and signal transduction machinery, to finally the repair or degradation of damaged proteins (Hendrick and Hartl, 1993; Gething and Sambrook, 1992). How can such a diverse set of functions be attributed to a relatively small number of molecular chaperone types? In many cases a single family of molecular chaperones appears to play roles in many different functions (Gething and Sambrook, 1992). As more is understood about how molecular chaperones are involved in these different processes several basic characteristics of molecular chaperone function are becoming clear. First, all molecular chaperones interact with unfolded or misfolded polypeptides, but the mechanisms of these interactions differ between chaperone families. Therefore, the molecular chaperone families are specialized in this fashion. For example, chaperones of the Hsp70 family preferentially interact with unfolded proteins in a fully extended conformation, whereas Hsp60 family members prefer partially folded alpha-helical structures, and Hsp40 family members prefer folded proteins with exposed hydrophobic patches (Langer et al., 1992). This differential recognition of substrates is the basis by which different molecular chaperones are limited to specific tasks in the cellular environment— i.e. Hsp70s will not detect certain substrates because they do not display the right type of conformation, but these proteins may be recognized by Hsp60s. An important point to note however, is that while different molecular chaperone 26 families play important roles in different cellular processes, in many cases these roles overlap. Hsp70 and Hsp100 families are both capable of initiating DNA replication by disassembling inactive replication complexes (Wickner et al., 1994; Wawrzynow et al., 1995). Hsp70 and Hsp60 families both shuttle precursor proteins to the plasma membrane for secretion in Escherichia coli. Second, in many cases, there are multiple members of a particular chaperone family which may play different roles in cellular metabolism. In eukaryotic cells this is particularly evident as each intracellular compartment appears to contain its own Hsp70 chaperone member (Gething and Sambrook, 1992). Third, molecular chaperones act as molecular machines in the cooperation with partner proteins. Molecular chaperones cooperate with one another to perform specific tasks within the cell. For example, during translation Hsp70$ interact with proteins as they are exposed as nascent chains from the translating ribosome (Nelson et al., 1992). Hsp70$ affinity for fully unfolded protein conformations allows it to interact, and stabilize proteins at this stage. In cooperation with Hsp40 this bound protein can then either be folded, or delivered to the Hsp60 chaperone for folding (Langer et al., 1992). A single molecular chaperone may be involved in multiple cellular processes depending on its partner proteins. Hsp100 chaperones act in degradation of associated proteins if they are associated with a protease subunit, but when deprived of this protease subunit as in the eukaryotic cytoplasm of yeast have folding/renaturation activity (Sanchez et al., 1992). 27 Hsp70 in the matrix of mitochondria act in translocation when associated with translocation components in the mitochondrial membrane, but deliver misfolded proteins to the matrix localized Ion protease, and act to fold protein depending on their protein partners (Manning-Krieg et al., 1991). THE CHLOROPLAST STROMA CONTAINS HOMOLOGUES OF SEVERAL MOLECULAR CHAPERONE FAMILIES Chloroplasts like other eukaryotic organelles contain their own requisite complement of molecular chaperones (VIerIing, 1991). The first of these to be identified was a member of the Hsp60 molecular chaperone family, also known as the GroEL chaperonins (Barraclough and Ellis, 1980; Ellis, 1988). These molecular chaperones have been identified in all eubacteria and are also found in those eukaryotic organelles that evolved from the endosymbiosis of prokaryotes. In all cases these Hsp60 family members are found associated with their co-chaperones, the Hsp10s or GroES chaperonins. These two proteins associate into large complexes and the presence of both subunits are required for folding activity (Gething and Sambrook, 1992). This protein was originally identified by the observation that prior to their assembly into holo- enzymes, Rubisco large subunits transiently associated with an additional stromal protein. From these observations it was hypothesized that the Rubisco large subunits were maintained in a folding-competent state by the plastid 28 Hsp60/10 complex until their association with Rubisco small subunit, a nuclear- encoded protein that must first be imported into the chloroplast from the cytoplasm (Chua and Schmidt, 1978). In addition to its interaction with the Rubisco large subunit, plastid Hsp60/10 also associates with other proteins imported into the chloroplast. The plastid Hsp60/10 chaperone has been identified in transient association with several different newly-imported proteins (Lubben et al., 1989; Maduetlo et al., 1993; Tsugeki and Nishimura, 1993). These observations have led to the hypothesis that the Hsp60/10 complex may be involved in the folding of these proteins in the stromal after their emergence from the translocation apparatus. Molecular chaperones of the Hsp70 family were first identified in chloroplasts by Marshall at al., 1990. In this study, immunoblotting techniques with antibodies raised to either prokaryotic or eukaryotic forms of Hsp703 were used to identify chloroplastic homologues. In addition to a major soluble Hsp70 homologue found in the stroma, possibly two other chloroplast-specific Hsp70 homologues located in the outer envelope membrane, and stroma were identified. Of these Hsp70$ only the major stromal form has been cloned, and Shown to be related to the prokaryotic Hsp70, DnaK (Marshall and Keegstra, 1992). The functions of these plastidic Hsp70 homologues has remained largely unknown. Recent studies have identified stromal complexes between newly- imported precursors and Hsp70s, and the stromal Hsp70 has been implicated in integration of several thylakoid membrane proteins (Madueho et al., 1993; 29 Tsugeki and Nishimura, 1993). Additionally, isolated translocation complexes from purified outer membranes displayed reactivity to antibodies against Hsp70 homologues, implying a possible role for the outer membrane Hsp70 in protein translocation (Soll and Waegemann, 1992). Finally, stromal members of the Hsp100 molecular chaperone family have been identified. The function of these proteins in any organism remains largely unknown. Originally identified as members of an ATP—dependent caseinolytic protease, these proteins are also commonly referred to as Clp proteins (Qaseinolytic protease). The caseinolytic protease consists of two distinct complexes, one containing the Hsp100 molecular chaperones, and the other containing the protease ClpP (Qaseinolytic protease Protease). Members of the Hsp100 protein family have recently been added to the long list of molecular chaperones, and can act independently from their proteolytic counterpart ClpP as ATP-dependent chaperones (Wickner et al., 1994; Wawrzynow et al., 1995). Interestingly, the chloroplastic Hsp100, called ClpC, has been independently isolated three times as an inner membrane associated protein (Moore and Keegstra, 1993; K0 et al., 1994; N. Hoffman personal communication to K. Keegstra). Subsequent in vitro import of this protein into isolated chloroplasts indicated this protein localized primarily to the stromal compartment however, and it remains to be seen if the interaction of ClpC with the chloroplastic inner envelope membrane is specific or a result of stromal contamination of isolated membrane fractions. 30 DO STROMAL CHAPERONES PLAY A ROLE IN PROTEIN IMPORT INTO CHLOROPLASTS? In both mitochondria and the endoplasmic reticulum, a class of proteins termed molecular chaperones have been shown to play important roles in the translocation processes of these two organelles. Additionally, molecular chaperones have been identified as important in delivery and movement of precursors across the bacterial plasma membrane. The function of these proteins in the import process has been proposed based on the predicted unfolded nature of the translocating precursors in all these systems, and are thought to provide unidirectional movement through the protein translocation channel The stroma of chloroplasts contain members of several molecular chaperone families (Barraclough and Ellis, 1980; Marshall at al., 1990; Moore and Keegstra, 1993). In many cases these proteins have already been shown to interact with newly imported proteins (Lubben et al., 1989; Madueflo et al., 1991; Nishimura and Tsugeki, 1991). Given their importance in the protein translocation mechanisms of other systems, and their presence in chloroplasts, there seemed to be a reasonable chance that one or more of these proteins might have a role in chloroplastic protein translocation. 31 STATEMENT OF PROBLEM AND ATTRIBUTION The majority of proteins localized to the chloroplastic interior are encoded by nuclear genes and translated on cytoplasmic ribosomes. These proteins must then be recognized by specific receptors in the outer envelope membrane of chloroplasts and translocated across the chloroplastic envelope membranes en route to their correct destinations inside chloroplasts. This process of recognition and translocation is carried out by a general protein import apparatus consisting of an undefined number of protein components. When I began my dissertation research, two protein components of the chloroplastic protein translocation apparatus, T0075 and Toc86, had recently been identified (Perry and Keegstra, 1994). As organellar molecular chaperones were known to be essential components of other protein transport systems (Kang et al., 1990; Brodsky et al., 1992), the initial goal of my dissertation research was to determine whether stromal molecular chaperones played a role in chloroplastic protein import. The rationale was that if stromal molecular chaperones were found in chloroplastic protein translocation complexes roles for these proteins in import could be proposed based on previously identified activities of molecular chaperones in other protein import systems. As more protein components of the chloroplastic import apparatus are identified and their cDNAs cloned, it will become essential to address their functions in the import process. Identification of translocation components, such as chaperones, with defined activities will aid in the assignment of functions to protein translocation components. 32 Chapter 2 describes the identification and partial characterization of detergent solubilized protein translocation components formed in limiting ATP concentrations. The experiments presented in this chapter were previously published as part of a manuscript [Nielsen, E., Akita, M., Davila-Aponte, J., and Keegstra, K. (1997) EMBO J., 16:635-646]. I wrote this manuscript, and performed all the experiments presented. Jennifer Davila—Aponte prepared antibodies against Toc75, and Mitsuru Akita contributed scientifically through sharing results of a parallel investigation of protein translocation complexes by chemical—crosslinking. Chapter 3 describes the evaluation of the roles of three different stromal molecular chaperones in chloroplastic protein import. I performed all the experiments in this chapter. Panels A and B of Figure 3.3, and Figure 3.5 were previously published as part of a manuscript [Nielsen, E., Akita, M., Davila- Aponte, J., and Keegstra, K. (1997) EMBO J., 162635-646]. The remainder of this chapter has not been previously published. Some of the information in this chapter may form the basis of a future published manuscript, in which case I will be listed either as an author or co-author. Chapter 4 describes the initial characterization of the manner by which ClpC interacts with translocation complexes and chloroplastic envelope membranes. I performed all the experiments in this chapter. Figures 4.1 and 4.2 were previously published as part of a manuscript [Nielsen, E., Akita, M., DaviIa-Aponte, J., and Keegstra, K. (1997) EMBO J., 162635-646]. The other results have not been previously published. Some of the information in this 33 chapter may form the basis of a future published manuscript, in which case I will be listed either as an author or co-author. Conclusions and possible avenues for future research are discussed in Chapter 5. In addition to the results presented in this thesis dissertation, l have been listed as a co-author on other publications: Based on a collaboration with members of the laboratory of Jilrgen Soll in Kiel, Germany I was listed as a co-author on a published manuscript [Ltibeck, J., Soll, J., Akita, M., Nielsen, E., and Keegstra, K. (1996) EMBO J., 15:4230-4238]. I provided evidence that precursors were coimmunoprecipitated with Tic110 immunoprecipitated complexes, and was involved in preparation of the manuscript. Based on a collaboration with Mitsuru Akita l was listed as a co-author on a published manuscript [Akita, M., Nielsen, E., and Keegstra, K. (1997) J. Cell Biol., 136:983-994]. I provided antibodies against the stromal Hsp70, S78, and Tic110, and contributed scientifically by sharing results of my parallel investigation of chloroplastic translocation complexes using mild detergent solubilization techniques. REFERENCES Baker, K.P., Schaniel, A., Vestweber, D. and Schatz, G. (1990) A yeast mitochondrial outer membrane protein essential for protein import and cell viability. Nature, 348, 605-609. Barraclough, R. and Ellis, R.J. (1980) Protein synthesis in chloroplasts IX. Assembly of newly-synthesized large subunits into ribulose bisphosphate carboxylase in isolated intact pea chloroplasts. BBA, 608, 19-31. Bassilana, M., and Wickner, W. (1993) Purified Escherichia coli preprotein translocase catalyzes multiple cycles of precursor protein translocation. Biochemistry, 32, 26262630. Baurle, C., Dorl, J., and Keegstra, K. (1991) Kinetic analysis of the transport of thylakoid lumenal proteins in experiments using intact chloroplasts. J. Biol. Chem, 266, 5884-5890. Blom, J., Kiibrich, M., Rassow, J., Voos, W., Dekker, P.J.T., Maarse, A.C., Meijer, M. and Pfanner, N. (1993) The Essential Yeast Protein MIM44 (encoded by MPI1) Is Involved in an Early Step of Preprotein translocation across the Mitochondrial Inner Membrane. Mol. Cell. Biol, 13, Brodsky, J.L. and Schekman, R. (1993) A Sec63p-BiP complex from yeast is required for protein translocation in a reconstituted proteoliposome. J. Cell Biol, 123, 1355-1363. Brundage, L., Hendrick, J.P., Schiebel, E., Driessen, A.J.M. and Wickner, VII. (1990) The purified E. coli integral membrane protein SecY/E is sufficient for reconstitution of SecA-dependent precursor protein translocation. Cell, 62, 649-657. Caplan, A.J., Cyr, 0M. and Douglas, MG. (1993) Eukaryotic homologues of Escherichia coli dnaJ: A diverse protein family that functions with HSP70 stress proteins. Mol. Biol. Cell, 4, 555-563. Cline, K., Werner-Washbburne, M., Lubben, T., and Keegstra, K. (1985) Precursors to two nuclear-encoded chloroplast proteins bind to the outer envelope before being imported into chloroplasts. J. Biol. Chem, 260, 3691- 3696. 35 Connolly, T. and Gilmore, R. (1989) The signal recognition particle receptor mediates the gtp-dependent displacement of srp from the signal sequence of the nascent polypeptide. Cell, 57, 599-610. Comwell, K., and Keegstra, K. (1987) Evidence that a chloroplast surface protein is associated with a specific binding site for the precursor to the small subunit of ribulose 1,5-biscarboxylase. Plant Physiol, 85, 780-785. Crowley, K.S., Reinhart, 6.0., and Johnson, A.E. (1993) The signal sequence moves through a ribosomal tunnel into a noncytoplasmic aqueous environment at the ER membrane early in translocation. Cell, 73, 1101-1115. Cunningham, K., Lill, R., Crooke, E., Rice, M., Moore, K., Wickner, W., and Oliver, D. (1989) SecA protein, a peripheral protein of the Escherichia coli plasma membrane, is essential for the functional binding and translocation of proOmpA. EMBO J., 8, 955-959. Cyr, D.M. and Douglas, MG. (1994) Differential regulation of Hsp70 subfamilies by the eukaryotic DnaJ homologue YDJ1. J. Biol. Chem, 269, 9798-9804. Cyr, D.M., Stuart, R.A. and Neupert, W. (1993) A matrix ATP requirement for presequence translocation across the inner membrane of mitochondria. J. Biol. Chem, 268, 23751-23754. Davis, S.J., Schockmel, G.A., Somoza, C., Buck, D.W., Healey, D.G., Rieber, E.P., Reiter, C., and Williams, A.F. (1992) Antibody and HIV-1 gp120 recognition of CD4 undermines the concept of mimicry between antibodies and receptors. Nature, 358, 76-79. De Boer, AD, and Weisbeek, P.J. (1991) Chloroplast protein topogenesis: import, sorting and assembly. Biochim. Biophys. Acta, 1071, 221-253. Dekker, P.J.T., Keil, P., Rassow, J., Maarse, A.C., Pfanner, N. and Meljer, M. (1993) Identification of MIM23, a putative component of the protein import machinery of the mitochondrial inner membrane. FEBS Lett, 330, 66-70. Deshales, R.J., Koch, B.D., Werner-Washburne, M., Craig, EA. and Schekman, R. (1988) A subfamily of stress proteins facilitates translocation of secretory and mitochondrial precursor polypeptides. Nature, 332, 800-805. Deshaies, R.J., Sanders, S.L., Feldheim, DA. and Schekman, R. (1991) Assembly of yeast Sec proteins involved in translocation into the endoplasmic reticulum into a membrane-bound multisubunit complex. Nature, 349, 806-808. 36 Douville, K., Price, A., Elchler, J., Economou, A., and Wickner, W. (1995) SecYEG and SecA are the stoichiometric components of preprotein translocase. J. Biol. Chem, 270, 20106-20111. Eilers, M., and Schatz, G. (1986) Binding of a specific ligand inhibits import of a purified precursor protein into mitochondria. Nature, 322, 228-232. Ellers, M., Oppliger, W. and Schatz, G. (1987) Both ATP and an energized inner membrane are required to import a purified precursor protein into mitochondria. EMBO J., 6, 1073-1077. Ellers, M. and Schatz, G. (1988) Protein unfolding and the energetics of protein translocation across biological membranes. Cell, 52, 481-483. Ellis, JR. (1981) Chloroplast proteins: synthesis, transport and assembly. Annu. Rev. Plant Physiol, 32, 1 1 1-137. Ellis, R.J. and Hemmingsen, SM. (1989) Molecular chaperones: Proteins essential for the biogenesis of some macromolecular structures. TIBS, 14, 339-342. Ellis, R.J. and van der Vies, SM. (1988) The rubisco binding protein. Photosynth. Res, 1 6, 101 -1 15. Feldhelm, D., Rothblatt, J. and Schekman, R. (1992) Topology and functional domains of Sec63p. an endoplasmic reticulum membrane protein required for secretory protein translocation. Mol. Cell. Biol, 12, 3288-3296. Fliigge, U-I., Fischer, K., Gross, A., Sebald, W., Lottspeich, F., and Eckerskom, C. (1989) The triose phosphate-3-phosphoglycerate-phosphate translocator from spinach chloroplasts: Nucleotide sequence of a full length cDNA clone and import of the in vitro synthesized precursor protein into chloroplasts. EMBO J., 8, 39-46. FIiIgge, U-I., Weber, A., Fischer, K., Lottspeich, F., Eckerskorn, E., Waegemann, K., and Soll, J. (1991) The major chloroplast envelope polypeptide is the phosphate translocator and not the protein import receptor. Nature, 353, 364-367. Friedman, A.L. and Keegstra, K. (1989) Chloroplast protein import: quantitative analysis of receptor mediated binding. Plant Physiol, 89, 993-999. Gambill, B.D., Voos, W., Kang, P.J., Miao, B., Langer, T., Craig, EA. and Pfanner, N. (1993) A dual role for mitochondrial heat shock protein 70 in membrane translocation of preproteins. J. Cell Biol, 123, 109-117. 37 Gething, M.J. and Sambrook, J. (1992) Protein folding in the cell. Nature, 355, 33-45. Gilmore, R. (1993) Protein translocation across the endoplasmic reticulum: A tunnel with toll booths at entry and exit. Cell, 75, 589-592. Gbrlich, D., and Rapoport, TA. (1993) Protein translocation into proteoliposomes reconstituted from purified components of the endoplasmic reticulum membrane. Cell, 75, 615-630. Gratzer, S., Lithgow, T., Bauer, R.E., Lamping, E., Paltauf, F., Kohlwein, S.D., Haucke, V., Junne, T., Schatz, G. and Horst, M. (1995) Mas37p, a novel receptor subunit for protein import into mitochondria. J. Cell Biol, 129, 25-34. Hachiya, N., Alam, R., Sakasegawa, Y., Sakaguchi, M., Mihara, K. and Omura, T. (1993) A mitochondrial import factor purified from rat liver cytosol is an ATP-dependent conformational modulator for precursor proteins. EMBO J., 12, 1579-1586. Hardy, S.J.S. and Randall, LL. (1991) A kinetic partitioning model of selective binding of nonnative proteins by the bacterial chaperone SecB. Science, 251, 439-443. Harkness, T.A.A., Nargang, F.E., Van der Klei, I., Neupert, W. and Lill, R. (1994) A crucial role of the mitochondrial protein import receptor MOM19 for the biogenesis of mitochondria. J. Cell Biol, 124, 637-648. Hartmann, E., Sommer, T., Prehn, S., Gdrlich, D., Jentsch, S. and Rapoport, TA. (1994) Evolutionary conservation of components of the protein translocation complex. Nature, 367, 654-657. Hartl, F-U., Locker, S., Schiebel, E., Hendrick, J.P., and Wickner, W. (1990) The binding cascade of SecB to SecA to SecY/E mediates preprotein targeting to the E. coli plasma membrane. Cell, 63, 269-279. Hendrick, J.P. and Hartl, F.-U. (1993) Molecular chaperone functions of heat-shock proteins. Ann. Rev. Biochem, 62, 349-384. Hendrick, J.P., and Wickner, W. (1991) SecA protein needs both acidic phospholipids and SecY/E protein for functional high-affinity binding to the Escherichia coli plasma membrane. J. Biol. Chem, 266, 24596-24600. High, 3., Gtirlich, D., Wiedmann, M., Rapoport, TA. and Dobbersteln, B. (1991) The identification of proteins in the proximity of signal-anchor sequences during their targeting to and insertion into the membrane of the ER. J. Cell Biol, 113, 35-44. 38 Hines, V., Brandt, A., Griffiths, G., Horstmann, H., Brtitsch, H. and Schatz, G. (1990) Protein import into yeast mitochondria is accelerated by the outer membrane protein MAS70. EMBO J., 9, 3191-3200. Hirsch, S., Muckel, E., Heemeyer, F., von Heijne, G. and Soll, J. (1994) A receptor component of the chloroplast protein translocation machinery. Science, 266, 1989-1992. Horst, M., Hilfiker-Rothenfluh, 8., Oppliger, W. and Schatz, G. (1995) Dynamic interaction of the protein translocation systems in the inner and outer membranes of yeast mitochondria. EMBO J., 14, 2293-2297. Horst, M., Jena, P., Kronidou, N.G., Bolllger, L., Oppliger, W., Scherer, P., Manning-Krieg, U., Jascur, T. and Schatz, G. (1993) Protein import into yeast mitochondria: The inner membrane import site protein ISP45 is the MPI1 gene product. EMBO J., 12, 3035-3041. Hwang, S., Jascur, T., Vestweber, D., Pon, L. and Schatz, G. (1989) Disrupted yeast mitochondria can import precursor proteins directly through their inner membrane. J. Cell Biol, 109, 487-493. Joly, J.C. and Wickner, W. (1993) The SecA and SecY subunits of translocase are the nearest neighbors of a translocating preprotein, shielding it from phospholipids. EMBO J., 12, 255-263. Kalies, K.U., Gbrlich, D., and Rapoport, TA (1994) Binding of ribosomes to the rough endoplasmic reticulum mediated by the Sec61p-complex. J. Cell Biol, 126, 925-934. Kang, P.-J., Ostennann, J., Shilling, J., Neupert, W., Craig, EA. and Pfanner, N. (1990) Requirement for hsp70 in the mitochondrial matrix for translocation and folding of precursor proteins. Nature, 348, 137-143. Kessler, F., Blobel, G., Patel, HA. and Schnell, DJ. (1994) Identification of two GTP-binding proteins in the chloroplast protein import machinery. Science, 266, 1035—1039. Kessler, F. and Blobel, G. (1996) Interaction of the protein import and folding machineries in the chloroplast. Proc. Natl. Acad. Sci. USA, 93, 7684-7689. Kiebler, M., Becker, K., Pfanner, N. and Neupert, W. (1993) Mitochondrial protein import: Specific recognition and membrane translocation of preproteins. J. Membr. Biol, 135, 191-207. Kiebler, M., Pfaller, R., Sbllner, T., Griffiths, G., Horstmann, H., Pfanner, N. 39 and Neupert, W. (1990) Identification of a mitochondrial receptor complex required for recognition and membrane insertion of precursor proteins. Nature, 348, 610-616. Kim, Y.J., Rajapandi, T., and Oliver, D. (1994) SecA protein is exposed to the periplasmic surface of the E. coli inner membrane in its active state. Cell, 78, 845-53. Ko, K., Bornemisza, C., Kourtz, L., Ko, Z.W., Plaxton, W.C. and Cashmere, AR. (1992) Isolation and characterization of a cDNA clone encoding a cognate 70-kDa heat shock protein of the chloroplast envelope. J. Biol. Chem, 267, 2986-2993. Ko, K., Doung, C. and Ko, Z.W. (1994) Nucleotide sequence of a Brassica napus Clp homolog. Plant Physiol, 104, 1087-1089. Kouranov, A. and Schnell, D.J. (1996) Protein translocation at the envelope and thylakoid membranes of chloroplasts. J. Biol. Chem, 271, 31009-31012. Kronidou, N.G., Oppliger, W., Bolliger, L., Hannavy, K., Glick, B.S., Schatz, G. and Horst, M. (1994) Dynamic interaction between lsp45 and mitochondrial hsp70 in the protein import system of the yeast mitochondrial inner membrane. Prec. Natl. Acad. Sci. USA, 91, 12818-12822. Kusukawa, N., Yura, T., Ueguchi, C., Akiyama, Y., and Ito, K. (1989) Effects of mutations in heat—shock genes groES and groEL on protein export in Escherichia coli. EMBO J., 8, 3517-3521. Ktibrich, M., Keil, P., Rassow, J., Dekker, P.J.T., Blom, J., Meijer, M. and Pfanner, N. (1994) The polytopic mitochondrial inner membrane proteins MIM17 and MIM23 operate at the same preprotein import site. FEBS Lett, 349, 222-228. Lamppa, G.K. (1988) The chlorophyll alb binding protein inserts into the thylakoids independent of its cognate transit peptide. J. Biol. Chem, 263, 14996-14999. Langer, T., Lu, C., Echols, H., Flanagan, J., Hayer, M.K., and Hartl, F-U. (1992) Successive action of DnaK, DnaJ and GroEL along the pathway of chaperone-mediated protein folding. Nature, 356, 683-689. Leheny, EA. and Theg, S.M. (1994) Apparent inhibition of chloroplast protein import by cold temperatures is due to energetic considerations not membrane fluidity. Plant Cell, 6, 427-437. 40 Li, H-m., Moore, T., and Keegstra, K. (1991) Targeting of proteins to the outer envelope membrane uses a different pathway than transport into chloroplasts. Plant Cell, 3, 709-717. Lill, R., Dowhan, W. and Wickner, W. (1990) The ATPase activity of SecA is regulated by acidic phospholipids, SecY, and the leader and mature domains of precursor proteins. Cell, 60, 271-280. Lithgow, T., Junne, T., Suda, K., Gratzer, S. and Schatz, G. (1994) The mitochondrial outer membrane protein Ma822p is essential for protein import and viability of yeast. Proc. Natl. Acad. Sci. USA, 91, 11973-11977. Lubben, T.H., Donaldson, G.K., Viitanen, PM and Gatenby, A.A. (1989) Several proteins imported into chloroplasts form stable complexes with the GroEL-related chloroplast molecular chaperone. Plant Cell, 1, 1223-1230. Luirink, J., ten Hagen-Jongman, C.M., van der Weijden, C.C., Oudega, B., High, 8., Dobberstein, B., and Kusters, R. (1994) An alternative protein targeting pathway in Escherichia coli: studies on the role of FtsY. EMBO J., 13, 2289-2296. Lllbeck, J., Soil, J., Akita, M., Nielsen, E. and Keegstra, K. (1996) Topology of IEP110, a component of the chloroplast protein import machinery present in the inner envelope membrane. EMBO J., 15, 4230-4238. Ma, Y., Kouranov, A., LaSaIa, S. and Schnell, D.J. (1996) Two components of the chloroplast protein import apparatus, IAP86 and IAP75, interact with the transit sequence during the recognition and translocation of precursor proteins at the outer envelope. J. Cell Biol, 134, 315-327. Maduefio, F., Napier, J.A. and Gray, J.C. (1993) Newly imported Rieske iron-sulfur protein associates with both Cpn60 and Hsp70 in the chloroplast stroma. Plant Cell, 5, 1865-1876. Manning-Krieg, U.C., Scherer, PE. and Schatz, G. (1991) Sequential action of mitochondrial chaperones in protein import into the matrix. EMBO J., 10, 3273-3280. Marshall, J.S., DeRocher, A.E., Keegstra, K. and Vierling, E. (1990) Identification of heat shock protein hsp70 homologues in chloroplasts. Proc. Natl. Acad. Sci. USA, 87, 374-378. Marshall, J.S. and Keegstra, K. (1992) Isolation and characterization of a cDNA clone encoding the major Hsp70 of the pea chloroplastic stroma. Plant Physiol, 100, 1048-1054. 41 Mayer, A., Lill, R. and Neupert, W. (1993) Translocation and Insertion of Precursor Proteins into Isolated Outer Membranes of Mitochondria. J. Cell Biol, 121, 1233-1243. McFarland, L., Francetic, 0., and Kumamoto, C.A. (1993) A mutation of Escherichia coli SecA protein that partially compensates for the absence of SecB. J. Bacteriol, 175, 2255-2262. Miller, J.D., Bernstein, H.D., and Walter, P. (1994) Interaction of E. coli thl4.5S ribonucleoprotein and FtsY mimics that of mammalian signal recognition particle and its receptor. Nature, 367, 657-659. Moore, T. and Keegstra, K. (1993) Characterization of a cDNA clone encoding a chloroplast-targeted Clp homologue. Plant Mol. Biol, 21, 525-537. Nelson, R.J., Ziegelhoffer, T., Nicolet, C., Werner-Washbume, M. and Craig, EA. (1992) The translation machinery and 70 Rd heat shock protein cooperate in protein synthesis. Cell, 71, 97-105. Neupert, W., Hartl, F-U., Craig, EA, and Pfanner, N. (1990) How do polypeptides cross the mitochondrial membranes? Cell, 63, 447-450. Oblong, J.E., and Lamppa, G.K. (1992) Precursor for the light-harvesting chlorophyll alb-binding protein synthesized in Escherichia coli blocks import of the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase. J. Biol. Chem, 267, 14328-14334. Ogg, S.C., Poritz, M.A., and Walter, P. (1992) Signal recognition particle receptor is important for cell growth and protein secretion in Sacchammyces cerevisiae. Mol. Biol. Cell, 3, 895-911. Olsen, L.J., Theg, S.M., Selman, B.R., and Keegstra, K. (1989) ATP is required for the binding of precursor proteins to chloroplasts. J. Biol. Chem, 264, 6724-6729. Osborne, R.S., and Silhavy, T.J. (1993) PrIA suppressor mutations cluster in regions corresponding to three distinct topological domains. EMBO J., 12, 3391- 3398. Paln, D., and Blobel, G. (1987) Protein import into chloroplasts requires chloroplast ATPase. Proc. Natl. Acad. Sci. USA, 84, 3288-3292. Pain, D., Kanwar, Y.S., and Blobel, G. (1988) Identification of a receptor for protein import into chloroplasts and its localization to envelope contact zones. Nature, 347, 444-449. 42 Perara, E., Rothman, RE. and Lingappa, V.R. (1986) Uncoupling translocation from translation: implications for transport of proteins across membranes. Science, 232, 348-352. Perry, S.E., Buvinger, W.E., Bennett, J., and Keegstra, K. (1991) Synthetic analogues of a transit peptide inhibit binding or translocation of chloroplastic precursor proteins. J. Biol. Chem, 266, 11882-11889. Perry, S.E., and Keegstra, K. (1994) Envelope membrane proteins that interact with chloroplastic precursor proteins. Plant Cell, 6, 93-105. Phillips, G.J., and Silhavy, T.J. (1990) Heat-shock proteins DnaK and GroEL facilitate export of LacZ hybrid proteins in E. coli. Nature, 344, 882-884. Rapoport, TA. (1992) Transport of proteins across the endoplasmic reticulum membrane. Science, 258, 931-936. Rassow, J., Hartl, F.-U., Guiard, B., Pfanner, N. and Neupert, W. (1990) Polypeptides traverse the mitochondrial envelope in an extended state. FEBS Lett, 275, 190-194. Rassow, J., Maarse, A.C., Krainer, E., Kubrlch, M., Muller, H., Meijer, M., Craig, EA. and Pfanner, N. (1994) Mitochondrial Protein Import: Biochemical and Genetic Evidence for Interaction of Matrix hsp70 and the Inner Membrane Protein MIM44. J. Cell Biol, 127, 1547-1556. Reid, G.A., and Schatz, G. (1982) Import of proteins into mitochondria. Extramitochondrial pools and post-translational import of mitochondrial protein precursors in vivo. J. Biol. Chem, 257, 13062-13067. Rollo, E.E., and Oliver, 0.8. (1988) Regulation of the Escherichia coli secA gene by protein secretion defects: analysis of secA, secB, secD, and secY mutants. J. Bacteriol, 170, 3281-3282. Rothblatt, J.A., Deshaies, R.J., Sanders, S.L., Daum, G. and Schekman, R. (1989) Multiple genes are required for proper insertion of secretory proteins into the endoplasmic reticulum in yeast. J. Cell Biol, 109, 2641-2652. Sanchez, Y., Taulien, J., Borkovich, K.A. and Lindquist, S. (1992) Hsp104 is required for tolerance to many forms of stress. EMBO J., 11, 2357-2364. Sanders, S.L. and Schekman, R. (1992) Polypeptide translocation across the endoplasmic reticulum membrane. J. Biol. Chem, 267, 13791-13794. Sanders, S.L., Whitfield, K.M., Vogel, J.P., Rose, MD. and Schekman, R.W. (1992) Sec61p and BiP directly facilitate polypeptide translocation into the ER. 43 Cell, 69, 353-365. Schlenstedt, G., Gudmundsson, G.H., Boman, H.G. and Zimmermann, R. (1990) A large presecretory protein translocates both cotranslationally, using signal recognition particle and ribosome, and post-translationally, without these ribonucleoparticles, when synthesized in the presence of mammalian microsomes. J. Biol. Chem, 265, 13960-13968. Schneider, H.-C., Berthold, J., Bauer, M.F., Dietrneler, K., Guiard, B., Brunner, M. and Neupert, W. (1994) Mitochondrial Hsp70/MIM44 complex facilitates protein import. Nature, 371, 768-774. Schnell, D.J., Blobel, G., and Pain, D. (1990) The chloroplast import receptor is an integral membrane protein of chloroplast envelope contact sites. J. Cell Biol, 111, 1825-1838. Schnell, D.J., Blobel, G., and Pain, D. (1991) Signal peptide analogs derived from two chloroplast precursors interact with the signal recognition system of the chloroplastic envelope. J. Biol. Chem, 266, 3335-3342. Schnell, D.J., and Blobel, G. (1993) Identification of intermediates in the pathway of protein import into chloroplasts and their localization to envelope contact sites. J. Cell Biol, 120, 103-1 15. Schnell, D.J., Kessler, F. and Blobel, G. (1994) Isolation of components of the chloroplast protein import machinery. Science, 266, 1007-1012. Schreier, P.H., Seftor, E.A., Schell, J., and Bohnert, H.J. (1985) The use of nuclear encoded sequences to direct the light regulated synthesis and transport of a foreign protein into plant chloroplasts. EMBO J., 4, 25-32. Seedorf, M., Waegemann, K and Soil, J. (1995) A constituent of the chloroplast import complex represents a new type of GTP-binding protein. Plant J., 7, 401-411. SeguI-Real, B., Kispal, G., Lill, R. and Neupert, W. (1993) Functional independence of the protein translocation machineries in mitochondrial outer and inner membranes: Passage of preproteins through the interrnembrane space. EMBO J., 12, 2211-2218. Shinozakl, K. and Sugiura, M. (1986) The complete nucleotide sequence of the tobacco chloroplast genome: its gene organization and expression. EMBO J., 5, 2043-2049. Simon, S.M. and Blobel, G. (1991) A protein-conducting channel in the endoplasmic reticulum. Cell, 65, 371-380. Soll, J. and Waegemann, K. (1992) A functionally active protein import complex from chloroplasts. Plant J., 2, 253-256. Stuart, R.A., Cyr, D.M., Craig, EA. and Neupert, W. (1994) Mitochondrial molecular chaperones: Their role in protein translocation. Trends Biochem. Sci, 19, 87-92. Stuart, R.A., Gruhler, A., Van der Klei, l., Guiard, B., Koll, H. and Neupert, W. (1994) The requirement of matrix ATP for the import of precursor proteins into the mitochondrial matrix and interrnembrane space. Eur. J. Biochem., 220, 9-18. Theg, S.M., Baurle, C., Olsen, L.J., Selman, B.R., and Keegstra, K. (1989) Internal ATP is the only requirement for the translocation of precursor proteins across chloroplastic membranes. J. Biol. Chem, 264, 6730-6736. Tranel, P.J., Froehllch, J., Goyal, A. and Keegstra, K. (1995) A component of the chloroplastic protein import apparatus is targeted to the outer envelope membrane via a novel pathway. EMBO J., 14, 2436-2446. Tsugeki, R. and Nishimura, M. (1993) Interaction of homologues of Hsp70 and Cpn60 with ferredoxin-NADP+ reductase upon its import into chloroplasts. FEBS Lett, 320, 198-202. Van den Broeck, G., Timko, M.P., Kausch, A.P., Cashmere, A.R., Van Montague, M., and Herrera-Estrella, L. (1985) Targeting of foreign protein to chloroplasts by fusion to the transit peptide from the small subunit of ribulose 1,5-bisphosphate carboxylase. Nature, 313, 358—363. Vierling, E. (1991) The roles of heat shock proteins in plants. Ann. Rev. Plant Physiol. Plant Mol. Biol, 42, 579-620. Vogel, J.P., Misra, L.M. and Rose, MD. (1990) Loss of BiP/GRP78 function blocks translocation of secretory proteins in yeast. J. Cell Biol, 110, 1885-1895. Voos, W., Gambill, B.D., Guiard, B., Pfanner, N. and Craig, EA. (1993) Presequence and mature part of preproteins strongly influence the dependence of mitochondrial protein import on heat shock protein 70 in the matrix. J. Cell Biol, 123, 119-126. Walter, P., and Johnson, A.E. (1994) Signal sequence recognition and protein targeting to the endoplasmic reticulum membrane. Annu. Rev. Cell Biol, 10, 87- 1 19. Wawrzynow, A., Wojtkowiak, D., Marszalek, J., Banecki, B., Jonsen, M., Graves, B., Georgopoulos, C. and Zylicz, M. (1995) The Cle heat-shock 45 protein of Escherichia coli, the ATP-dependent substrate specificity component of the ClpP-Cle protease, is a novel molecular chaperone. EMBO J., 14, 1867-1877. Wickner, 8., Gottesman, S., Skowyra, D., Hoskins, J., McKenney, K. and Maurizi, MR. (1994) A molecular chaperone, CIpA, functions like DnaK and DnaJ. Proc. Natl. Acad. Sci. USA, 91, 12218-12222. Wickner, W., Driessen, A.J.M. and Hartl, F.-U. (1991) The enzymology of protein translocation across the Escherichia coli plasma membrane. Ann. Rev. Biochem., 60, 101-124. Wienhues, U., Becker, K., Schleyer, M., Guiard, B., Tropschug, M., HoMich, A.L., Pfanner, N. and Neupert, W. (1991) Protein folding causes an arrest of preprotein translocation into mitochondria in vivo. J. Cell Biol, 115, 1601-1609. Wild, J., Altman, E., Yura, T. and Gross, C.A. (1992) DnaK and DnaJ heat shock proteins participate in protein export in Escherichia coli. Genes Dev., 6, 1165-1172. Willey, D.L., Fischer, K., Wachter, E., Link, T.A., and Fltlgge, F-U. (1991) Molecular cloning and structural analysis of the phosphate translocator from pea chloroplasts and its comparison to the spinach phosphate translocator. Planta, 183, 451-461. Wu, C., Siebert, F.S. and Ko, K. (1994) Identification of chloroplast envelope proteins in close proximity to a partially translocated chimeric precursor protein. J. Biol. Chem, 269, 32264-32271. Chapter 2 DETERGENT SOLUBILIZATION OF CHLOROPLASTIC PROTEIN TRANSLOCATION COMPLEXES 46 47 INTRODUCTION Most chloroplastic proteins are encoded by nuclear genes and are translated on cytoplasmic ribosomes. To reach their correct position, these proteins must be transported across the double membrane system surrounding chloroplasts (Chua and Schmidt, 1978; Highfield and Ellis, 1978). These proteins are synthesized as precursors containing an N-tenninal transit-peptide responsible for their targeting (Schmidt et al., 1979). Translocation of precursors into chloroplasts can be divided into two discernible steps based on their differing energy requirements. The first is association of a precursor with the chloroplastic translocation apparatus, and the second is transport across the membranes. Stable association of precursors with the translocation apparatus requires low levels of ATP or other NTPs (Olsen et al., 1989), and results in the irreversible interaction of precursors with the chloroplastic envelopes. At this stage the precursor remains susceptible to exogenous protease and the transit-peptide is not cleaved by the stromal processing peptidase, indicating that the precursor has not completely traversed the envelope membranes (Cline et al., 1985). Translocation of precursors across the envelope membranes can be initiated by raising stromal ATP concentrations (Pain and Blobel, 1987; Theg et al., 1989). After a precursor has traversed the envelope membranes, the transit peptide is proteolytically removed by a stromal processing peptidase, producing a mature-sized protein in the stromal compartment (Reed et al., 1990). 48 Translocation of precursors across the two chloroplastic envelope membranes is thought to occur simultaneously at “contact sites” (Schnell and Blobel, 1993), a term given to regions where both envelope membranes are found in close physical proximity. By analogy with mitochondria, where precursors must also cross two membranes, precursors at contact sites are thought to interact with proteinaceous complexes from both the inner and outer membranes (for review, see Schatz and Dobberstein, 1996). In mitochondria, translocation complexes from the outer and inner membranes can act independently from one another, forming contact sites only when precursors associate with both complexes simultaneously (Horst et al., 1995; Segui-Real et al., 1993). Whether simultaneous engagement is required in chloroplasts is presently unknown. Recent work on the chloroplastic protein-import apparatus has resulted in the identification of several components of the envelope-based translocation complex (for reviews, see Gray and Row, 1995; Schnell, 1995). Several of these translocation complex members including Toc86, Toc75, Toc34, and Tic110, have been identified, and their corresponding cDNAs have been cloned (Schnell et al., 1994; Tranel et al., 1995; Hirsch et al., 1994; Seedorf et al., 1995; Ltlbeck et al., 1996). All of these proteins are integral membrane proteins, and only Toc86 and Toc34 show any sequence homology with other proteins that could provide insight to their functions. Both Toc86 and Toc34 contain GTP-binding motifs and can bind GTP (Kessler et al., 1994; Hirsch et al., 1994; Seedorf et al., 1995). The exact functions of these proteins have yet to be determined, but 49 based on biochemical and structural features, Toc86 has been proposed to be the transit peptide receptor protein, and Toc75 may represent a translocation pore (Perry and Keegstra, 1994). Using a combination of mild detergent solubilization, coimmunoprecipitation, and sucrose gradient fractionation techniques we have identified and partially characterized a chloroplastic protein translocation complex. This complex contains translocation components of both the outer and inner envelope membranes, as well as a stromal Hsp100 homologue, ClpC. We observed this complex in low ATP conditions, even in the absence of added precursors. Based on these observations a working model of the ATP- dependent binding stage of chloroplastic protein import is discussed. MATERIALS AND METHODS Isolation of chloroplasts Chloroplasts were isolated from 8 to12-day-old pea seedlings (Pisum sativum var. little marvel) as previously described (Bruce et al., 1994), and suspended In import buffer (50 mM HEPES—KOH pH 8.0, 300 mM Sorbitol) at a concentration of 1 mg chlorophyll/ml. 50 In vitro translation of precursor proteins Transcription of mRNA was performed as previously described (Bruce et al., 1994). Plasmid containing cDNA clone of precursor to Rubisco small-subunit (pRBCS, PstI-cut; Olsen and Keegstra, 1992) was linearized using the appropriate restriction enzyme, and then transcribed into mRNA. mRNAs were translated and labeled with 35S-Methionine (NEN-DuPont) as previously described (Bruce et al., 1994). After translation, residual nucleotides were removed by gel-filtration as previously described (Olsen et al., 1989). Chloroplastic binding and import reactions 50 pl of isolated, intact chloroplasts (1mg chlorophyll/ml) that had been pre- treated with 5pM nigericin to inhibit photophosphorylation were mixed with in vitro translated precursors (1 x 106 dpm) in 150 pl import buffer supplemented with 100 pM ATP. Reactions were incubated at room temperature in the dark for 10 minutes. The reactions were terminated by reisolation of intact chloroplasts with associated precursors by sedimentation through a 40% Percoll cushion. Immunoprecipitation Repur’rfied, intact chloroplasts were hypotonically Iysed in 200 pl lysis buffer (25 mM HEPES—KOH pH 8.0, 4 mM MgClz). The lysis reaction was incubated on ice for 5 minutes in the dark, and the supernatant and membrane fractions were separated by centrifugation (5 minutes, 100,000g, Sorvall RP100-AT2). Isolated chloroplastic membranes and supernatant fractions were suspended in 1 ml of 51 lPES-DM (25 mM HEPES-NaOH pH 7.5, 50 mM NaCl, 2 mM EDTA pH 8.0, 2 mM EGTA pH 8.0, 1 mM PMSF, 1% wlv decylmaltoside), and incubated for 5 minutes on ice in the dark. Insoluble material was removed from the detergent- solubilized fractions by a centrifugation step (5 minutes, 100.0009, Sorvall RP100-AT2). The resulting supernatant was immunoprecipitated with the appropriate antisera (10 pl OEP75, OEP34, IEP110, IEP35, $78, or LHCP), or affinity-purified lgGs (15 pg ClpC) , and 10 mg (dry weight) lPES-DM pre-washed Protein A Sepharose CL-4B (Phannacia, lnc.). Immunoprecipitation was carried out for 2 hours at 4°C, in the dark. lmmunoprecipitated pellets were washed three times with 1 ml lPES-OM, and once with 1 ml lPES (without decylmaltoside), and resuspended in SOS-PAGE sample buffer (Laemmli, 1970). Preparation of antibodies All antibodies were polyclonal and raised in rabbits. Antisera and preimmune sera to OEP34, OEP86, and IEP35 (Schnell et al., 1994) were a gift from D. Schnell. Affinity-purified, anti-ClpC IgG (Shanklin et al., 1995) was a gift from J. Shanklin. Antiserum to LHCP (Payan and Cline, 1991) was a gift from K. Cline. Antiserum to OEP75 was raised against the mature region of the OEP75 protein over-expressed in E. coli using the pET expression system (Novagen, lnc.). Antisera to IEP110 were raised as discussed in Akita, et al., 1997). Production of antiserum that specifically recognized S78 is discussed in Chapter 3. 52 Electrophoresis and immunoblotting All electrophoresis was performed as previously described (Laemmli, 1970) using the Hoefer gel-electrophoresis system (BioRad, Inc.). lmmunoblotting consisted of transferring proteins onto lmmobilon-P membrane (Millipore, Inc.), in transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol, 0.05% SDS) as described by Towbin and Gordon, (1984). Detection of immunoblotted proteins was performed with horseradish peroxidase conjugated to goat. anti-rabbit Fabs (Kirkegaard and Perry, Inc.), and were visualized by Westem-ECL chemiluminescence (Amersham, Inc.) exposed to x-ray film (Eastman—Kodak, Inc.). RESULTS Translocation components of the outer membrane, inner membrane, and stroma form a stable complex with the precursor under binding conditions. In vitro import of precursors into intact chloroplasts can be halted at the envelope membranes in the presence of low ATP concentrations (Olsen et al., 1989). These bound precursors form a stable interaction with the chloroplastic protein transport machinery, but can be fully imported when internal ATP concentrations are increased to adequate levels (Cline et al., 1985; Olsen and Keegstra,1992). We sought to determine whether precursor proteins trapped at this early stage of 53 translocation were sufficiently stably associated with translocation components to survive detergent solubilization and analysis. Three different methods of analysis were used to detect complexes. The first was coimmunoprecipitation of radiolabeled precursors with antibodies directed against individual translocation components. To accomplish this, radiolabeled precursor to the small-subunit (prSS) of Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was allowed to interact with isolated, intact chloroplasts in the presence of 100 pM ATP. After reisolation, intact chloroplasts were Iysed hypotonically and separated into membrane and supernatant fractions by centrifugation. Chloroplastic membranes were solubilized with buffer containing decylmaltoside and subjected to centrifugation to remove aggregates and incompletely solubilized membranes. Using decylmaltoside approximately 80 to 90% of precursors and translocation components remained in the supernatant after centrifugation (data not shown). The solubilized membrane proteins were subjected to immunoprecipitation using antibodies to specific components of the chloroplastic protein translocation machinery. Sufficient antiserum was added in each case to insure that more than 80% of each translocation component was immunoprecipitated (data not shown). lmmunoprecipitates were analyzed by SDS-PAGE and fluorography to detect coimmunoprecipitation of radiolabeled prSS (Figure 2.1). An aliquot of the solubilized membrane fraction was analyzed by SDS-PAGE prior to immunoprecipitation to assess the amount of prSS that was bound to chloroplasts (Figure 2.1, lane 1). Antibodies specific for Toc75 and Toc34, two outer membrane translocation components, were capable of 54 coimmunoprecipitating prSS (Figure 2.1, lanes 3 and 4), while preimmune serum to Toc75 (Figure 2.1, lane 2) and Toc34 (data not shown) were not. In addition, antibodies specific to Tic110, an inner membrane translocation component, were also capable of coimmunoprecipitating prSS (Figure 2.1, lane 5), whereas the corresponding preimmune serum was not (data not shown). As the amount of protein present in the total solubilized membrane fraction is equivalent to one fifth of that added to each immunoprecipitation, we estimated that approximately ten percent of the prSS added to each immunoprecipitation was associated with translocation complexes (compare lane 1 with lanes, 3, 4, and 5). These coimmunoprecipitation efficiencies are comparable to those observed with mitochondrial transport complexes (Manning-Krieg et al., 1991; Ungerrnann et al., 1994). To evaluate the possibility that coimmunoprecipitation of prSS was due to non-specific interaction of precursors with membrane proteins, immunoprecipitation was performed using antibodies raised against two membrane proteins that are not part of the translocation apparatus. Antibodies raised against either the inner envelope membrane protein, IEP35 (Schnell et al., 1994), or the thylakoid membrane Iight-harvesting-complex protein (LHCP) family (Payan and Cline, 1991), were incapable of coimmunoprecipitating prSS (Figure 2.1, lanes 6 and 8), demonstrating the specificity of the coimmunoprecipitation procedure. 55 Figure 2.1. Association of prSS with known translocation components. Gel-filtered, 35S-labeled, prSS was incubated with isolated chloroplasts in 100 pM ATP. After incubation for 10 minutes at room temperature, intact chloroplasts were repurified, Iysed hypotonically, and the chloroplastic membranes were resuspended in buffer containing decylmaltoside. Immunoprecipitation reactions were performed with anti-Toc75 (lane 3), anti— T0034 (lane 4), anti-Tic110 (lane 5), or anti-ClpC (lane 7) antibodies, and Toc75 preimmune control (lane 2). Ten percent of each reaction was removed before immunoprecipitation (R, a representative sample is shown in lane 1); the remaining 90% was split into equal portions to which immune (I lanes) or preimmune serum (P lanes) to Toc75, Toc34, Tic110, or ClpC were added. Only a representative preimmune control for Toc75 is shown (lane 2); other preimmune controls were similar. Instead of a preimmune control, an anti-IEP35 (lane 6) immunoprecipitation was performed on the other half of the anti-LHCP (lane 8) immunoprecipitation. Samples were analyzed by SDS-PAGE and fluorography. 56 n_o_._._- 8 08.0-8 mmmmw 8 or For..- 8 «much. 8 mnooh- 8 oczEE_oE R 57 Because soluble molecular chaperones have been shown in other systems to interact with translocation complexes (Schatz and Dobberstein, 1996), we investigated whether any chloroplastic chaperones were present in this complex. Antibodies to ClpC, a chloroplastic molecular chaperone of the Hsp100 family, were capable of coimmunoprecipitating prSS (Figure 2.1, lane 7), but no association was detected with preimmune serum (data not shown). S78 could also be detected in association with translocation complexes, but this interaction displayed characteristics that required further investigation (see below). Collectively, the results described above suggested that prSS forms a stable association with known translocation components from both the outer envelope membrane and the inner envelope membrane, as well as a stromally localized chaperone. The most likely explanation for the results described above was that all of these translocation components were associated with one another in a large complex. To evaluate this prediction, the solubilized complex was analyzed via a second strategy. The putative complexes were first immunoprecipitated with anti-Toc75 or anti-ClpC antibodies. Again, sufficient antibodies were added to ensure that more than 80% of these components were immunoprecipitated (data not shown). The composition of the lmmunoprecipitates was then analyzed by SDS-PAGE and immunoblotting (Figure 2.2). Antibodies against these two proteins were chosen because, based on the known location of Toc75 and ClpC 58 Figure 2.2. Complexes immunoprecipitated by anti-Toc75 and anti-ClpC contain other translocation components. Radiolabeled prSS was incubated with isolated chloroplasts in 100 pM ATP. After a 10 minute incubation, intact chloroplasts were repurified, Iysed hypotonically, and isolated chloroplastic membranes were solubilized in buffer containing decylmaltoside. Immunoprecipitation reactions were performed with anti-Toc75, anti-ClpC antibodies, or their corresponding preimmune controls. Ten percent of each reaction was removed for direct analysis on SDS-PAGE (R lanes); the remaining 90% was split into two equal fractions and immunoprecipitated with either anti-Toc75 serum (I lanes), or the corresponding preimmune control (P lanes). Similar reactions were analyzed with anti-ClpC antibodies. Three replicates of the immunoprecipitations were analyzed by SDS- PAGE, and transferred to Immobilon-P membrane. After immunoblotting, each membrane was divided into two parts, above (A-C) and below (D-F) the position of IgG, and probed with antibodies against Tic110 (A), Toc75 and Toc86 (B), ClpC and S78 (C), Toc34 (D), LHCP (E), and IEP35 and ClpP (F). A anti-TIC110 r————I (II-ClpC a-Toc75 R'I P” I P| Tic110-) Toc34» IFIIP l I P anti-T0034 B Toc86-9 Toc75-) E LHCPI 59 anti-T0086 anti-Toc75 a-ClpC a-Toc75 R' I P"I P' IRIPIP' anti-LHCP C anti-ClpC anti-S78 a—ClpC or-Toc75 a WWI IE P35-) IFiIPIPI anti-IEP35 anti-ClpP 60 within chloroplasts, they should be situated at opposite sides of a putative translocation complex. To demonstrate the relative amounts of the proteins present before immunoprecipitation, a sample of the solubilized membranes from the chloroplast-binding reaction was analyzed (Figure 2.2, R lanes). The complexes associated with both Toc75 and ClpC contained Tic110 (Figure 2.2A, l lanes), Toc34 (Figure 2.20, I lanes), and Toc86 (Figure 2.23, l lanes). In addition, complexes associated with ClpC contained Toc75 (Figure 2.2B, I lanes), and ClpC was found in complexes containing Toc75 (Figure 2.20, l lanes). By comparing the amounts of the translocation components coimmunoprecipitated with amounts present in the total soluble membrane fractions we estimated that between ten and twenty percent of most translocation components remained associated with ClpC, or Toc75 after detergent solubilization and immunoprecipitation. Lower levels of Toc86 were associated with immunoprecipitated ClpC complexes, and both Toc86 and Toc34 were coimmunoprecipitated with Toc75 at higher levels. These immunoprecipitations were specific, as the samples immunoprecipitated with the corresponding preimmune serums did not contain any of these proteins (Figure 2.2, P lanes). No association of light-harvesting- complex protein (LHCP), or the inner envelope-membrane protein (IEP35), were found in lmmunoprecipitates of Toc75 or ClpC (Figures 2E and 2F, respectively). Several proteins of differing molecular weights were detected by the LHCP antiserum, reflecting the presence of members of an antigenically related family of proteins (Payan and Cline, 1991). 61 ClpC is only one class of stromal chaperones identified in chloroplasts (Moore and Keegstra, 1993; Shanklin et al., 1995; Gething and Sambrook, 1992). Thus, we sought to determine whether molecular chaperones of the Hsp70 family were associated with the translocation complex, because they have been shown to interact with translocating precursors in other protein transport systems (Kang et al., 1990; Scherer et al., 1990; Vogel et al., 1990). Antibodies raised against the stromal Hsp70, S78, were used to probe the immunoblot of the immunoprecipitated complexes. The presence of S78 in the solubilized membrane fraction demonstrated that some stromal $78 was present in the isolated membranes (Figure 2.20, R lane). However no association of $78 with the complexes immunoprecipitated with anti-Toc75 or anti-ClpC antibodies could be detected (Figure 2.2C, l lanes). We concluded that $78 was not stably associated with the solubilized translocation complex in these immunoprecipitations. In prokaryotes, the Hsp100 chaperone family can function as subunits of the Ti protease (for review see, Squires and Squires, 1992). This protease is active as a hetero-oligomer containing the Hsp100 homologue and a separate subunit, ClpP, which contains protease activity (Hwang et al., 1988; Maurizi et al., 1990). Because both of these proteins have homologues in chloroplasts (Shanklin et al., 1995), it was possible that the association between ClpC and translocation complex members reflected association with a protease, and did not indicate that ClpC itself was a translocation component. To evaluate this possibility, complexes immunoprecipitated by anti-Toc75 and anti-ClpC 62 antibodies were probed with antibodies to stromal ClpP (Figure 2.2F). A reactive band at the correct molecular weight (Figure 2.2F, R lane) demonstrated the presence of ClpP in the membranes, presumably from stromal contamination. No significant association of this protein with the ClpC immunoprecipitate was detected (Figure 2.2F, I lanes) even when longer exposures were examined (data not shown). We concluded that the majority of the ClpC associated with translocation components was not associated with stromal ClpP, and therefore the association was not involved in proteolysis of these complexes. To obtain further evidence that solubilized translocation components were present in complexes, a third method of analysis was used. Solubilized chloroplastic envelope membranes containing translocation components and prSS were layered over a sucrose gradient and fractionated to enable study of their sedimentation patterns (Figure 2.3). Radiolabeled prSS, analyzed by scintillation counting, appeared in two peaks, with the majority (~80% of total) found near the top of the gradient in fractions 5 to 9, and a smaller peak (~20% of total) migrating further into the gradient at fraction 23. Because prSS is rapidly processed and assembled into Rubisco holoenzyme after import into chloroplasts (Archer and Keegstra, 1993), it was necessary to determine whether both peaks contained full-length precursor. This was measured by SDS-PAGE followed by fluorography and all radioactivity was found in prSS (Figure 2.3B). Silver staining indicated that some Rubisco holoenzyme and Cpn60/10 were present, presumably due to stromal contamination, but these complexes sedimented primarily in fractions 11 and 15, respectively (data not shown). 63 Figure 2.3. Translocation components and prSS cosediment as a complex. Radiolabeled prSS was incubated with isolated chloroplasts in 100 pM ATP. After incubating for 10 minutes, intact chloroplasts were repurified, Iysed hypotonically, and isolated membranes were solubilized in buffer containing decylmaltoside. The solubilized membrane fraction was layered over a 10 to 30% linear sucrose density gradient and sedimented at 150,0009 for 18 hours. Fractions were removed and the sedimentation patterns were analyzed by scintillation counting (A), SOS-PAGE and fluorography (B), and SOS-PAGE and transferring to Immobilon-P for immunoblotting with antibodies against the indicated proteins (C). 1000 800 600 400 SSS-prSSU (DPM) 200 Fraction Fraction T0086- T0075- T0034- Tic1 10- ClpC- IEP35- LCHP- Irrrew V I J J l LJJLJ l I J L l l 1 l L .IJ I I l I I I 1 1 ,1?- il- ”,_ -: >-' 40 5 10 15 20 25 Fraction number (10%-30% sucrose) 5 7 91113151719 2123 252729 3 'e'\ "12.71 21'? I" L'L” 11!“ p.52 1, "M': ' 3T 4' ‘2‘ *L ,1!“ .E‘h,‘ 3] -‘ .4 “w w ww- 1:55 3 tar-25.11 '1' ’r‘v‘” ‘. ..s ..... P'vw-p ‘12.“ 5h“. era's-7m. ._ :- 1 u -r-_ a . ' - - _sl ." . i , ‘ - ,‘ > . ,' I . _- ' V'ml'fll'" "" 1 ‘ . ' l I l - 1 ' _v ~ ,1 i I .A ‘I. r' c . , ‘. _i W L l 5:1 ‘51:. . :3. e . . , ~. . j, v . u'-T‘ '. fir. 4' x. _‘l, ' '- > ... .- ‘ . . ‘efi'. ' .‘ ¢ . _ t It .’- . . - . \ - - r 1: .-. .. ~ . . - . . e. .- .5 .. .I‘ ‘ l u r'l . ...—.... .W'M‘xbs 2351’ if. 3 e ---:;-.~-t .*' ‘ ...; \‘\e'.v."l'h:,!,"v‘ ""2'rx'-‘ .j,_(.‘h .‘ei‘v 3": ‘ltv 1'1. _ . . . 3 x-- ~ #2 l.’ *3. . .- £154.; 65 To determine the sedimentation patterns of the translocation components Toc86, Toc75, Toc34, Tic110, and the chaperone ClpC, the gradient fractions were analyzed by SDS-PAGE, followed by immunoblotting with specific antibodies (Figure 2.30). These components sedimented in two distinct peaks, similar to the sedimentation pattern of prSS (compare Figure 2.3, panels B and C). However, two control proteins, IEP35 and LHCP, were observed to have different sedimentation patterns (Figure 2.3C). From these results, we conclude that a significant portion of the radiolabeled precursor had been incorporated into a large complex containing components of the translocation apparatus from the outer and inner membranes as well as the stromal Hsp100 chaperone, ClpC. This complex sedimented far into the gradient at fraction 23, with the majority of the various translocation components being present in this fraction. Interestingly, Toc86 and Toc34 migrated no further into the gradient than fraction 23, while Toc75, Tic110, and ClpC were also found in fraction 25. Whether these differences represented different complexes was beyond the resolution of the sucrose gradient and could not be determined. A significant portion of both the radiolabeled precursors and translocation components were observed at the top of the gradient. One possible explanation for the presence of translocation components near the top of the gradient is that they represent individual components that were not part of translocation complexes. Another, more likely explanation, is that some of the translocation complexes dissociated into individual components during experimental manipulation and could have been maintained as complexes if milder, or more 66 stabilizing conditions had been used. Further work will be needed to distinguish between these two possibilities. A translocation complex can form in the absence of added precursors. Toc86, Toc75, and Toc34 have previously been shown to form a complex even in the absence of added precursors (Waegemann and Soll, 1991, Ma et al., 1996). We have shown that in the presence of added precursors ClpC and Tic110 can also interact with these outer membrane translocation components (Figures 2.1-2.3). We therefore wanted to determine if ClpC and Tic110 could associate with outer membrane translocation components in the absence of added precursors. Chloroplasts were incubated with 100 (M ATP in the presence or absence of prSS. Intact chloroplasts were recovered, lysed, and membranes isolated and solubilized as described above for the experiment shown in Figure 2.2. After a portion was removed for direct analysis on SDS- PAGE, the remaining samples were split into equal fractions and immunoprecipitated with anti-ClpC or anti-Toc75 antibodies and their respective preimmune sera. Sufficient antiserum was added to ensure at least 80% of the ClpC and Toc75 were immunoprecipitated (data not shown). The immunoprecipitates were analyzed by SOS-PAGE and immunoblotting (Figure 2.4). Complexes that coimmunoprecipitated with ClpC contained Tic110, Toc86, Toc75. and Toc34 regardless of whether precursor was present or not (Figure2.4A, compare I lanes, + and - prSS). 67 Figure 2.4. Translocation complexes containing ClpC and Toc75 form in the absence of added precursor. Isolated chloroplasts were incubated in 100 pM ATP, either in the presence (+prSS), or absence (-prSS) of radiolabeled precursor. After a 10 minute incubation, intact chloroplasts were repurified, Iysed hypotonically, and isolated membranes were solubilized in decylmaltosode buffer. Ten percent of each reaction was removed for direct analysis by SDS-PAGE (R lanes, -prSS not shown); the remaining 90% was split into two equal fractions and immunoprecipitated either with anti-ClpC (aClpC), or anti-Toc75 (aToc75) antibodies (l lanes) and their corresponding preimmune serum (P lanes). Samples were analyzed by SOS-PAGE and immunoblotting with antibodies to Toc86, Toc75, Toc34, Tic110, or ClpC. The experiment was repeated three times with similar results each time. All the panels shown were taken from a single set of anti-ClpC or anti-Toc75 immunoprecipitations and were developed for equivalent times except for the anti—ClpC immunoprecipitated, anti-Toc86 probed panel which was from a separate experiment and was developed four times longer to visualize the Toc86 present in the total membrane fraction. 68 a—ClpC or-Toc75 + prSS - prSS + prSS - prSS ngnavmw 1.5:; :.mrx:.'.xycaalr-'-a‘zu. .‘ u ..' -.., ) . f ......‘t . ...“; ,V .1‘ f H t‘ - 'I 7 ' s T0086 . - ' ‘ ' . . ' , ‘ z ” . . . . ‘ ‘ » hhuuazm ' mph-van. m- :7: :umMuwrw»--. ‘.. m'plwujr LP.‘|‘.‘\' 1 A“ ~'. ., L121 9;;- ‘51'4- "271$ ri‘. ‘ t.“ ‘ - ‘2 t _a 2 l Toc75 l l u: . ‘l 5, . ‘ ‘ _ ~ , . .‘- v , g" , . . _ ’ . . \ u—v. ,.‘.-..--.-.-. ..‘Jmmm“mh1m,varrs Li‘ié fix .- ' g 1;, . . ,. * ‘2}. . ‘- , ’. . .’,...-.. “‘3‘. """" “‘ ' ' "‘ I‘...g‘v“2;‘_':$h2!‘.z:‘c {"4 gmqmm. ., _._ . _, __ ‘ .flfz'fizi‘)’ ‘ y .‘ . , ~ . . . ‘~ . v .“ ~ . . ‘ . \ ‘ . . . w .‘ ' . V . ' . '~ 1-. 'r n t '5. ‘.:'-'s’.'1'.l., I " ..z- ... ' ‘ 'fi- ;.+. . . . _ (A _ ‘ ' . '- “ '“';‘;'vmi'.l3v‘.‘.“f1T‘ZL),'|'K.YL4_‘:l‘u;-f .._. .. ~ ... . - _ «. _ 9241' __ . ”Dy-Vs..».a-Ar-,\V.--ry.--.ni -~- - a. . ». ... .... -. _...- - . . . n . . . » . _I‘ . ‘8; ~_ '. . ‘- ' It .‘ ‘4."_.'_V_.v 7“,. ( . < ,- t : , ‘ 3. ~ ‘ ,. \ ¢v ‘ .’ . .... ‘ . J 69 Conversely, complexes associated with Toc75 also contained Toc86, Toc34, Ticl 10, and ClpC whether precursor was present or not (Figure 2.48, compare l lanes, + and - prSS). Interestingly, when immunoprecipitation was performed with anti-Toc75 antibodies, the levels of Toc34 and Toc86 present in the complexes were significantly higher than that observed in ClpC immunoprecipitates. This may indicate that the majority of Toc86, T0034, and Toc75 can associate in a separate complex that does not contain inner membrane and stromal components, and that only a portion of these complexes associate with inner membrane and stromal components. In the presence of precursors, about ten percent of Tic110 and Toc75 proteins, and lower levels of Toc86 and Toc34, were found associated with ClpC precipitated complexes as measured by comparing with the amount of the proteins present in the total solubilized membrane fractions (Figure 2.4A, compare R and l lanes). However, it should be noted that only about twenty percent of the prSS associated with chloroplasts could be found in high molecular weight complexes after separation on a sucrose gradient (Figure 2.3). The remaining 80% of the prSS was found in fractions consistent with partially or completely destabilized complexes, and might explain the low levels of coimmunoprecipitation of complexes containing both outer and inner membrane components. While the proportion of Toc86, T0034 and Toc75 associated with ClpC precipitated complexes remained unchanged in the absence of added precursors, the amount of Tic110 present in ClpC precipitated complexes was reduced. However, we are uncertain whether this is significant because the level 70 of reduction varied in different experiments (data not shown), and the amounts of Tic110 associated with Toc75 remained about the same in the presence or absence of added precursors (Figure 2.43). DISCUSSION Recent identification of several members of the chloroplastic protein translocation machinery has allowed for further refinement of our understanding of the mechanism by which precursors are transported into chloroplasts. We have attempted to define the composition of complexes that form during translocation using coimmunoprecipitation techniques with antibodies specific to translocation components. We have observed that precursors could be found in stable association with translocation complexes after solubilization with a mild detergent, decylmaltoside. Characterization of these complexes has led to two conclusions: (1), that under limiting ATP conditions, precursors associated with translocation complexes containing components of the outer and inner envelope membranes; (2), that the chaperone ClpC, a stromal Hsp100 homologue, was associated with precursor-containing complexes under these limiting ATP conditions. On the basis of data presented here and previous work, we offer a revised hypothesis for the topology of the precursor during ATP-dependent association 71 with chloroplasts (Figure 2.5). Earlier views depicted precursors as associated simply with outer membrane components in the presence of low ATP concentrations (for reviews see, Gray and Row, 1995; Schnell, 1995). The data presented here argue that these precursors also engage inner membrane and stromal components of the transport apparatus either directly (Figure 2.5A) or indirectly (Figure 2.58). Although other models cannot be excluded, the two possibilities presented are simplest and are consistent with our current observations. Previous models, showing bound precursors associated only with outer envelope membranes, were based on observations that, in limiting ATP conditions, precursors were not sufficiently inserted into the translocation machinery to be protected from exogenous protease, or to allow cleavage of the transit peptide by stromal processing peptidase (for reviews, see Archer and Keegstra, 1990; Soll and Alefsen, 1993; Tian et al., 1995). This ATP-dependent stage of import, where precursors associate stably with chloroplastic membranes, has been termed binding. While binding has traditionally referred to a reversible process, the ATP-dependent association of precursors with chloroplastic membranes is not reversible, and these precursors are no longer in equilibrium with free precursor proteins (Olsen et al., 1989; Schnell and Blobel, 1993). Indeed, these precursors are even partially resistant to extraction by high salt and carbonate (Waegemann and Soll, 1991). Additionally, these models do not explain why bound precursors associate with chloroplastic membranes in "patches," correlated with contact sites, nor do they explain why, upon lysis of 72 Figure 2.5. A model for formation of translocation complexes during ATP- dependent docking to chloroplasts. In this scheme, the initial interaction of cytoplasmically synthesized precursors with receptor proteins occurs in the absence of added energy. At this stage the association of precursors with receptor proteins remains reversible, and is not necessarily confined to contact sites. Upon the addition of ATP the precursor transit peptide is inserted across both envelope membranes (A), or across the outer membrane only (B), forming a contact-site, and allowing interaction of the transit peptide with inner envelope membrane, and stromal translocation components. 73 74 intact chloroplasts and separation of chloroplastic membranes, bound precursors migrate with mixed membrane fractions containing contact sites rather than with purified outer membranes (Schnell and Blobel, 1993; Perry and Keegstra, 1994; J. Ostrom and K. Keegstra, unpublished results). In light of these observations, as well as the work presented here and by others (Wu et al., 1994, Akita et al., 1997), we propose that a more appropriate term for this stage in chloroplastic protein translocation is “docking.” We define docked precursors as having progressed to a discrete step following initial recognition that would occur during binding, but is halted prior to full translocation of the precursor into the stroma. At this stage in import, docked precursor would associate at contact sites in an ATP-dependent fashion forming complexes containing translocation components of both outer and inner envelope membranes. The association of precursors with these complexes would be stable, as opposed to the reversible association of precursors with receptor proteins that would occur during binding. The hypothesis presented in Figure 2.5 shows the formation of a single translocation complex consisting of components of several compartments of the chloroplast. Using both coimmunoprecipitation (Figures 1 and 2) and differential centrifugation techniques (Figure 2.3), we detected translocation complexes containing precursors and translocation components of the outer envelope membrane, inner envelope membrane, and stromal compartments. Similar complexes have been identified when cross-linked complexes containing docked precursors are analyzed (Wu et al., 1994; Akita et al., 1997). In addition, ATP- dependent association of precursors with chloroplasts localize in a punctate 75 pattern in intact chloroplasts correlating with regions of close association of the inner and outer chloroplast membranes (Schnell and Blobel, 1993). Furthermore, these precursors migrate with chloroplastic membrane fractions containing contact sites (Schnell and Blobel, 1993; Perry and Keegstra, 1994; J. Ostrom and K. Keegstra, unpublished results). All these results are consistent with formation of a translocation complex containing components of both outer and inner envelope membranes. The observation that a portion of the protein translocation components of the outer and inner membranes associated in complexes even in the absence of added precursors (Figure 2.4) presents an intriguing difference from protein import into mitochondria, in which outer and inner membrane translocation complexes can act independently, and are only connected by translocating precursors (Segui-Real et al., 1993; Horst et al., 1995). Kessler and Blobel - (1996), reported that in the absence of precursors Tic110 was not found in association with outer membrane translocation components either by detection with Coomassie blue or immunoblotting techniques. We clearly observe Tic110 in association with Toc75 precipitated complexes (Figure 2.4). At present the reasons for these discrepancies are not clear. Detergents and salt concentrations used in our experiments differed from those used by Kessler and Blobel (1996). Additionally, we incubated chloroplasts in 100 pM ATP prior to lysis and solubilization which may have affected the association of inner and outer membrane complexes. 76 Because the complex we characterized does not represent reversible binding, the question arises of whether there is an earlier binding step in chloroplastic protein import. Several lines of evidence support the formation of a different binding complex earlier than the docking step. While stable association of precursor with intact chloroplasts requires ATP, T0086 and to a lesser degree Toc75, can be specifically cross-linked to prSS in the absence of nucleotide (Perry and Keegstra, 1994; Ma et al., 1996). This complex migrates with purified outer envelopes rather than contact sites, and upon addition of ATP can be chased into the contact sites; where it interacts with Toc75, and an inner membrane protein (Ma et al., 1996). Since, the docking complex includes inner envelope membrane and stromal components, it is unlikely it would migrate with purified outer envelopes. Outer envelope membrane translocation complexes may not always be associated with inner envelope membrane translocation components during import of precursors into chloroplasts. A slowly translocating precursor can be found in a translocation complex that includes only outer envelope membrane translocation components (Schnell et al., 1994). A membrane protein complex isolated from purified outer membranes has been shown to interact with precursors in an ATP-dependent, and transit peptide specific manner (Soll and Waegemann, 1992). Also, when chloroplasts are subjected to hypertonic conditions to separate the outer and inner envelope membranes, precursors can accumulate in the inter-membrane space during an import reaction (Scott and Theg, 1996). These results suggest that an outer membrane complex is capable 77 of recognizing and translocating precursors independently of inner membrane components. We detected Tic110 and ClpC in Toc75 precipitated complexes, but T0086 and T0034 were present at significantly higher levels (Figure 2.4). We interpret these results as an indication that, although some Toc75 can interact with inner membrane and stromal components, even in the absence of added precursors, much more is found associated only with T0034 and T0086. This result is consistent with the work of Ma et al. (1996), who showed T0075, T0034, and T0086 were associated with one another even in the absence of added precursors. While the authors observed no association of inner membrane components under these conditions, antibodies to inner membrane components were not tested, and Coomassie blue staining detection methods employed may have not have been sufficiently sensitive to detect lower amounts of inner membrane components. 78 REFERENCES Akita, M., Nielsen, E. and Keegstra, K. (1997) Identification of protein transport complexes in the chloroplastic envelope membranes via chemical cross-linking. J. Cell Biol., 136, 983-994. Archer,E.K. and Keegstra,K. (1990) Current views on chloroplastic protein import and hypotheses on the origin of the transport mechanism. J. Bioenerg. Biomemb., 22, 789-810. Archer,E.K. and Keegstra,K. (1993) Analysis of chloroplast transit peptide function using mutations in the carboxyl-terminal region. Plant Mol. Biol., 23, 105-1115. Bruce,B., Perry,S., Froehlich,J. and Keegstra,K. (1994) In vitro import of proteins into chloroplasts. Plant Molecular Biology Manual J1. Kluwer Academic Publishers, Belgium. pp. 1-15. Chua,N.H. and Schmidt,G.W. (1978) Post-translational transport into intact chloroplasts of a precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase. Proc. Natl. Acad. Sci. USA, 75, 6110-61 14. Cline, K., Werner-Washbbume, M., Lubben, T., and Keegstra, K. (1985) Precursors to two nuclear-encoded chloroplast proteins bind to the outer envelope before being imported into chloroplasts. J. Biol. Chem, 260, 3691- 3696. Gething, MN. and Sambrook, J. (1992) Protein folding in the cell. Nature, 355, 33-45. Gray,J.C. and Row,P.E. (1995) Protein translocation across chloroplast envelope membranes. Trends Cell Biol., 5, 243-247. l-Iighfleld,P.E. and Ellis,R.J. (1978) Synthesis and transport of the small subunit of chloroplast ribulose bisphosphate carboxylase. Nature, 271, 420-424. Hirsch, 8., Muckel, E., Heemeyer, F., von Heijne, G. and Soll, J. (1994) A receptor component of the chloroplast protein translocation machinery. Science, 266, 1989—1992. 79 Horst,llll., Hilfiker-Rothenfluh,$., Oppllger,W. and Schatz,G. (1995) Dynamic interaction of the protein translocation systems in the inner and outer membranes of yeast mitochondria. EMBO J., 14, 2293—2297. Hwang, B.J., Woo, K.M., Goldberg, A.L. and Chung, C.H. (1988) Protease Ti, a new ATP-dependent protease in Escherichia coli, contains protein-activated ATPase and proteolytic functions in distinct subunits. J. Biol. Chem, 263, 8727- 8734. Kang, P.-J., Ostermann, J., Shilling, J., Neupert, W., Craig, EA. and Pfanner, N. (1990) Requirement for hsp70 in the mitochondrial matrix for translocation and folding of precursor proteins. Nature, 348, 137-143. Kessler, F., Blobel, G., Patel, HA. and Schnell, D.J. (1994) Identification of two GTP-binding proteins in the chloroplast protein import machinery. Science, 266, 1035-1039. Kessler, F. and Blobel, G. (1996) Interaction of the protein import and folding machineries in the chloroplast. Proc. Natl. Acad. Sci. USA, 93, 7684-7689. Laemmli,U.K. (1970) Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature, 227, 680-685. Ltibeck, J., Soll, J., Akita, M., Nielsen, E. and Keegstra, K. (1996) Topology of IEP110, a component of the chloroplast protein import machinery present in the inner envelope membrane. EMBO J., 15, 4230-4238. Ma, Y., Kouranov, A., LaSaIa, S. and Schnell, D.J. (1996) Two components of the chloroplast protein import apparatus, IAP86 and IAP75, interact with the transit sequence during the recognition and translocation of precursor proteins at the outer envelope. J. Cell Biol., 134, 315-327. Manning-Krieg, U.C., Scherer, PE. and Schatz, G. (1991) Sequential action of mitochondrial chaperones in protein import into the matrix. EMBO J., 10, 3273-3280. MaurizI,M.R., Clark,W.P., Katayama,Y., Rudikoff,S., Pumphrey,J., Bowers,B. and Gottesman,s. (1990) Sequence and structure of Clp P, the proteolytic component of the ATP-dependent Clp protease of Escherichia coli. J. Biol. Chem, 265, 12536-12545. Moore, T. and Keegstra, K. (1993) Characterization of a cDNA clone encoding a chloroplast-targeted Clp homologue. Plant Mol. Biol., 21, 525-537. 80 Olsen, L.J., Theg, S.M., Selman, B.R., and Keegstra, K. (1989) ATP is required for the binding of precursor proteins to chloroplasts. J. Biol. Chem, 264, 6724-6729. OIsen,L.J. and Keegstra,K. (1992) The binding of precursor proteins to chloroplasts requires nucleoside triphosphates in the interrnembrane space. J. Biol. Chem, 267, 433-439. Pain, D., and Blobel, G. (1987) Protein import into chloroplasts requires chloroplast ATPase. Proc. Natl. Acad. Sci. USA, 84, 3288-3292. Payan,L.A. and CIine,K. (1991) A stromal protein factor maintains the solubility and insertion competence of an imported thylakoid membrane protein. J. Cell Biol., 112, 603-613. Perry, S.E., and Keegstra, K. (1994) Envelope membrane proteins that interact with chloroplastic precursor proteins. Plant Cell, 6, 93-105. Reed,J.E., CIine,K., Stephens,L.C., Bacot,K.O. and Vlitanen,P.V. (1990) Early events in the import/assembly pathway of an integral thylakoid protein. Eur. J. Biochem., 194, 33-42. Shanklin,J., DeWitLND. and Flanagan,J.M. (1995) The stroma of higher plant plastids contain ClpP and ClpC, functional homologs of Escherichia coli ClpP and CIpA: An archetypal two-component ATP—dependent protease. Plant Cell, 7, 1713-1722. Schatz,G. and Dobberstein,B. (1996) Common principles of protein translocation across membranes. Science, 271 , 1519-1526. Scherer,P.E., Krieg,U.C., Hwang,S.T., Vestweber,D. and Schatz,G. (1990) A precursor protein partly translocated into yeast mitochondria is bound to a 70 kd mitochondrial stress protein. EMBO J., 9, 4315-4322. SchmIdLG.W., Devillers-Thiery,A., Desruisseaux,H., Blobel,G. and Chua,N.H. (1979) NH2-terminal amino acid sequences of precursor and mature forms of the ribulose-1,5-bisphosphate carboxylase small subunit from Chlamydomonas reinhardtii. J. Cell Biol., 83, 615-622. Schnell, D.J., and Blobel, G. (1993) Identification of intermediates in the pathway of protein import into chloroplasts and their localization to envelope contact sites. J. Cell Biol., 120, 103-115. Schnell, D.J., Kessler, F. and Blobel, G. (1994) Isolation of components of the chloroplast protein import machinery. Science, 266, 1007-1012. 81 Schnell,D.J. (1995) Shedding light on the chloroplast protein import machinery. Cell, 83, 521-524. Scott,S.V. and Theg,S.M. (1996) A new chloroplast protein import intermediate reveals distinct translocation machineries in the two envelope membranes: Energetics and mechanistic implications. J. Cell Biol., 132, 63-75. Seedorf, M., Waegemann, K and Soll, J. (1995) A constituent of the chloroplast import complex represents a new type of GTP-binding protein. Plant J., 7, 401-411. Segui-Real, B., Kispal, G., Lill, R. and Neupert, W. (1993) Functional independence of the protein translocation machineries in mitochondrial outer and inner membranes: Passage of preproteins through the intennembrane space. EMBO J., 12, 2211-2218. Soll,J. and Alefsen,H. (1993) The protein import apparatus of chloroplasts. Physiol, Plant. 87, 433-440. Soll, J. and Waegemann, K. (1992) A functionally active protein import complex from chloroplasts. Plant J., 2, 253-256. Squires,C. and Squires,C.L. (1992) The Clp proteins: proteolysis regulators or molecular chaperones?. J. Bacteriol, 174, 1081-1085. Theg, S.M., Baurle, C., Olsen, L.J., Selman, B.R., and Keegstra, K. (1989) lntemal ATP is the only requirement for the translocation of precursor proteins across chloroplastic membranes. J. Biol. Chem, 264, 6730-6736. Tian,F., Ma,Y., Kouranov,A., LaSaIa,S.E. and Schnell,D.J. (1995) Molecular dissection of the mechanism of protein import into chloroplasts. Cold Spring Harbor Symposia on Quantitative Biology, LX, Cold Spring Harbor Laboratory Press, pp.629-636. Towbin, H. and Gordon, J. (1984) lmmunoblotting and dot immunoblotting— current status and outlook. J. Immunol. Methods, 72, 313-340. Tranel, P.J., Froehlich, J., Goyal, A. and Keegstra, K. (1995) A component of the chloroplastic protein import apparatus is targeted to the outer envelope membrane via a novel pathway. EMBO J., 14, 2436-2446. Ungerrnann,C., NeuperLW. and Cyr,D.M. (1994) The role of hsp70 in conferring unidirectionality on protein import into mitochondria. Science, 266, 1250-1253. Vogel, J.P., Misra, L.M. and Rose, MD. (1990) Loss of BiP/GRP78 function 82 blocks translocation of secretory proteins in yeast. J. Cell Biol., 1 10, 1885-1895. Waegemann,K. and Soll,J. (1991) Characterization of the protein import apparatus in isolated outer envelopes of chloroplasts. Plant J., 1 , 149-158. Wu, C., Siebert, F.S. and K0, K. (1994) Identification of chloroplast envelope proteins in close proximity to a partially translocated chimeric precursor protein. J. Biol. Chem, 269, 32264-32271. Chapter 3 EVALUATION OF THE ROLES OF THREE STROMAL MOLECULAR CHAPERONES IN CHLOROPLASTIC PROTEIN IMPORT 83 34 INTRODUCTION Molecular chaperones perform important functions in protein translocation systems (Schatz and Dobberstein, 1996). In both mitochondria and the endoplasmic reticulum, molecular chaperones have been identified as essential members of the translocation apparatus (Kang at al., 1990; Sanders at al., 1992). These chaperones are thought to allow for the unidirectional movement of a largely unfolded precursor through a protein import channel by successive binding and release of the precursor as it emerges into the interior of the organelle, possibly through the generation of a pulling force (Glick, 1995). In bacterial secretion the story is slightly different, as a specific translocation component is thought to generate a pushing force at the cytoplasmic face of the protein export channel (Economou and Wickner, 1994). However, in this system molecular chaperones have been demonstrated as important components in the delivery of precursors to the translocation apparatus (Luirink et al., 1994; Miller at al., 1994; Hendrick etal., 1993). In Chapter 2 we had determined that ClpC, a stromal Hsp100 homologue, was present in chloroplastic protein translocation complexes. The presence of this chaperone in detergent solubilized protein complexes containing precursors and other known protein translocation components indicated that perhaps it played a role in chloroplastic protein import. However, in addition to ClpC, members of two other molecular chaperone families, Hsp70 and Hsp60, are known to be present in the stromal compartment of chloroplasts. Both of these molecular chaperone families had been previously identified as playing important 85 roles during protein transport into chloroplasts (Madueho at al., 1994; Tsugeki and Nishimura, 1993) as well as in other protein translocation systems (Gething and Sambrook, 1992). We therefore wished to use our methods to confirm roles for the stromal forms of these two molecular chaperone families in protein import into chloroplasts. Additionally, if stromal Hsp70s and Hsp60s were detected in association with importing precursors we wished to define the stage(s) of protein translocation in which they interacted. Homologues of three different molecular chaperone families have been identified in the stromal compartment of chloroplasts. Hsp60, also known as chaperonin—60, or Rubisco-binding protein, is present in the stromal compartment of chloroplasts. Two different forms, the alpha and beta forms, have been identified, but are thought to be present in stoichiometric amounts in the holoenzyme (Hemmingsen et al., 1988). In this respect the stromal Hsp60 differs from Hsp603 found in Escherichia coli and the mitochondrial matrix, but it is unclear at the moment if this difference is significant to the function of the chloroplastic Hsp60. In Escherichia coli, Hsp60 was shown to play a role in delivery of proteins to the secretory apparatus (Bochkareva at al., 1988; Lecker et al., 1989; Kusukawa at al., 1987). In chloroplasts Hsp603 have been shown to interact transiently with several different newly imported proteins (Lubben at al., 1989; Madueflo at al, 1994; Tsugeki and Nishimura, 1993). However in all these studies only soluble complexes were studied, and no information regarding the possibility of Hsp60 interaction with precursor proteins was determined. Hsp60 has recently been reported in association with the inner membrane translocation 86 component, Tic110, and newly-imported mSS in detergent solubilized chloroplasts (Kessler and Blobel, 1996), but whether these complexes were membrane-associated was not determined. We therefore wished to evaluate whether chloroplastic Hsp60 was capable of interacting with importing precursors still associated with the envelope membranes and presumably therefore still in association with the translocation apparatus. Chloroplasts contain three separate Hsp70 homologues, one associated with the outer envelope membrane (E75), and two soluble stromal forms (S78 and S75; Marshall at al., 1990). Recent experiments have indicated a possible role for the E75 homologue in the binding stages of protein import into chloroplasts (Schnell et al., 1994). The major stromal form (S78) has been cloned and has high sequence similarity with the mitochondrial Hsp70 form, and even higher similarity with the DnaK homologue of Synechocystis (Craig at al., 1989, Marshall and Keegstra, 1992). Given the central role Hsp70s play in protein translocation into organelles in other systems (Kang et al., 1990; Sanders at al., 1992), we wished to evaluate whether the stromal Hsp70 homologues played a role in protein import into chloroplasts. Additionally, chloroplasts contain members of a recently identified class of molecular chaperones, the Hsp100 family (also known as Clp proteins). The chloroplastic homologue of this family is known as ClpC, and has been identified as a soluble stromal protein (Moore and Keegstra, 1993). These proteins, originally identified as members of an ATP-dependent protease in Escherichia coli, have recently been found to be molecular chaperones in their own right 87 (Wickner at al., 1994; Wawrzynow at al., 1995). In mitochondria, mutation of the matrix-Hsp70 and over-expression of the matrix-localized Hsp100 homologue allowed a partial restoration of import into the mitochondria (Schmitt et al., 1995). Because we had observed these proteins as members of translocation complexes formed under binding conditions, we wished to determine if these chaperones only associated with translocation complexes under these conditions, or whether they associated during import of precursors into chloroplasts as well. Using coimmunoprecipitation techniques in conjunction with protein import timecourses we demonstrated that both stromal forms of Hsp100 (ClpC) and Hsp70 (S78) molecular chaperone families interacted with precursor-containing complexes. These complexes were associated with the membrane fractions, and disappeared in a time-dependent fashion as import progressed. While ClpC associated with solubilized precursor-containing complexes, S78 was found associated with precursors only in particulate complexes. We therefore favor a role for ClpC in chloroplastic protein import. Additionally, stromal Hsp60 could be observed in association with mSS in complexes present only in soluble protein fractions after hypotonic lysis of chloroplasts. These results are consistent with a role during folding or assembly of newly imported proteins rather than a direct role in the translocation event. 88 MATERIALS AND METHODS Isolation of chloroplasts Chloroplasts were isolated from 8 to12-day-0Id pea seedlings (Pisum sativum var. little marvel) as previously described (Bruce at al., 1994), and suspended in import buffer (50 mM HEPES-KOH pH 8.0, 300 mM Sorbitol) at a concentration of 1 mg chlorophyll/ml. In vitro translation of precursor proteins Transcription of mRNA was performed as previously described (Bruce et al., 1994). Plasmids containing cDNA clones of precursor to Rubisco small-subunit (pRBCS, Pstl-cut; Olsen and Keegstra, 1992) was linearized using the appropriate restriction enzyme, and then transcribed into mRNA. mRNAs were translated and labeled with 35S-Methionine (NEN-DuPont) as previously described (Bruce at al., 1994). After translation, residual nucleotides were removed by gel-filtration as previously described (Olsen et al., 1989). Chloroplastic binding and import reactions 50 pl of isolated, intact chloroplasts (1mg chlorophyll/ml) that had been pre- treated with 5uM nigericin to inhibit photophosphorylation were mixed with in vitro translated precursors (1 x 106 dpm) in 150 pl import buffer supplemented with 100 uM ATP. Reactions were incubated at room temperature in the dark for 10 minutes. The reactions were terminated by reisolation of intact chloroplasts with 89 associated precursors by sedimentation through a 40% Percoll cushion. If import was carried out, the above steps were performed as a batch reaction corresponding to the number of timepoints in the import reaction (i.e. 6 timepoints = 6 x 150 pl reaction vol.). After reisolation of intact chloroplasts, import was initiated by resuspending chloroplasts in import buffer (50 uI/tlmepoint) supplemented with 4 mM ATP. The batch import reaction was carried out at room temperature, and 50 pl aliquots removed at given timepoints and import halted by reisolation of intact chloroplasts by sedimentation through a 40% Percoll cushion. Immunoprecipitation Except where specified, the repurified, intact chloroplasts were hypotonically lysed in 200 pl lysis buffer (25 mM HEPES-KOH pH 8.0, 4 mM MgClz). The lysis reaction was incubated on ice for 5 minutes in the dark, and the supernatant and membrane fractions were separated by centrifugation (5 minutes, 100.0009, Sorvall RP100-AT2). Isolated chloroplastic membranes and supernatant fractions were suspended in 1 ml of lPES-DM (25 mM HEPES-NaOH pH 7.5, 50 mM NaCl, 2 mM EDTA pH 8.0, 2 mM EGTA pH 8.0, 1 mM PMSF, 1% wlv decylmaltoside), and incubated for 5 minutes on ice in the dark. Except where specified, insoluble material was removed from the detergent-solubilized fractions by a centrifugation step (5 minutes, 100.0009, Sorvall RP100-AT2). The resulting supernatant was immunoprecipitated with the appropriate antisera (10 pl OEP75, OEP34, IEP110, IEP35, $78, or LHCP), or affinity-purified lgGs 90 (15 pg ClpC) , and 10 mg (dry weight) lPES-DM pre-washed Protein A Sepharose CL-4B (Pharrnacia, lnc.). Immunoprecipitation was carried out for 2 hours at 4°C, in the dark. lmmunoprecipitated pellets were washed three times with 1 ml lPES-DM, and once with 1 ml lPES (without decylmaltoside), and resuspended in SDS-PAGE sample buffer (Laemmli, 1970). Construction of the GST-S78 fusion protein Mutagenesis of the C-tenninal portion of the S78 cDNA (Marshall and Keegstra, 1992) was necessary in order to fuse this region of the S78 cDNA in-frame with the Glutathione—S-Transferase (GST) gene present in the over-expression plasmid pGEX-2T (Novagen, Inc.). This mutagenesis was performed using a PCR-based strategy. Two mutagenic oligonucleotides were designed so that a PCR—amplified DNA fragment corresponding to the C-tenninal 142 amino acids of the S78 cDNA would have the necessary restriction sites for sub-cloning into the pGEX-2T plasmid. At the 5’-end, an in-frame BamHl-cleavage site was introduced into the wild-type S78 cDNA sequence using the mutagenic oligonucleotide primer KEE46: 5’-ATTACCATTACTGGATCCAGCACTTTG-3’. At the 3’-end, and an EcoRl-cleavage site was introduced into the 3’- untranslated region of the S78 cDNA using the mutagenic oligonucleotide primer KEE17: 5’-GCGACTGAATTCACAAAAATCCCTAAG-S’. After PCR- amplification, the resulting DNA fragment was purified by gel electrophoresis (Maniatis, 1982), digested with BamHI, and EcoRI restriction enzymes and 91 ligated into the BamHI and EcoRI sites of pGEX-2T creating the plasmid pGEX2T-GST::S78. Over-expression and purification of the GST-S78 fusion protein The GST-S78 fusion protein was purified using protocols described in the pGEX protein purification kit (Novagen, lnc.). Briefly, the plasmid pGEX2T-GST::S78 was transformed into Escherichia coli DH5a. Cells were grown at 37°C to an OD595 of 0.7 in 250 mL of Luria broth (Maniatis, 1982), and expression of the GST-S78 fusion protein was induced with 1 mM lPTG for three hours. Cells were pelleted by centrifugation in a Sorvall SS-34 rotor for 10 minutes at 8000 rpm. The pelleted cells were resuspended in 10 mL of phosphate buffered saline (PBS; Maniatis, 1982) and lysed in a French Press at 17,000 p.s.i. Triton X-100 was added to the Iysed cells to a final concentration of 1% (volume/volume) and mixed for 30 minutes on ice. Unbroken cells and insoluble material was removed from the lysate by centrifugation for ten minutes in an SS- 34 rotor. The resulting supernatant was passed over a Glutathione CL-4B affinity column (200 pL bed volume) three times, and washed three times with 2 mL of PBS. The GST-S78 fusion protein was eluted from the Glutathione CL-4B column with 200 pL of PBS + 10 mM reduced Glutathione. The elution was repeated three times. Purity of the GST-S78 fusion protein was assessed by analysis by SDS-PAGE followed by silver-staining. Preparation of antibodies 92 All antibodies were polyclonal and raised in rabbits. Affinity-purified, anti-ClpC IgG (Shanklin at al., 1995) was a gift from J. Shanklin. Antiserum to T0075 and Tic110 were raised as discussed in Chapter 2. Antiserum to S78 was raised against purified GST-S78 fusion protein. The purified GST-S78 fusion protein was mixed with Titre-Max adjuvant (Vaxcel, Inc.) and injected into rabbits. Electrophoresis and immunoblotting All electrophoresis was performed as previously described in Chapter 2. RESULTS Preparation of antibodies that specifically recognize and Immunoprecipitate 878. Both Hsp60 and Hsp100 homologues were detected only in the chloroplastic stromal compartment (Gatenby and Ellis, 1990; Moore and Keegstra, 1993), and specific antibodies against both were already available. However, because chloroplasts contain three separate Hsp70 homologues (Marshall at al., 1990), the use of antibodies to determine if the major stromal Hsp70 was involved in protein import into chloroplasts required antibodies specific for the S78 homologue. Specifically, because the envelope associated Hsp70, E75, had been implicated in the binding reaction (Schnell et al., 1994) antibodies that could distinguish between the stromal and envelope 93 Hsp70 forms would be necessary to separate possible roles for these two Hsp70 homologues in chloroplastic protein import. All Hsp70 proteins are organized into two distinct functional domains (Gething and Sambrook, 1992). The N-terrninal ATPase domain of Hsp70 proteins is highly conserved but this family of proteins display higher levels of variability in the C-terrninal peptide-binding domain (Figure 3.1A). We therefore decided to attempt to create specific antibodies to S78 by raising them only against the variable C-ten'ninal domain. S78 had previously been shown to be most closely related to the DnaK homologue of Synechocystis (Marshall and Keegstra, 1992). We therefore lined up the C-tenninal regions of these proteins and selected a non-homologous region encoding the final 142 amino acid residues (Figure 3.18). Appropriate restriction enzyme sites were incorporated into the S78 gene by PCR-based mutagenesis and this altered PCR fragment was placed behind the Glutathione-S-Transferase gene as a C-terminal fusion in the pGEX-2T over-expression vector. This fusion protein was over-expressed in Escherichia coli, purified, and presented to rabbits for antibody production. To determine the specificity of anti-S78 antiserum (anti-S78) immunoblotting techniques were used with Hsp70 homologues present in various chloroplastic fractions. Anti-S78 specifically recognized a single 78 kDa protein in chloroplast and stromal fractions, but did not react with purified outer membranes (Figure 3.2A), confirming that the antiserum was specific for stromal members of the Hsp70 family. Confirmation that the immunoreactive protein 94 Figure 3.1. The C-tenninal peptide-binding domain of S78 is not highly conserved. (A) A linear diagram of a generalized Hsp70 protein indicating two major domains: a highly conserved N-terminal ATPase domain, and a less conserved C-terrninal peptide-binding domain. (8) The C-terrninal 210 amino acids of the peptide binding domain of 878 displayed low-conservation when aligned with the protein sequences of Synechocystis PCC7942 DnaK, and Pisum sativum cytosolic Hsp70. Alignment was performed using the CLUSTAL algorithm in the DNAStar sequence analysis program (DNAstar, Madison, WI, USA). 95 High cons. Low cons. , Pept. Dom. . l l ATPase WW VREKVEAKLGELKEAITQ---§3TQTEKEALAAENEEVMQLEQSLENEPEAAéQAEfiTPP . w fiGLIKDLKEAVAQ-—-EDDAKIQTVMfiELQpVLYSEGSNMYQQAGAEAdVGAP:A ER§§IEKAV3§AIQWLEENQLEEVEEFEfixomggEGMCNpgIAmEXg--EGAWQD a SESEPSESSGKEfiEEGBVTDfiDFTDS-§ P. sativum chloroplastic Hsp70 ooooo PgAGTssch ----- DDVIDQEfiSEPEg synechocystis PCC6803 DnaK EMPGfiGgNgggP WIEEVQ P. sativum cytoplasmic Hsp70 96 Figure 3.2. Specificity of anti-S78 antiserum. Purified outer envelope membrane proteins (0), stromal proteins (S), and whole chloroplast proteins (Cp) were separated by SDS-PAGE, transferred to Immobilon-P membranes, and probed with anti-S78 antibodies (A), SPA-820 antibodies (B), or a mixture of both (C). All lanes contained 10 pg of protein. (D) lmmunodetection of Hsp705 in immunoprecipitations. 125 pg of whole chloroplast protein (lanes 1 and 4), or anti-S78 immunoprecipitated protein (lanes 2, 3, 5, and 6) from control chloroplasts (lanes 2 and 5) or chloroplasts to which prSS had been bound (lanes 3 and 6) were separated on SDS-PAGE, transferred to lmmobilon-P membranes, and probed with anti-S78 antibodies (lanes 1, 2, and 3), or SPA-820 antibodies (lanes 4, 5, and 6). Arrowheads indicate the positions of the S78 and E75 Hsp70s in the protein fractions. 97 A B . anti-S78 + anti-S78 SPA-820 SPA-820 MWO 80p 0 SCpOSCp 200- 115- <— S78 80' 4—E75 50- F ront- anti-S78 SPA-820 98 was $78 and not 875 was obtained by co-migration of radiolabeled S78 imported into chloroplasts (data not shown). To determine if the lack of a reaction in the outer membrane fraction was due to the specificity of anti-S78 or the absence of the outer membrane Hsp70 (E75), chloroplastic fractions were probed with a monoclonal antibody (SP-820, Stress-Gen, Inc.) that reacted specifically with E75 (Schnell at al., 1994). SPA-820 reacted with a 75 kDa protein in the outer membrane fraction but not the stromal fraction (Figure 3.2B). E75 was not detected in the whole chloroplast fraction, however we believe this was due to the low abundance of outer membrane proteins as compared to stromal proteins, in whole chloroplast fractions. Figure 3.2C showed the relative signal when chloroplast fractions were probed with a mixture of anti-S78 and SPA-820. We conclude that anti-S78 is capable of recognizing the S78 stromal Hsp70 but displays specificity for this form, and does not react with the envelope associated Hsp70, E75, or with a 75 kDa stromal Hsp70, S75. Since our goal was to utilize anti-S78 for immunoprecipitation experiments we wanted to establish its specificity in immunoprecipitation conditions where antibody and Hsp70 concentrations are much higher. Also, immunoprecipitation might reveal higher-order complexes between $78 and other Hsp70s that would not be detected with SDS-PAGE immunoblotting techniques. Anti-S78 immunoprecipitated $78 from detergent solubilized chloroplasts while preimmune serum did not (data not shown). To determine if $78 was the only Hsp70 immunoprecipitated from chloroplasts, products were run on SDS-PAGE, immunoblotted and then probed with anti-S78 or SPA-820 (Figure 3.2D). First, 99 to make sure Hsp703 could be detected using this immunoblotting protocol a lane of total protein chloroplast equivalent to that introduced into the immunoprecipitation reactions was run (Figure 3.20, lanes 1 and 4). Both S78 and E75 could be detected. Next, to determine if anti-S78 was capable of directly immunoprecipitating E75, chloroplasts from an import reaction to which no precursor had been added were immunoprecipitated. Under these conditions S78 was precipitated, but no E75 could be detected (compare lanes 2 and 5). We conclude that anti-S78 does not directly immunoprecipitate E75. Because E75 has been found as a member of a solubilized complex containing bound precursors (Schnell et al., 1994) we wanted to see if both S78 and E75 could be coimmunoprecipitated if precursor was bound to chloroplasts before immunoprecipitation. Under these conditions $78 was detected, but again no E75 was detected with the immunoprecipitated complex (compare lanes 3 and 6). From these results we concluded that anti-S78 specifically immunoprecipitated S78 chloroplastic extract derived from protein import assays. ClpC, $78, and Hsp60 each associate with precursors or newly imported proteins during an import timecourse. ClpC was detected in a complex with other translocation components and prSS, but using similar conditions, association of the stromal Hsp70, S78, could not be detected (Figure 2.2). Because Hsp70, and Hsp60 homologues are important members of other protein translocation systems (Kang at al., 1990; Scherer at al., 1990; Vogel et al., 1990; Manning-Krieg, 1991), their involvement in protein import into chloroplasts was 100 examined in more detail. Previous experiments were performed with complexes prepared from isolated membranes only in binding conditions. The other stromal molecular chaperones, Hsp70 and Hsp60, might have roles at later stages of import. The interaction of Hsp100 (ClpC), Hsp70 (S78), and Hsp60 chaperones with translocation complexes was further characterized by investigating these interactions during an import timecourse (Figure 3.3). Whole chloroplasts were solubilized to analyze possible interactions of chaperones with newly-imported proteins in the stromal compartment in addition to precursors translocating across membranes. First, radiolabeled precursors were allowed to bind to isolated chloroplasts in the presence of low levels of ATP. After removal of unbound precursors, chloroplasts were resuspended in the presence of high levels of ATP, thereby allowing import to occur. Analysis of the import timecourses demonstrated that mSS accumulated in significant levels only after a 2.5-min lag, but accumulation then continued for 30 minutes (Figure 3.3A, C, and. E). Precursor, but not mSS, was detected in the anti-ClpC immunoprecipitated fractions (Figure 3.3B). Anti- CIpC antibodies immunoprecipitated complexes at high levels at time 0, and decreased as import progressed (Figure 3.3B). Precursors were detected in the anti-S78 immunoprecipitated fractions (Figure3. 3D). No mSS associated with anti-ClpC or anti-S78 precipitated fractions. No precursor was detected in association with anti-Hsp60 immunoprecipitates at any point during the import reaction, but mSS was observed at later timepoints in the import reaction 101 Figure 3.3. Association of ClpC, S78, and Hsp60 with translocation complexes during import. Radiolabeled prSS was incubated with isolated chloroplasts in 100 pM ATP. After a 10 minute incubation, intact chloroplasts were repurified, import was initiated by addition of 4 mM ATP at time=0, and aliquots were removed at the given timepoints. Intact chloroplasts were again repurified, and then solubilized in buffer containing decylmaltoside. Ten percent of each timepoint was removed for direct analysis on SDS-PAGE (A, C, and E). The remaining 90% of each timepoint was immunoprecipitated with either anti-ClpC (B), anti-S78 (D), or anti- Hsp60 (F) antibodies. Samples were analyzed by SDS-PAGE and fiuorography. 102 A Import anti- -C|pC I02.55103060Inin I02.55103060Inin <—prSS—> <—mSS—> Import anti- S78 I.0255103060Inin I02.55103060Inin -+prss_>- <—mSS—> Import anti- -Hsp60 I02.55103060Inin I02.55103060Inin d—prSS—b <— mSS—b 103 (Figure 3.3F). Anti-Hsp60 immunoprecipitated complexes containing mSS were first observed in the 2.5-min timepoint, levels of association increased in the 5- min timepoint, and then decreased in the 10-min timepoint. We concluded that both ClpC and S78 interacted with productively bound precursors because these complexes disappeared in later timepoints. Additionally, the time-dependent association of mSS with anti-Hsp60 immunoprecipitated complexes is consistent with interaction of the stromal Hsp60 homologue with newly-imported mSS prior to its incorporation into Rubisco holoenzyme. ClpC and S78 Interact with membrane-associated precursor, but Hsp60 interacts with soluble mSS. Stromal Hsp100 (ClpC), Hsp70 (S78), and Hsp60 homologues all interacted with either prSS or mSS during an import reaction. The question was, are these interactions relevant to protein translocation? If these chaperones interacted with translocating precursor then the complexes should be membrane-associated. To determine whether chaperones interacted with precursors in soluble or membrane-associated complexes, chloroplasts were lysed hypotonically and membrane and soluble protein fractions separated prior to detergent treatment and immunoprecipitation. Because Hsp60 did not interact with prSS at the earliest timepoints in the protein import reaction, but rather displayed greatest interaction with mSS in the 5-min and 10-min timepoints (Figure 3.3F), both binding (Figure 3.4A) and 10-min import reactions were analyzed (Figure 3.4B). First, to assess the relative amounts of prSS in the binding and import reactions 104 aliquots of the membrane and soluble protein fractions were removed prior to immunoprecipitation and analyzed directly by SDS-PAGE (Figure 3.4, R lanes). No import had occurred under the binding conditions as all prSS was associated with the membrane fractions and none had been processed to mature-size (Figure 3.4A, R lanes). Under these conditions, prSS was contained in anti-ClpC and anti-S78 immunoprecipitated complexes only in the membrane fractions, but no prSS could be detected in the anti-Hsp60 immunoprecipitated complexes in either the membrane or soluble protein fractions (Figure 3.4A, l lanes). After ten minutes of import, significant levels of prSS was imported and processed to mSS in the soluble protein fraction (Figure 3.48, compare R lanes). Both anti-ClpC and anti-S78 immunoprecipitated complexes again contained only prSS in the membrane fractions, but anti-Hsp60 immunoprecipitated complexes contained high levels of mSS in the soluble protein fractions (Figure 3.48, I lanes). Interestingly, anti-Hsp60 immunoprecipitated complexes in the soluble protein fraction contained low amounts of prSS in addition to mSS (Figure 3.48, anti-Hsp60 lane). Precursor was not observed in association with anti-Hsp60 immunoprecipitated complexes in the import timecourse (Figure 3.3). Because the prSS is present in the soluble protein fraction we speculate that this interaction reflects a transient interaction of newly imported precursor with Hsp60 prior to its processing to mSS by stromal processing protease. This explanation is consistent with previous results where newly-imported prSS with mutations in the stromal processing protease cleavage-site could be observed in Rubisco holoenzyme in an unprocessed form (Archer and Keegstra, 1993). 105 Figure 3.4. ClpC and S78 interact with membrane-associated prSS, but Hsp60 interacts with mSS in the soluble protein fraction. Radiolabeled prSS was incubated with isolated chloroplasts in 100 pM ATP. After a 10 minute incubation, intact chloroplasts were repurified, and chloroplasts were either Iysed in hypotonic conditions immediately (A), or import was initiated by addition of 4 mM ATP and allowed to proceed for ten minutes prior to reisolation of intact chloroplasts and hypotonic lysis. After lysis, membrane and soluble fractions were separated by centrifugation at 150.0009 for 5 minutes and then solubilized in buffer containing decylmaltoside. Ten percent of the resulting membrane and soluble protein fractions were removed for direct analysis on SDS-PAGE (R lanes, a representative R lane shown only for anti-ClpC immunoprecipitated samples). The remaining 90% of each sample was immunoprecipitated with either anti-ClpC, anti-S78, or anti-Hsp60 antibodies (I lanes). Samples were analyzed by SDS-PAGE and analysis on a phosphorimager (Molecular Dynamics, Inc.). 106 Binding Supernatant Pellet 8601.8 _Hl Ems—HI oeosT R Iomamxé _Hl EwsmI 0a_0-a_HI R 10’ Import Supernatant Pellet 88:8 _HI EwsmI oeosmI H locum—+6 _HI mnmsml oeosmI _H 107 The observation that Hsp60 interacted with mSS in chloroplastic soluble protein fractions indicated that this association most likely was not directly relevant to the protein translocation event, but rather represented a folding or assembly step of newly imported proteins. Such a role for stromal Hsp60 would be consistent with results obtained by others in chloroplasts (Lubben at al., 1989; Maduetlo et al., 1994; Tsugeki and Nishimura, 1993), and mitochondria (Manning-Krieg et al., 1991 ; Osterrnann at al., 1989). ClpC and 878 both interact with translocation complexes, but only the association with ClpC is stable in solubilized complexes. Both ClpC and S78 immunoprecipitated complexes containing prSS were associated with membrane fractions (Figure 3.4), and therefore were potentially relevant to the protein translocation event. Interestingly, earlier experiments had not shown an association of S78 with translocation complexes, whereas now prSS could be detected in anti-S78 immunoprecipitated complexes (compare Figures 2.2 with Figures 3.3 and 3.4). Two differences between the earlier and later experiments were hypotonic lysis of chloroplasts to separate membrane and soluble protein fractions, and centrifugation of the detergent solubilized membrane fractions to remove insoluble material. Since hypotonic lysis of chloroplasts and separation of membrane and soluble protein fractions did not disrupt the ability to detect prSS in anti-S78 immunoprecipitated complexes (Figure 3.4) the presence of a 108 centrifugation step to remove insoluble material might have sedimented complexes containing S78 and prSS. To determine if anti-S78 immunoprecipitated complexes containing prSS were indeed sedimented during centrifugation to remove insoluble material, precursors were allowed to bind to chloroplasts. Intact chloroplasts were re- isolated and immediately solubilized in detergent-containing buffer and then immunoprecipitation was either performed immediately (Figure 3.5A), or after a sedimentation step (Figure 3.5B). The absence of processed mature-sized Rubisco small-subunit (mSS) in aliquots of the solubilized chloroplasts indicated that import had not occurred (Figure 3.5A, lanes 1, 4, and 7). Antibodies to T0075, ClpC, and S78 were able to coimmunoprecipitate prSS from the solubilized chloroplasts (Figure 3.5A, lanes 2, 5, and 8, respectively). No prSS could be detected with the corresponding preimmune controls (Figure 3.5A, lanes 3, 6, and 9), demonstrating the specificity of the immunoprecipitation reactions. If insoluble material was sedimented prior to immunoprecipitation prSS could still be coimmunoprecipitated with anti-Toc75 and anti-ClpC antibodies, but not with anti-S78 antibodies (Figure 3.5B, compare lanes 2 and 6, with lane 10). When the pellet fraction was analyzed, significant amounts of prSS were present (Figure 3.58, lanes 4, 8, and 12). 109 Figure 3.5. Translocation complexes containing S78 are insoluble. Radiolabeled prSS was incubated with isolated chloroplasts in 100 pM ATP. After a 10 minute incubation, intact chloroplasts were repurified and immediately solubilized in buffer containing decylmaltoside. (A) Immunoprecipitation reactions were performed directly after solubilization with anti-Toc75 (lane 2). anti-ClpC (lane 5), or anti-S78 (lane 8) antibodies, and their corresponding preimmune controls (lanes 3, 6, and 9). Ten percent of each reaction was removed for direct analysis by SDS-PAGE (R, lanes 1, 4, and 7); the remaining 90% was split into two equal fractions and immunoprecipitated with immune (I lanes) or their corresponding preimmune sera (P lanes). (B) Solubilized chloroplasts were centrifuged at 150.0009 for 5 minutes before immunoprecipitation with anti-Toc75 (lane 2). anti-ClpC (lane 6). or anti-S78 (lane 10) antibodies, and their corresponding preimmune controls (lanes 3, 7. and 11). Ten percent of each reaction was removed for direct analysis by SDS- PAGE (R, lanes 1, 5, and 9); the remaining 90% was split into equal fractions and immunoprecipitated with immune (l lanes). or their corresponding preimmune sera (P lanes). After centrifugation. the pellet was resuspended in SDS-PAGE buffer and analyzed by SDS-PAGE (Pell, lanes 4. 8, and 12). Samples were analyzed by SDS-PAGE and fluorography. 110 A no centrifugation oc-Toc75 oc-CIpC oc-S78 R | P R l P R' | P prSS-) 123456789 B with centrifugation or-Toc75 oc-CIpC oc-S78 R I PPeII.R | PPeIl.R | PPeII. prSS—) 123456789101112 111 $78 displays different solubilization characteristics than precursor and translocation components. Because complexes precipitated by anti-S78 antibodies sedimented by centrifugation, we concluded that it was either very large or represented an aggregation of translocation complexes or incompletely solubilized membrane fragments. To distinguish between these possibilities additional experiments were performed. First, when visually inspecting the pellet fraction after sedimentation. the pellets appeared greenish—we interpreted this as a possible indication that chloroplastic membranes were being incompletely solubilized by detergent treatment. If this were the case. some precursor bound to translocation complexes might co-fractionate with these insoluble membranes, and be present in the pellet fraction. Presumably. additional detergent washes should further solubilize the membrane fragments thereby releasing precursor and translocation components to the soluble fraction after sedimentation. To see if this was the case. prSS was allowed to bind to chloroplasts in the presence of low ATP conditions. Intact chloroplasts were re-isolated solubilized in detergent-containing buffer, and sedimented by centrifugation. After sedimentation, the supernatant was removed and not analyzed further. A set of isolated pellet fractions were then subjected to a series of sequential washes with either detergent-free buffer (Figure 3.6A). 0r detergent-containing buffer (Figure 3.68). These washes were followed by re-sedimentation and separation of insoluble from soluble fractions which were then solubilized in Laemmli buffer for subsequent SDS-PAGE analysis. Pellet and supernatant 112 Figure 3.6. S78 displays different solubilization characteristics than precursor and translocation components. Radiolabeled prSS was incubated with isolated chloroplasts in 100 pM ATP. After incubation for 10 minutes at room temperature, intact chloroplasts were repurified, Iysed hypotonically, and the chloroplastic membranes were resuspended in buffer containing decylmaltoside. Solubilized chloroplasts were centrifuged at 150.0009 for 5 minutes, and the soluble fraction was removed. Insoluble pellet fractions were then suspended in 200 pL of buffer (A), or buffer containing decylmaltoside (B) from one to five times (lanes 1 to 5). After each resuspension the soluble and insoluble fractions were separated by centrifugation at 150.0009 for 5 minutes and resuspended in SDS-PAGE buffer for analysis by SDS-PAGE. The presence or absence of various proteins were detected by phosphorimager analysis (Molecular Dynamics. Inc.) (prSS), or transferal to lmmobilon-P for immunoblotting with anti-Tic110 (110). anti-ClpC (ClpC). anti-Toc75 (75). anti-S78 (S78), or anti-Rubisco large-subunit (LSU) antibodies. 110 ClpC S78 LSU prSS 1 10 ClpC 75 S78 LSU prSS Pellet 113 lPES Supernatant x 3 4 -' r" 5.33de ‘ ' I’mf.1mi .' rxr‘w- 'GI’Aétk'L'F". . 11::- - firs-3min:- - ll1 . 0.!ifi ‘L’wfllz‘cp" - “pv ~ - - .2 LLL’E'ENIW"? .'~ 11‘ 103‘7-«33. Pellet 3 ..4 . I" :i‘l‘fk'é'u‘_:f:_‘2'g'.’. q '.’_ --- "1. ._ ‘. . .- ,. '1» H. ;« 1‘ r . * II . . . _ ‘ .... . . « , .- .. . - -,;' \ ' a . _ s . . a..'.v...-. N}.-- r‘ (.4 ‘9 .mencrm.¢§n~a-ez W '%.I e%u'nfilms;-.u:wu,;. 33;... . _.,,,,.‘._~ g. ._. .... ... '-..I£~'~a;—~s--- a -'. - -‘-'.“.- 3 i’lfl'.\'.‘ .'o1.'l.'\"~‘f-'-‘M‘- 4 32.33.94. . ,, .... . Jr 1&3- I: . ‘ 1.. . . t ,1. . . .' A mn- fr; . 1. inn-saut- .ma‘v m . .... . ..- .r .‘_.".'.' v.13." 10931593133.»Emmett:- rum-2:37;! I-LII‘. 5 l . ~ . ~..---- . .3“. ‘\ '. . I V‘l‘l .- . e‘ki-.-.~a6r.“ u d.‘ I?;s‘;:‘;tt;ifi;d‘flsn" ...... s * If" . I . IRE: i'iffi'fl- A'* vim-ap- gmm’nrz "-‘f 1.15- fink“. itmz’i’"} this ‘1‘. .v: I 1 - j. - 4.» .. . _ B.JIJI.‘ Ian-Ingmar I'M“...IDAQ¥‘0<’1~ Iv‘r‘n ms. ~~ l ‘-“.:'.-: ‘fql-l’ -:'I~i‘~n.“‘!3 Bah")? ‘- "Z’Z‘L‘PlAJ; $51“; 1:53:32“. 3.3.". ".."f .3 A 116.6»: :3? - '. a“: NWT-1:5.”- " ,_ - 7a.}. .,...>._.;..q..v,rM-.. ‘37,.11.._-_t;.,-.; s.t..~- - an. «'iJ-‘I‘K-L;¢:-‘=-1‘.-'-'-:I'Ii ‘ . ..- . _ ., - -~ I . 3’ a. I“. ~ ... .I. . .. i‘lt'l'difl'l'tfi quiz”... .I._._ k - . '" " ’ " bf. . , "T .1 . «u—-’ ‘C v."_ :fr'r "‘ :. 5w; yq- ‘t‘tfibiffw‘ ~ 3' WIFE-J - : _ Jan: - , . ..., “[41“, _ : ::." ' , ' . ' ..‘ ' " 4"."‘I\.~ '. . ‘3']. ... _. I -‘ ‘. : t ‘,a '1‘m£$'wxr-'v‘.- y ‘ r u I v ‘- ~-~-.~va-utrvu~ - . new E-z-mmxmrufi . '.: . .. .- I: i if , ., w M 3 . .9 j j . ‘ _. I... . ...: {shquen‘cv Ln. . libi..4‘.‘- Lathe ".3: ...;q.& finer“ .1. ... - ~ by ".,,.,.,.,..._. V'I'V‘l' '1 91.“ 114 fractions were analyzed for the presence of precursor by autoradiography, and for the presence of integral membrane translocation components. T0075 and Tic110, the molecular chaperones ClpC. and S78, and Rubisco large-subunit by immunoblotting techniques. If detergent was left out of the washes. the translocation components T0075 and Ti0110. the chaperones ClpC and S78, Rubisco large-subunit, and prSS all remained in the pellet fraction even after five washes (Figure 3.6A). Significant amounts of all these proteins were observed in the initial supernatant fractions (Figure 3.6A. supernatant lanes). however since no previous washes had been performed it is likely these represented contaminating soluble membrane proteins and soluble proteins that were not completely removed after the first sedimentation step. If detergent-containing buffer was used to wash the pellets Toc75. Tic110. prSS. and ClpC appeared to be almost completely extracted from the pellet to the supernatant fraction after the fourth wash, but both S78 and Rubisco large-subunit remained primarily in the pellet fractions (Figure 3.6B. compare pellet and supernatant lanes). The observation that additional detergent washes removed prSS and translocation components from the pellet fraction was consistent with prSS and translocation components being present in the pellet fractions due to incomplete solubilization of chloroplastic membranes during the initial detergent solubilization step. However. while ClpC co-fractionated with these translation components, S78 appeared not to. and was found primarily in detergent insoluble complexes along with Rubisco large-subunit, even after repeated 115 detergent washes (Figure 3.6B). One reason for this might be the presence of these two proteins in aggregates. Hsp70s have been observed in association with protein aggregates in many systems (Gething et al., 1986; Pelham, 1990; Skowrya et al., 1990). In many cases this association is ATP—dependent (Gething and Sambrook, 1992). If S78 and LSU are present in distinct complexes from prSS and the membrane-associated translocation complexes. it might be possible to separate them by addition of ATP in detergent-free buffer. Since prSS and the translocation complexes are membrane-associated. they should remain in the pellet fraction whereas. S78 and possibly also Rubisco large-subunit would be released to the supernatant. Pellet fraction from detergent solubilized membranes of chloroplasts to which precursor was bound were washed three times with detergent-free buffer to remove any soluble proteins. These pellet fractions were then incubated for 30 minutes in the absence (Figure 3.7A) or presence (Figure 3.7B) of Mg'ATP. Following this incubation insoluble material was sedimented by centrifugation. The pellet and SUpematant fractions were suspended in Laemmli buffer and analyzed by SDS-PAGE. The presence of prSS was detected by autoradiography, and the presence of Toc75, Ti0110. ClpC, S78, and Rubisco large-subunit were detected by immunoblotting techniques (Figure 3.7). Precursor as well as the translocation components T0075 and Tic110, and the molecular chaperone ClpC, remained in the pellet fractions regardless of whether ATP was present or not (Figures 3.7A and 3.78. compare P lanes). A portion of the molecular chaperone S78 could be released from the pellet 116 Figure 3.7. Some S78 is released from detergent-insoluble material by addition of ATP. Radiolabeled prSS was incubated with isolated chloroplasts in 100 pM ATP. After incubation for 10 minutes at room temperature. intact chloroplasts were repurified, Iysed hypotonically. and the chloroplastic membranes were resuspended in buffer containing decylmaltoside. Solubilized chloroplasts were centrifuged at 150.0009 for 5 minutes, and the soluble fraction was removed. Insoluble pellet fractions were then suspended in 200 pL of buffer (A), or buffer containing 10 mM Mg'ATP (B) and incubated at 4°C for one hour. The soluble and insoluble fractions were separated by centrifugation at 150.0009 for 5 minutes and resuspended in SDS-PAGE buffer for analysis by SDS-PAGE. The presence or absence of various proteins were detected by phosphorimager analysis (Molecular Dynamics, Inc.) (prSS). or transferal to lmmobilon-P for immunoblotting with anti-Tic110 (110), anti-ClpC (ClpC), anti-Toc75 (75), anti- S78 (S78), or anti-Rubisco large-subunit (LSU) antibodies. 117 Tic1 10- ClpC- Toc75- S78- LSU- - . c , ‘4." ' ‘1! HI ..‘I . {. 3: prSS- ' .. > > I ‘ f. r ‘ - I l .1“ ‘. . — ». 118 fraction to the supernatant when in the presence of ATP (Figures 3.7A and 3.7B. compare S lanes). Rubisco large-subunit remained in the pellet fraction whether ATP was present or not (Figures 3.7A and 3.7B. P lanes). Since prSS. the translocation components T0075 and Tic110. and ClpC all follow the same detergent-extraction. and ATP insensitivity patterns this is consistent with their association into translocation complexes. However S78 is not solubilized by detergent, and is released from the pellet fraction by ATP. These different migration patterns are consistent with the explanation that $78 and translocation complexes are both present in the insoluble fraction after initial detergent solubilization of chloroplasts, but are not associated with one another. To evaluate alternative explanations for the presence of molecular chaperones in the solubilized complexes additional control reactions were performed. First. radiolabeled prSS was not associated with complexes immunoprecipitated with anti-ClpC or anti-S78 in the absence of chloroplasts (data not shown). Therefore. molecular chaperones present in the wheat-gerrn translation system were not responsible for the association of prSS with the anti- ClpC or anti-S78 immunoprecipitates. Second, when immunoprecipitation of prSS bound to chloroplasts was performed after the proteins were denatured by boiling in two percent SDS. no prSS could be found in complexes precipitated by anti-ClpC or anti-S78 antibodies (data not shown). This experiment demonstrated that the antisera did not cross-react with prSS, causing direct immunoprecipitation. Third, chloroplasts were solubilized in immunoprecipitation buffer before radiolabeled precursor was added. When comparable amounts of 119 precursor were present. coimmunoprecipitation of prSS was not observed (data not shown). We concluded that formation of complexes required intact chloroplasts. and did not occur as a result of mixing of the stromal compartments with the exterior of the chloroplast during solubilization. These results, combined with the observation that presolubilized chloroplasts did not support association between ClpC and prSS, further supported an interaction between ClpC and translocation complexes during binding that is relevant to protein import. DISCUSSION Chloroplasts contain members of several molecular chaperone families in their stromal compartments. In addition to the stromal Hsp100 homologue. ClpC, whose presence in chloroplastic protein translocation complexes was discussed in Chapter 2. three homologues of the Hsp70 molecular chaperone family have been identified in chloroplasts (Marshall at al., 1990). two in the stroma (S78 and S75), and one associated with the outer envelope membrane (E75). Two homologues of the Hsp60 molecular chaperone family (alpha and beta) are present in the stroma of chloroplasts (Hemmingsen at al., 1988). These Hsp60 homologues associate with one another, and together act with a chloroplastic form of their co-chaperone Hsp10 as a folding machinery for proteins newly synthesized on the chloroplastic translation machinery, as well as 120 several newly imported proteins synthesized on the cytoplasmic translation machinery (Lubben at al., 1989; Madueilo at al., 1994; Tsugeki and Nishimura, 1993). In Chapter 2 we utilized detergent solubilization techniques in conjunction with immunoprecipitation and sucrose density fractionation techniques to identify chloroplastic protein translocation complexes formed in the presence of low ATP concentrations. During characterization of the components of these chloroplastic protein translocation complexes we detected the presence of a stromal Hsp100 molecular chaperone, ClpC. In this Chapter we have further characterized the association of ClpC with translocation complexes during precursor import into chloroplasts. In addition we have expanded our analysis of the role of stromal molecular chaperones in chloroplastic protein translocation to include two additional molecular chaperones, namely stromal members of the Hsp70 (S78), and Hsp60 chaperone families. The experiments in this Chapter resulted in three significant observations. First, both S78 and ClpC could be found associated with prSS after detergent solubilization of chloroplastic membranes, but only the complex containing ClpC and prSS remained in the soluble fraction after sedimentation was performed to remove insoluble material. Second, further examination of the insoluble fraction suggested that although both S78 and translocation complexes containing prSS were present, that these two complexes were not directly associated with one another. Third. Hsp60 could be found associated in a soluble complex with newly-imported mSS in the stromal compartment. 121 The data presented in this paper further support a role for the stromal Hsp100 homologue. ClpC. as a component of the translocation complex. Three experimental results provided additional evidence that the association of ClpC with the chloroplastic protein translocation apparatus was physiologically significant. First, the amount of ClpC associated with precursor-containing translocation complexes decreased in a time-dependent manner during an import reaction (Figure 3.3). We interpreted this in terms of a dynamic interaction of ClpC and chloroplastic translocation components with the translocating precursor. In this scheme. dissociation occurs upon release of the precursor into the stroma resulting in decreasing coimmunoprecipitation of precursor as the import reaction progresses. Second, the association of precursor with anti-ClpC precipitated complexes occurred only in the membrane fractions of Iysed chloroplasts during both binding and import conditions (Figure 3.4). In other words. ClpC interacted only with precursors translocating across membranes. This implied a tight linkage between ClpC and membrane-localized protein translocation machinery. and was consistent with the observation in Chapter 2 that ClpC was found in protein translocation complexes even in the absence of added precursor (Figure 2.4. Chapter 2). Finally, although a portion of precursor associated with ClpC was pelleted with insoluble material after initial detergent solubilization of chloroplastic membranes, the ClpC present in this fraction demonstrated similar detergent extraction profiles to both precursor and the integral membrane translocation components. T0075 and Tic110 (Figure 3.5). We interpret this result as indication that the majority of the ClpC present in 122 the insoluble fraction after an initial detergent solubilization is in association with protein translocation complexes present in residual membrane fragments. Members of the Hsp70 molecular chaperone family are essential components of the protein translocation systems of both mitochondria and the endoplasmic reticulum (Kang at al., 1990; Sanders et al., 1992). Is the stromal Hsp70 homologue S78 involved in protein import into chloroplasts? Precursor could be coimmunoprecipitated by anti-S78 antibodies under binding and import conditions (Figures 3.3 and 3.4), but upon further examination these complexes were sedimented by a centrifugation step to remove insoluble material (Figure 3.5). This coimmunoprecipitation was not simply due to a non-specific precipitation of insoluble protein aggregates or association of S78 with residual membrane fragments. as no precursor was detected in the preimmune control. This result indicated the formation of a complex that contained both S78 and precursor must have occurred either during the import reaction, or during the detergent solubilization of the reisolated chloroplasts. Additionally. precursor coimmunoprecipitated by anti-S78 antibodies appeared to be a true import intermediate (Figure 3.3), and was dependent on intact chloroplasts (data not shown). For these reasons. we cannot at this point completely eliminate the possibility of an interaction between $78 and translocating precursor. However. while ClpC associated with detergent-soluble translocation complexes, the complexes containing S78 and precursor were sedimented after a centrifugation step. The relevance of this S78-precursor interaction to protein import was called into question by the observation that non-translocation 123 components were also found in this pellet (Figure 3.5). When this pellet fraction was subjected to further detergent extraction steps precursor, known translocation components T0075 and Tic110. and ClpC were all released to the supernatant fraction. but S78 remained largely in the pellet (Figure 3.6). Also, in the absence of detergent S78’s association with the pellet fraction was susceptible to added ATP, but precursor and translocation components all remained in the pellet (Figure 3.6). Finally, S78 did not cosediment with prSS and other translocation components on a sucrose gradient (data not shown). These results are all consistent with the interpretation that both precursor- containing translocation complexes and S78 are present in the pellet fraction, but that the majority of S78 is not directly associated with precursor-containing translocation complexes. Previously. outer membrane Hsp70 homologues have been identified as members of translocation complexes (Schnell et al., 1994; Waegemann and Soll, 1991; K0 at al., 1992). It was proposed that these proteins might play a role in insertion of precursors through the outer membrane translocation complex. Because the S78 antiserum was specific to the stromal Hsp70 and did not cross- react with the outer membrane Hsp70 (Figure 3.2; Akita at al., 1997) we were unable to confirm or refute these findings in this study. Stromal Hsp60 does not appear to be directly involved in protein translocation in chloroplasts. Several experimental results provided evidence that Hsp60 interacted with newly-imported proteins in a manner consistent with a folding. or assembly function rather than as a chloroplastic protein translocation 124 component. First. precursor could be detected in association with Hsp60 only after import had been initiated by addition of high levels of ATP (Figure 3.4). This association was only observed after significant levels of mSS had accumulated. presumably by completion of import and subsequent processing in the stromal compartment (Figure 3.3). Second. Hsp60 associated with both mSS and a small amount of prSS in a complex that was located in the soluble protein fraction of Iysed chloroplasts. not the membrane fraction (Figure 3.4). These observations were consistent with an interaction between Hsp60 and importing protein that occurred only after translocation was complete. Hsp60s have recently been detected in association with the inner membrane translocation component. Tic110 and newly-imported mSS (Kessler and Blobel. 1996). As Tic110 is an integral membrane protein. these results were interpreted as evidence that Hsp60 interacted with the membrane- associated protein translocation complex. Whether these complexes were truly membrane-associated was not demonstrated though. as separation of chloroplastic membrane and soluble protein fractions was not performed prior to immunoprecipitation of these complexes (Kessler and Blobel. 1996). Although Hsp60 was detected in association with mSS in this study. mSS interacted with Hsp60 in a complex which migrated in the soluble fraction, not the membrane fraction of chloroplasts (Figure 3.4). The presence of mSS in envelope membranes has not been previously reported. but Hsp60 has been detected in association with newly-imported proteins in chloroplastic soluble fractions 125 (Gatenby at al., 1988; Lubben at al., 1989; Madueno at al., 1994; Tsugeki and Nishimura. 1993). The observation that the association between Hsp60 and mSS disappeared in a time-dependent manner suggested that either mSS was degraded. or assembled into Rubisco holoenzyme. We favor the latter possibility as analysis of the immunoprecipitation supernatant fraction showed high levels of mSS (data not shown). This interpretation is consistent with the previous observation that newly-imported mSS is incorporated into Rubsico holoenzyme after a transient association with stromal Hsp60 (Lubben et al., 1989; Gatenby at al., 1988). Additionally, others have demonstrated that Hsp60 interacts with newly-imported stromal intermediates of several thylakoid-targeted proteins (Madueflo at al., 1994; Tsugeki and Nishimura, 1993). Using coimmunoprecipitation techniques. we evaluated the roles of three stromal molecular chaperones. ClpC, S78. Hsp60. in chloroplastic protein translocation. Based on results of these experiments ClpC, a stromal Hsp100 homologue, appears to associate with precursor-containing complexes in a manner consistent with it having a physiologically significant role in chloroplastic protein translocation. S78 also could be found associated with precursor, however these proteins probably did not directly interact with one another. Rather their coimmunoprecipitation probably occurred as a result of an interaction of insoluble protein aggregates and residual membrane fragments containing protein translocation complexes. Finally, Hsp60 appears to associate 126 with newly-imported prSS and processed mSS in a manner consistent with a role in folding or assembly rather than protein translocation. 127 REFERENCES Akita, M., Nielsen, E. and Keegstra, K. (1997) Identification of protein transport complexes in the chloroplastic envelope membranes via chemical cross-linking. J. Cell Biol., 136, 983-994. Bochkareva, E.S., Lissin, NM. and Girhovich, A.S. (1988) Transient association of newly-synthesized unfolded proteins with the heat-shock GroEL protein. Nature, 336, 254-257. Bruce,B., Perry,S., Froehlich,J. and Keegstra,K. (1994) In vitro import of proteins into chloroplasts. Plant Molecular Biology Manual J1. Kluwer Academic Publishers, Belgium. pp. 1-15. Craig, E.A., Kramer, J., Shilling, J., Wemer-Washbume, M., Holmes, 3., Kosic-Smithers, J. and Nicolet, C.M. (1989) SCCI, an essential member of the yeast HSP70 multigene family, encodes a mitochondrial protein. Mol. Cell. Biol., 9, 3000-3008. Economou, A. and Wickner, W. (1994) SecA promotes preprotein translocation by undergoing ATP-driven cycles of membrane insertion and deinsertion. Cell, 78, 835-843. Gatenby, A.A., Lubben. T.H., Ahhlquist, P. and Keegstra, K. (1988) Imported large subunits of ribulose bisphosphate carboxylase/oxygenase, but not import B-ATP synthase subunits. are assembled into holoenzyme in isolated chloroplasts. EMBO J., 7, 1307-1314. Gatenby, A.A. and Ellis, R.J. (1990) Chaperone function: the assembly of ribulose bisphosphate carboxylase-oxygenase. Annu. Rev. Cell Biol., 6, 125- 149. Gething, M.J., McCammon, K. and Sambrook, J. (1989) Protein folding and intracellular transport: evaluation of conformational changes in nascent exocytic proteins. J. Math. Cell. Biol., 32, 185-206. Gething, M.J. and Sambrook, J. (1992) Protein folding in the cell. Nature, 355, 33—45. Glick, 8.3. (1995) Can Hsp70 proteins act as force generating motors? Cell, 60, 1 1-14. 128 Hemmlngsen, S.M., Woolford, C., van der Vies, S., Tilly, K., Dennis, D.T., Georgopoulous, C.P., Hendrix, R.W. and EIIis,R.J. (1988) Homologous plant and bacterial proteins chaperone oligomeric protein assembly. Nature, 333, 330- 334. Hendrlck, J.P. and Hartl, F.-U. (1993) Molecular chaperone functions of heat-shock proteins. Ann. Rev. Biochem., 62, 349-384. Kang, P.-J., Ostennann, J., Shilling, J., Neupert, W., Craig, EA. and Pfanner, N. (1990) Requirement for hsp70 in the mitochondrial matrix for translocation and folding of precursor proteins. Nature, 348, 137-143. Kessler, F. and Blobel, G. (1996) Interaction of the protein import and folding machineries in the chloroplast. Proc. Natl. Acad. Sci. USA. 93, 7684-7689. K0, K., Bornemisza, O., Kourtz, L., K0. Z.W., Plaxton, W.C. and Cashmore, AR. (1992) Isolation and characterization of a cDNA clone encoding a cognate 70-kDa heat shock protein of the chloroplast envelope. J. Biol. Chem, 267, 2986-2993. Kusukawa, N., Yura, T., Ueguchi, C., Akiyama, Y., and Ito, K. (1989) Effects of mutations in heat-shock genes groES and groEL on protein export in Escherichia coli. EMBO J., 8. 3517-3521. Laemmli,U.K. (1970) Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature, 227, 680-685. Lecker, S., Lill, R., Ziegelhoffer, T., Georgopoulous, C., Bassford, P.J., Kumamoto, C.A. and Wickner, W. (1989) Three pure chaperone proteins of Escherichia coli—SecB, trigger factor, and GroEL—form soluble complexes with precursor proteins in vitro. EMBO J., 8, 2703-2709. Lubben, T.H., Donaldson, G.K., Viitanen, P.V. and Gatenby, A.A. (1989) Several proteins imported into chloroplasts form stable complexes with the GroEL-related chloroplast molecular chaperone. Plant Cell, 1, 1223-1230. Luirink, J., ten Hagendongman, C.M., van der Weijden, C.C., Oudega, B., High, 8., Dobberstein, B., and Kusters, R. (1994) An alternative protein targeting pathway in Escherichia coli. studies on the role of FtsY. EMBO J., 13. 2289-2296. Maduel‘lo, F., Napier, J.A. and Gray, J.C. (1993) Newly imported Rieske iron-sulfur protein associates with both Cpn60 and Hsp70 in the chloroplast stroma. Plant Cell, 5, 1865-1876. Manning-Krieg, U.C., Scherer, PE. and Schatz, G. (1991) Sequential action of 129 mitochondrial chaperones in protein import into the matrix. EMBO J., 10, 3273-3280. Marshall, J.S., DeRocher, A.E., Keegstra, K. and Vierling, E. (1990) Identification of heat shock protein hsp70 homologues in chloroplasts. Proc. Natl. Acad. Sci. USA, 87, 374-378. Marshall, J.S. and Keegstra, K. (1992) Isolation and characterization of a cDNA clone encoding the major Hsp70 of the pea chloroplastic stroma. Plant Physiol, 100, 1048-1054. Miller, J.D., Bernstein, H.D., and Walter, P. (1994) Interaction of E. coli thl4.5$ ribonucleoprotein and FtsY mimics that of mammalian signal recognition particle and its receptor. Nature, 367. 657-659. Moore, T. and Keegstra, K. (1993) Characterization of a cDNA clone encoding a chloroplast-targeted Clp homologue. Plant Mol. Biol., 21, 525-537. Olsen, L.J., Theg, S.M., Selman, B.R., and Keegstra, K. (1989) ATP is required for the binding of precursor proteins to chloroplasts. J. Biol. Chem, 264, 6724-6729. OIsen,L.J. and Keegstra,K. (1992) The binding of precursor proteins to chloroplasts requires nucleoside triphosphates in the intermembrane space. J. Biol. Chem, 267, 433-439. Ostermann, J., Horwlch, A.L., Neupert. W. and Hartl, F.-U. (1989) Protein folding in mitochondria requires complex formation with hsp60 and ATP hydrolysis. Nature. 341, 125-130. Pelham, H.R.B. (1989) Heat shock and the sorting of ER lumenal proteins. EMBO J., 8, 3171-3176. Sanders, S.L., Whitfield, K.M., Vogel, J.P., Rose, MD. and Schekman, R.W. (1992) Se061p and BiP directly facilitate polypeptide translocation into the ER. Cell, 69, 353-365. Schatz,G. and Dobberstein,B. (1996) Common principles of protein translocation across membranes. Science, 271, 1519-1526. Scherer,P.E., Krieg,U.C., Hwang,S.T., Vestweber,D. and Schatz,G. (1990) A precursor protein partly translocated into yeast mitochondria is bound to a 70 kd mitochondrial stress protein. EMBO J., 9, 4315-4322. 130 SchmitLM., NeuperLW. and Langer,T. (1995) Hsp78, a Clp homologue within mitochondria, can substitute for chaperone functions of mt-hsp70. EMBO J., 14, 3434-3444. Schnell, D.J., Kessler, F. and Blobel, G. (1994) Isolation of components of the chloroplast protein import machinery. Science, 266, 1007-1012. Shanklin,J., DeWitLND. and Flanagan,J.M. (1995) The stroma of higher plant plastids contain ClpP and ClpC. functional homologs of Escherichia coli ClpP and CIpA: An archetypal two-component ATP-dependent protease. Plant Cell, 7, 1713-1722. Skowyra, D., Georgopoulous,C. and Zylicz, M. (1990) The E. coli dnaK gene product, the hsp70 homolog. can reactivate heat inactivated RNA polymerase in an ATP hydrolysis-dependent manner. Cell, 62, 939-944. Towbin, H. and Gordon, J. (1984) lmmunoblotting and dot immunoblotting— current status and outlook. J. Immunol. Methods, 72, 313-340. Tsugeki, R. and Nishimura, M. (1993) Interaction of homologues of Hsp70 and Cpn60 with ferredoxin-NADP+ reductase upon its import into chloroplasts. FEBS Left, 320, 198-202. Vogel, J.P., Misra, L.M. and Rose, MD. (1990) Loss of BiP/GRP78 function blocks translocation of secretory proteins in yeast. J. Cell Biol., 110, 1885-1895. Waegemann,K. and Soll,J. (1991) Characterization of the protein import apparatus in isolated outer envelopes of chloroplasts. Plant J., 1, 149-158. Chapter 4 CLPC INTERACTS WITH THE CHLOROPLASTIC PROTEIN IMPORT MACHINERY IN A PHYSIOLOGICALLY RELEVANT MANNER 131 132 INTRODUCTION Molecular chaperones have a diverse array of roles in biological systems including an essential role in the transport of proteins across biological membranes. Molecular chaperones also display considerable structural diversity, with members of the Hsp60 and Hsp70 families being the best characterized of several different protein families. Recently members of the Hsp100 protein family have been demonstrated to be molecular chaperones (Wickner at al., 1994; Wawrzynow at al., 1995). The results we obtained in Chapters 2 and 3 suggested that the stromal Hsp100, ClpC, might be involved in protein translocation into chloroplasts. Because the involvement of members of the Hsp100 protein family in protein translocation is a new role for this class of proteins we wished to examine the role of this protein in the chloroplastic protein import process in further detail. Comparison of Hsp100 protein sequences has revealed at least three distinct subfamilies, i.e. the CIpA, ClpB. and ClpC families. Each of these groups contain two nucleotide-binding domains, and in all cases where tested. Hsp100s have ATPase activity (Woo et al., 1992; Wawrzynow et al., 1994; Wilson et al., 1995). These two nucleotide-binding domains are flanked by amino-terminal, middle. and carboxy-terrninal regions (Squires and Squires, 1992). The three Clp subfamilies are distinguished by their structural organization and consensus sequence features. the most important of these features being the size of the middle region (Schirmer et al., 1996). 133 The first member of this protein family to be functionally characterized was the ClpA protein of Escherichia coli. This protein was discovered as a component of an ATP-dependent protease, and it was named for its ability to proteolyze casein, hence the name Clp (QaseinoLytic Erotease). Upon further characterization of the Escherichia coli protease activity indicated that the ClpA protein had no intrinsic protease activity. and ATP-dependent proteolysis of casein was supported only when ClpA was associated with the unrelated protease subunit. ClpP (Hwang et al., 1987). However, as more Hsp100s have been identified, it now seems likely that most members of this protein family are not involved in proteolysis (Schirmer at al., 1996). Since the identification of this first Hsp100, the number of roles attributed to the Hsp100 molecular chaperone family has expanded greatly. ClpA has subsequently been shown to act independently of the ClpP protease subunit in promoting the DNA-binding activity of the P1 plasmid origin-binding replication factor (Wickner at al., 1994). Hsp104. a cytoplasmic Hsp100 homologue found in eukaryotic cells, plays essential roles in heat-stress tolerance and translational termination in Saccharomyces cerevisea (Sanchez and Lindquist, 1990; Chemoff et al., 1995). In Bacillus subtilis. a C-type Hsp100 is required for salt-stress tolerance and genetic competence (Kruger at al., 1994; Msadek at al., 1994). In mitochondria. a matrix-localized Hsp100 can partially substitute for a temperature-sensitive mutations in matrix-Hsp70 and restore mitochondrial protein translocation competence in this organelle (Schmitt et al., 1995). 134 Chloroplasts contain at least one member of the Hsp100 molecular chaperone family, ClpC (Gottesmann at al., 1990; Moore and Keegstra, 1993). This protein has subsequently been shown to interact with a stromal ClpP subunit to form an ATP-dependent protease (Shanklin at al., 1995). Whether the chloroplastic Hsp100, ClpC. had other activities than in proteolysis was unknown. Using coimmunoprecipitation techniques we demonstrated that ClpC interacted with multiple precursors utilizing the protein import apparatus of chloroplasts. and that the interaction of ClpC with precursors is ATP-dependent (Nielsen at al., 1997). We have also examined in more detail the nature of the association of ClpC with chloroplastic membranes. The results of these observations indicated that ClpC specifically interacts with chloroplastic envelope membranes. and that this interaction involved an association that was resistant to extraction conditions typically sufficient to remove peripherally-associated membrane proteins. A model for the action of ClpC in chloroplastic protein translocation is presented. MATERIALS AND METHODS Isolation of chloroplasts Chloroplasts were isolated from 8 to12-day-old pea seedlings (Pisum sativum var. little marvel) as previously described (Bruce at al., 1994), and suspended in import buffer (50 mM HEPES-KOH pH 8.0. 300 mM Sorbitol) at a concentration of 1 mg chlorophyll/ml. 135 In vitro translation of precursor proteins Transcription of mRNA was performed as previously described (Bruce at al., 1994). Plasmids containing cDNA clones of precursors to Rubisco small-subunit (pRBCS, PstI-cut; Olsen and Keegstra, 1992). plastocyanin (pPPC, EcoRI-cut; Bauerle at al., 1991). Iight-harvesting-complex-protein (pAB80. EcoRl-cut; Payan et al., 1991). and 14-kDa outer envelope protein (p14kom. EcoRI-cut; Li et al., 1991) were linearized using the appropriate restriction enzymes, transcribed into mRNA. mRNAs were translated and labeled with 35S-Methionine (NEN-DuPont) as previously described (Bruce at al., 1994). After translation, residual nucleotides were removed by gel-filtration as previously described (Olsen at al., 1989) Chloroplastic binding reactions 50 pl of isolated. intact chloroplasts (1mg chlorophyll/ml) that had been pre- treated with 5pM nigericin to inhibit photophosphorylation were mixed with in vitro translated precursors (1 x 10° dpm) in 150 pl import buffer supplemented with 100 pM ATP. Reactions were incubated at room temperature in the dark for 10 minutes. The reactions were terminated by reisolation of intact chloroplasts with associated precursors by sedimentation through a 40% Percoll cushion. 136 Immunoprecipitation Repurified. intact chloroplasts were hypotonically lysed in 200 pl lysis buffer (25 mM HEPES-KOH pH 8.0. 4 mM MQCIz). The lysis reaction was incubated on ice for 5 minutes in the dark, and the supernatant and membrane fractions were separated by centrifugation (5 minutes, 100.0009, Sorvall RP100-AT2). Isolated chloroplastic membranes and supernatant fractions were suspended in 1 ml of IPES-DM (25 mM HEPES—NaOH pH 7.5. 50 mM NaCl. 2 mM EDTA pH 8.0, 2 mM EGTA pH 8.0, 1 mM PMSF, 1% wlv decylmaltoside), and incubated for 5 minutes on ice in the dark. If immunoprecipitations were performed in the presence of nucleotides, Mg'ATP or Na'GTP (supplemented with equimolar MgClz) were included in the lysis and IPES-DM buffers at a concentration of 10 mM. Insoluble material was removed from the detergent-solubilized fractions by a centrifugation step (5 minutes. 100.0009. Sorvall RP100-AT2). The resulting supernatant was immunoprecipitated affinity-purified lgGs (15 pg ClpC) . and 10 mg (dry weight) lPES-DM pre-washed Protein A Sepharose CL-4B (Pharrnacia. Inc.). Immunoprecipitation was carried out for 2 hours at 4°C, in the dark. lmmunoprecipitated pellets were washed three times with 1 ml lPES-DM. and once with 1 ml lPES (without decylmaltoside). and resuspended in SDS-PAGE sample buffer (Laemmli. 1970). 137 Isolation of envelope and thylakoid membrane fractions Purified envelope membrane. and thylakoid membrane fractions were obtained using a previously published protocol (Keegstra and Yousef. 1986). with some modifications. Briefly, chloroplasts were lysed in hypotonic conditions at a concentration of 0.3 mglml in lysis buffer. and frozen at -20°C to ensure chloroplastic lysis. After the Iysed chloroplasts were thawed, thylakoids and crude envelope fractions were separated by centrifugation in an HB-6 rotor at 5250 rpm for 10 minutes. The supernatant from this spin was then removed and subjected to centrifugation at 22,250 rpm for 30 minutes in a 88-34 rotor. The resulting crude envelope membrane pellet was resuspended in 0.3 M sucrose in lysis buffer and overlayed on a sucrose step gradient and centrifuged in an SW28.1 rotor at 28,000 rpm overnight. Purified thylakoid membranes were obtained by resuspending the crude thylakoid membrane pellet obtained from above at the HB-6 centrifugation step. The thylakoid pellet was resuspended in 2.0 M Sucrose in lysis buffer, and this was overlaid with a sucrose step gradient consisting of 4 mL 0.46 M sucrose in lysis buffer/ 4 mL 1.2 M sucrose in lysis buffer! 4 mL 1.6 M sucrose in lysis buffer! 4 mL resuspended thylakoid membranes in 2.0 M sucrose in lysis buffer. After flotation centrifugation for 14 hours at 25,000 rpm in a SW28.1 rotor. purified thylakoid membranes were isolated from the 1.2 MI 1.6 M sucrose interface. diluted three-fold in lysis buffer and pelleted in an RP-AT4 at 100,000 rpm for 20 minutes. The resulting thylakoid membrane pellet was resuspended in lysis buffer. 138 Extraction of membrane proteins 50 pl of isolated. intact chloroplasts (1mg chlorophyll/ml) that had been pre- treated with 5pM nigericin to inhibit photophosphorylation were mixed with in vitro translated precursors (1 x 106 dpm) in 150 pl import buffer supplemented with 100 pM ATP. Reactions were incubated at room temperature in the dark for 10 minutes. The reactions were terminated by reisolation of intact chloroplasts with associated precursors by sedimentation through a 40% Percoll cushion. Repurified, intact chloroplasts were hypotonically lysed in 200 pl lysis buffer. The lysis reaction was incubated on ice for 5 minutes in the dark, and the supernatant and membrane fractions were separated by centrifugation (10 minutes, 100.0009. Sorvall RP100-AT2). Chloroplastic membranes were extracted three times in 200 pL of lysis buffer, 1 M KCI, or 100 mM Na2C03 pH 11.5 with reisolation of extracted membranes by centrifugation (10 minutes. 100.0009, Sorvall RP100-AT2) after each extraction. Extracted proteins (supernatant fractions) were acetone precipitated at -20°C overnight and suspended in lysis buffer. Preparation of antibodies All antibodies were polyclonal and raised in rabbits. Affinity-purified, anti-ClpC IgG (Shanklin et al., 1995) was a gift from J. Shanklin. Antiserum to LHCP (Payan and Cline, 1991) was a gift from K. Cline. Antisera to T0075. Tic110, and S78 were raised as discussed in Chapter 3. 139 Electrophoresis and lmmunoblotting All electrophoresis was performed as previously described in Chapter 2. RESULTS A translocation complex containing ClpC is formed with several chloroplast-targeted precursors, and is destabilized by ATP. If ClpC has a general role in protein import it should associate with translocation complexes formed during import of precursors other than prSS. To test this prediction, several precursors, targeted to different chloroplastic compartments were allowed to associate with chloroplasts under low ATP conditions to form putative translocation complexes. The putative complexes were then analyzed by coimmunoprecipitation with anti-ClpC antibodies. In addition to prSS. we examined the precursor to plastocyanin (prPC), a thylakoid lumenal protein. and the precursor to LHCP (prLHCP), a thylakoid membrane protein. Association of the various precursors with chloroplasts during binding was analyzed (Figure 4.1, prSS. prPC, prLHCP. R lanes). All three precursors could be coimmunoprecipitated with anti-ClpC antibodies (Figure 4.1, prSS, prPC, prLHCP. l lanes). To demonstrate that the association 140 Figure 4.1. ClpC interacts with translocation complexes formed by other precursors that use the general translocation apparatus. Radiolabeled OEP14, prSS. prPC, and prLHCP were incubated with isolated chloroplasts in 100 pM ATP. After a 10 minute incubation. intact chloroplasts were repurified, Iysed hypotonically. and the isolated chloroplastic membranes were solubilized in buffer containing decylmaltoside. Twenty percent of each reaction was removed for direct analysis by SDS-PAGE (R lanes); the remaining 80% was immunoprecipitated by anti-ClpC antibodies (l lanes). Samples were analyzed by SDS-PAGE and fluorography. 141 OEP14 prSS prPC prLHCP IR .IIR IIIR IIIR II <—prLHCP OEP14-b 142 of ClpC was specific to precursors associated with the chloroplastic protein- import apparatus, radiolabeled OEP14 was allowed to associate with chloroplasts before solubilization and immunoprecipitation with anti-ClpC antibodies. This protein inserts into the outer membrane of chloroplasts, but does not have a cleavable N-terminal transit peptide and does not utilize the general import apparatus (Li at al., 1991). This protein associated with chloroplasts in amounts comparable to the other precursors tested (Figure 4.1. compare OEP14 R lane with other R lanes), but no association with immunoprecipitated ClpC could be detected (Figure 4.1. compare OEP14 l lane with other I lanes). Thus we conclude that ClpC associated with several precursors that used the general import pathway, but not with OEP14, a protein that does not use this pathway. The interaction of members of the Hsp100 chaperone family with substrate proteins has been shown to be regulated in an ATP-dependent manner (Wickner et al., 1994; Wawryznow etal., 1995). To analyze the effect of ATP on the interaction between ClpC and translocation complexes, chloroplasts were bound with precursor as described above. but were then Iysed and chloroplastic membranes were solubilized and immunoprecipitation reactions performed in the presence of Mg-ATP, or Mg-GTP (Figure 4.2). The ClpC- containing complex was destabilized if immunoprecipitation was performed in the presence of Mg-ATP, but only slight destabilization was seen if Mg-GTP was included instead (Figure 4.2). indicating that this particular step was specific for 143 Figure 4.2. ATP destabilizes the association of ClpC and translocation complexes. Radiolabeled prSS was incubated with isolated chloroplasts in 100 pM ATP. After a 10 minute incubation. intact chloroplasts were repurified. Iysed hypotonically, and isolated membranes were solubilized in unsupplemented decylmaltoside buffer (Control). or decylmaltoside buffer supplemented with either 10 mM ATP (ATP) or 10 mM GTP (GTP). Ten percent of each reaction was removed for direct analysis by SDS-PAGE (R lane); the remaining 90% was split into two equal fractions and immunoprecipitated by anti-ClpC antibodies (I lanes), or preimmune serum (P lane). Representative reaction (R), and preimmune (P) lanes are shown only for the control experiment. Samples were analyzed by SDS-PAGE and fluorography. (B) Quantitation of lanes from (7A) were conducted by direct analysis on a phosphorimager (Molecular Dynamics, Inc.). 144 A «Q ’3 V (9 Control V39 3% IR | Pll|ll|j prSS * B 100 - 75 t 50 ' 25 Percent of Control l' F’ll'll'l Control Q <2 94‘ 6‘ s s9 145 ATP. The addition of Mg-ATP did not simply stimulate import in the Iysed chloroplast membranes as no mSS could be detected in the total protein control (data not shown). ClpC interacts specifically with chloroplastic envelope membranes in an ATP-independent manner. The observation that ClpC associated with protein translocation complexes even in the absence of added prSS implied that this soluble protein stably associated with chloroplastic membranes. Interestingly, this protein was identified as an inner envelope membrane protein in several studies (Moore and Keegstra, 1993; K0 at al., 1994; N. Hoffman. personal communication to K. Keegstra). When the deduced protein sequences of cDNA clones of this protein were examined no obvious membrane-spanning domains were detected, and it was found predominantly in the stromal compartment after its in vitro import into isolated, intact chloroplasts (Moore and Keegstra, 1993). The membrane localization of this protein was therefore attributed to contamination of the membrane fraction with stromal proteins (Shanklin at al., 1995). We detected ClpC in a membrane-associated protein translocation complex, and ClpC could be observed in this complex in the absence of another major stromal protein (S78). We therefore wished to re-examine the question of whether membrane-associated ClpC was due to a specific interaction with protein translocation components or whether its presence was due to stromal contamination of membrane fractions. 146 The chloroplastic protein translocation apparatus is located in the envelope membranes, and we detected ClpC in detergent-solubilized protein translocation complexes. Therefore if the membrane-associated form of ClpC interacts with membranes in a specific manner, i.e. if it is a member of the translocation apparatus, it should fractionate specifically with envelope membranes. If ClpC did not specifically fractionate with envelope membranes but rather was found in both envelope and thylakoid membranes this would support the argument that ClpC was present in membranes as a stromal contaminant. To determine if membrane-associated ClpC interacted specifically with envelope membranes purified stromal, envelope membrane, and thylakoid membrane fractions were obtained using a published procedure (Keegstra and Yousef. 1986), with some modifications (See Materials and Methods). and equivalent quantities of protein were separated by SDS-PAGE followed by immunoblotting with antibodies specific to ClpC, and various marker proteins. Anti-ClpC antibodies specifically recognized a single 90 kDa protein in both envelope and stromal fractions but not in thylakoids (Figure 4.3A). Antibodies specific to the outer envelope membrane protein T0075. and the inner envelope membrane protein Tic110 each detected only proteins in the envelope fraction (Figure 4.38 and 3C. respectively). This eliminated the possibility that the ClpC detected in the stromal fraction was due to contamination by envelope proteins. Antibodies specific to the stromal Hsp70, S78. reacted with a 70-kDa protein only in the stromal fraction (Figure 4.30). 147 Figure 4.3. ClpC is present in chloroplast envelope membrane and stromal fractions. Purified stromal proteins (S). envelope membrane proteins (E), and thylakoid membrane proteins (T) were separated by SDS-PAGE. transferred to Immobilon- P membranes, and probed with anti-Toc75 antibodies (A). anti-ClpC antibodies (B), anti-Ti0110 antibodies (C), anti-S78 antibodies (D), or anti-LHCP antibodies (E). All lanes contained 10 pg of protein. 148 -ATP +ATP +Tx-1 00 |P SIIP slIP sI 1 10 ClpC S78 149 This demonstrated that the isolated envelope membranes were essentially free of stromal contamination. Finally. to demonstrate that proteins were present in the thylakoid fraction, antibodies to the thylakoid light-harvesting-complex protein, LHCP detected proteins in this fraction (Figure 4.3E). We concluded that the membrane-localized form of ClpC was specifically associated with the envelope membranes of chloroplasts. and did not interact with thylakoid membranes. The association of matrix-Hsp70 with the inner membrane translocation component. TIM44, is regulated by the ATP-bound state of the matrix-Hsp70. Additionally. the lumenal Hsp70 of the endoplasmic reticulum. BiP, associates with the translocation component. Sec63 in an ATP-dependent fashion. Since added Mg-ATP disrupted the association between prSS and ClpC-containing complexes (Figure 4.2). perhaps Mg-ATP also dissociated the association of ClpC with translocation components located in the inner envelope membrane. To test this chloroplasts were lysed hypotonically either in the absence (Figure 4.4A) or presence (Figure 4.48) of Mg-ATP and incubated on ice. Membranes were then sedimented by centrifugation and the soluble protein fraction removed. The membrane fractions were subjected to several additional washes with lysis buffer to remove contaminating soluble proteins. Protein concentrations in the resulting membrane and soluble protein fractions were determined and equal quantities were solubilized in Laemmli buffer and analyzed by SDS-PAGE followed by immunoblotting techniques to determine the presence 150 Figure 4.4. ClpC’s association with chloroplastic membranes is not ATP- dependent lsolated, intact chloroplasts were lysed in 100 pL of Lysis buffer (-ATP). Lysis buffer containing 10 mM Mg‘ATP (+ATP). or Lysis buffer containing the detergent Triton X-100 (+TX-100). After lysis, membrane and soluble fractions were separated by centrifugation at 150.0009 for 5 minutes. 10 pg of protein from each of these fractions was resuspended in SDS-PAGE buffer and analyzed by SDS-PAGE followed by transferal to lmmobilon-P and immunoblotting with anti- Tic110 (110), anti-ClpC (ClpC), and anti-S78 (S78) antibodies. 151 A Anti-Toc75 B Anti-ClpC C Anti-Tic110 110 D Anti-S78 MWSET S78 152 of ClpC. and the controls for integral membrane proteins (T i0110). and soluble proteins (S78). ClpC was detected in both membrane and supernatant fractions (Figures 4.4A and 4.4B, compare 8 and P lanes). The relative amount of ClpC present in the membrane protein fractions did not appear to change significantly versus the amount of inner envelope membrane protein, Tic110, upon the inclusion of Mg-ATP in the lysis buffer (Figures 4.4A and 4.48, compare P lanes). The ClpC present in the membrane fraction was not simply due to contamination by stromal proteins as significant amounts of S78 were detected only in the soluble protein fractions (Figures 4.4A and 4.4B, compare S and P lanes). To make sure that the majority of the ClpC present in the isolated membrane fractions was due to a true interaction with membranes and not simply due to an association with insoluble aggregates the isolated chloroplastic membranes were solubilized in the non-denaturing detergent Triton X-100 (Figure 4.4C). After detergent treatment. solubilized proteins were separated from insoluble material by centrifugation and analyzed in the same manner as described above. Upon detergent treatment both membrane-associated ClpC and the integral membrane control, Tic110, were efficiently solubilized (Figure 4.40, compare S and P lanes). The conclusion from these experiments was that the majority of the ClpC present in chloroplastic membrane fractions was not due to its presence in insoluble aggregates. Additionally, Mg-ATP did not appear to affect the association of ClpC with chloroplastic membranes. 153 ClpC is tightly associated with chloroplastic membranes. ClpC associated specifically with envelope membranes, and this interaction was not dependent upon the presence of Mg-ATP. To further explore the nature of the association of this protein with chloroplastic membranes, the association of ClpC with membranes was examined using various extraction conditions. The extraction profiles of ClpC under these conditions was compared to those of proteins of known. or previously characterized extraction profiles. Tic110 was used as an integral membrane control, S78 was used as a soluble protein control. and prSS bound in the presence of low ATP concentrations was used as a peripherally- associated membrane protein (T ranel at al., 1995). First the localization of the various proteins was determined after extraction in the presence of lysis buffer alone (Figure 4.5A, 5B. and 5C, lysis panels). In these wash conditions. membrane proteins should remain associated with the membrane. thereby allowing the level of soluble proteins that contaminated the membrane fraction to be estimated. Equivalent quantities of the membrane pellet. soluble protein, and extracted protein fractions were analyzed by SDS-PAGE followed by immunoblotting to detect Tic110. ClpC and S78 (Figure 4.5A, Lysis panel). autoradiography to detect prSS (Figure 4.5B. Lysis panel), and silver-staining to visualize protein profiles (Figure 4.50. Lysis paneD. Under these conditions ClpC was detected in both membrane pellet and soluble fractions, and to a much lesser degree in the extracted protein fraction 154 Figure 4.5. ClpC is tightly associated with chloroplastic membranes. Radiolabeled prSS was incubated with isolated chloroplasts in 100 pM ATP. After a 10 minute incubation, intact chloroplasts were repurified, Iysed hypotonically. and the membrane and soluble fractions (S) were separated by centrifugation at 150.0009 for 5 minutes. The membrane pellet fraction was then extracted with Lysis buffer (Lysis). buffer containing 1M KCI (KCI). or buffer 100mM Na2003 pH 11.5 (Na2C03). After reisolation of the membrane (P) and extracted soluble protein fractions (E) by centrifugation 10 pg of protein from each of the three protein (P, S, and E) fractions was resuspended in SDS-PAGE buffer and analyzed by SDS-PAGE. The SDS-PAGE was further analyzed either by transferal t0 lmmobilon-P and immunoblotting with anti-Tic110 (110), anti- ClpC (ClpC), and anti-S78 (S78) antibodies (A). fluorography to detect prSS (B). or silver-staining to visualize each fractions protein profile (C). Proteins efficiently extracted by Lysis buffer (arrows). 1M KCI (arrowheads). or 100mM Na2003 pH 11.5 (asterisks), are indicated. 155 A Lysis KCI NaZCO3 lPSEIIPSEHPSEl 156 (Figure 4.5A, Lysis panel). Tic110. and prSS were detected only in the membrane pellet fraction (Figure 4.5A and 5B Lysis panels, respectively), and $78 was detected only in the soluble protein fraction (Figure 4.5A, lysis panel). When the membrane pellet and extracted protein fractions were analyzed by silver-staining methods, most protein species remained in the pellet fraction. but several prominent protein bands could be found in the extracted protein fraction (Figure 4.5C Lysis panel. arrows). The major protein band observed at roughly 50 kDa. was identified as Rubisco large-subunit based on its abundance and migration. I therefore reasoned that the other proteins removed most likely represented contaminating stromal proteins. Protein-protein interactions occurring primarily at the surface of the membrane are susceptible to high-salt washes. Therefore if ClpC interacted with the solvent-accessible portion of an integral membrane protein it should be extracted from membranes by a high-salt treatment. To test if this was the case, intact chloroplasts were Iysed and then extracted three times with 1M KCI, and the resulting membrane pellet. soluble protein, and extracted protein fractions were isolated and analyzed as described above. Although a small amount of ClpC was removed by high-salt washes. this was not significantly more than observed after extraction with lysis buffer (Figure 4.5A, compare E lanes of Lysis and KCI panels) and the majority of ClpC remained associated with the membrane pellet (Figure5A KCI panel, compare P and E lanes). Tic110 remained in the membrane pellet fraction, and S78 fractionated with soluble 157 proteins. Under these conditions prSS bound to chloroplasts also remained largely resistant to high-salt extraction (Figure 4.5B. KCI panel). To ensure that the high-salt extraction procedure had worked the protein profiles of extracted proteins were examined by SDS-PAGE followed by silver- staining. High-salt extraction conditions efficiently removed several proteins from chloroplastic membranes (Figure 4.50, arrowheads). It was concluded from these experiments that ClpC remained associated with chloroplastic membranes even though some loosely-associated membrane proteins were efficiently removed by the high-salt extraction conditions. Peripherally-associated membrane proteins can be extracted from membranes upon treatment with strongly basic conditions (T ranel at al., 1995). Since ClpC has no obvious membrane spanning domains any association this protein has with membranes would be predicted to be peripheral in nature. To test this, intact chloroplasts were lysed and the isolated membranes were extracted in 100 mM sodium carbonate pH 11.5 as in the above two experiments (Figures 4.5A. 4.5B. and 4.50, Na2C03 panels). After carbonate treatment ClpC was observed in both the membrane pellet and soluble protein fractions (Figure 4.5A, Na2C03 panel, compare P and S lanes). About a third to half of the membrane-associated ClpC was removed in these extraction conditions (Figure 4.5A, Na2C03 panel, compare P and E lanes). Ti0110, as an integral membrane protein was retained in the membrane fraction after extraction, and $78 was again observed in the soluble protein fraction (Figure 4.5A, Nazco. panel). Precursors bound to chloroplasts prior to 158 the lysis step were partially sensitive to carbonate extraction (Figure 4.5B Nazco. panel. compare P and E lanes). About sixty percent of the bound precursor was released to the extracted protein fraction when the amounts of radiolabeled prSS present in the membrane pellet and extracted protein fractions were quantified by analysis on a phosphoimager (data not shown). This amount of extraction is consistent with that observed by others (T ranel at al., 1995). When the protein profiles of the membrane pellet and extracted protein fractions were analyzed by silver-staining several protein species, that had not been removed by either lysis buffer or high-salt conditions, were observed to be efficiently removed by carbonate treatment (Figure 4.50, Na2C03 panel, asterisks). We concluded that the carbonate extraction conditions were sufficient to remove some peripherally-associated membrane proteins. but that ClpC remained partially resistant to extraction under these conditions. A significant proportion of the membrane-associated ClpC is most likely deeply- embedded in the membrane, or firmly associated with integral membrane proteins. DISCUSSION In Chapter 2 we observed that the stromal Hsp100 homologue, ClpC. associated with translocation complexes formed under binding conditions. In Chapter 3, the examination of the role of molecular chaperones in the protein import system of chloroplasts was extended, and ClpC was also observed in 159 association with prSS under import conditions. This association disappeared with similar kinetics to the disappearance of prSS during the import reaction. These results implied that ClpC was involved in the import of precursors. In this chapter the manner in which ClpC interacts with the chloroplastic protein translocation apparatus has been examined in further detail. These results further support a role for the stromal Hsp100, ClpC, as a protein translocation component. Support for this hypothesis stems from two observations. First, multiple precursors could be coimmunoprecipitated with ClpC under binding conditions. indicating that ClpC interacted as a component of the general import apparatus and associated with the various precursors. This association with precursors during binding depended upon their utilization of the general import apparatus (Figure 4.1). Second. the ability of ATP to destabilize the association of ClpC from precursor-containing complexes (Figure 4.2) is consistent with the previous observations that ClpC interacts with substrate proteins in an ATP-dependent manner (Wickner etal., 1994; Wawryznow et al., 1995) Based on the results obtained in this chapter a working model for the interaction of ClpC with chloroplastic protein translocation complexes is proposed (Figure 4.6). In this model ClpC remains associated with the inner envelope membrane regardless of whether ATP is present (see Figure 4.4). Upon addition of ATP, the association of ClpC with precursor-containing complexes is disrupted (Figure 4.2). This observation could be interpreted in 160 Figure 4.6. Working model for the ATP-dependent dissociation of ClpC and prSS-containing translocation complexes. In this scheme, cytoplasmically synthesized precursors added to chloroplasts in the presence of low ATP concentrations (Docking conditions) interact with protein translocation complexes containing both outer envelope membrane, and inner envelope membrane translocation components, as well as membrane- associated ClpC. Upon the addition of ATP at high concentrations, either ClpC remains associated with the inner envelope membrane. but is released from precursor-containing protein translocation complexes (A). or ClpC remains associated with protein translocation complexes, but precursor is released and remains associated with chloroplastic membranes (8). 161 Docking 162 two ways. First, ClpC could be released from the complex, leaving precursor associated with other protein translocation components (Figure 4.6A). Second. precursor could be released from the protein translocation complex that still contains ClpC (Figure 4.68). Because complexes containing ClpC can be detected in the absence of added precursors (Chapter 2. Figure 2.4), we favor this second explanation. This implies that it is a precursor-ClpC interaction that stabilizes the complexes observed here. Further work is needed to confirm this conclusion and to explore the implications of this hypothesis for the mechanism of protein transport into chloroplasts. The proposal that the stromal Hsp100 homologue ClpC acts as a translocation component in a manner relevant to protein import is consistent with findings in other laboratories. Clp homologues were originally identified as members of the ATP-dependent Ti protease in E. coli (Hwang at al., 1988; Maurizi at al., 1990). Recent experiments have demonstrated these proteins can act as chaperones in the absence of the proteolytic subunit ClpP (Wickner et al., 1994). Because no stromal ClpP was detected in our immunoprecipitations we concluded that ClpC interacted with precursor as a chaperone and not as part of a protease (Chapter 2. Figure 2.4). This raised the possibility that ClpC interacts with precursors in a manner analogous to that observed for Hsp70 homologues in the mitochondrial and ER import systems (for review see, Schatz and Dobberstein, 1996). In mitochondria, a matrix Hsp100 homologue can interact with translocation intermediates, though only under specific conditions which limited levels of functional Hsp70 (Schmitt et al., 1995). Based on our results, Hsp100 homologues would appear to be the 163 dominant molecular chaperones interacting with precursors during chloroplastic protein import. In mitochondria, TIM44, an inner membrane translocation component has been observed to interact with the matrix-Hsp70 during import of precursors (Blom et al., 1993; Schneider at al., 1994; Kronidou at al., 1994; Rassow at al., 1994). In the endoplasmic reticulum. the lumenal Hsp70 has been found to form a complex with Se063p, a membrane protein component of the translocation machinery (Sanders et al., 1992). In both systems. these interactions can occur with or without associated precursor and depend on the ATP-bound state of the Hsp70 homologue (Brodsky and Schekman. 1993; von Ahsen et al., 1995). Perhaps an interaction similar to that observed for Hsp70 homologues in mitochondria and ER is occurring with the Hsp100 homologue ClpC in chloroplasts. However, in both mitochondria and ER the interaction of Hsp70 homologues in the interior of the organelle become associated with the membrane in an ATP-dependent manner (Schneider at al., 1994; Brodsky and Schekman. 1993). On the other hand. the association of ClpC with the envelope membranes is not ATP-dependent (Figure 4.4). In fact, despite the lack of obvious membrane-spanning alpha helixes or highly hydrophobic regions, ClpC appears to be quite tightly associated with membranes (Figure 4.5). It Is possible that ClpC does dissociate from membranes in a nucleotide-dependent manner. and that the proper conditions to reconstitute this activity have not been found. Still. the partial insensitivity of the membrane-associated ClpC even to 164 carbonate extraction conditions tends to argue against an interaction of ClpC with membranes through a protein-protein interaction that was labile enough to be disrupted solely by the nucleotide-bound state of ClpC. In bacteria, SecA, the protein translocation component responsible for providing the motive force to translocate secretory precursors across the plasma membrane. is also tightly associated with the membranes (Arkowitz et al., 1993). The SecA protein, interacting with other translocation components. utilizes ATP hydrolysis to insert a portion of itself across the plasma membrane thereby driving precursor associated with this domain through the translocation channel (Economou and Wickner. 1994). In this system, ATP does not disrupt the association of SecA with the plasma membrane. but does cause it to become more susceptible to extraction by high concentrations of urea (Cabelli at al., 1991; Ulbrandt at al., 1992). Although SecA is partially resistant to extraction even by high urea concentrations. cross-linking studies have demonstrated that this protein is not found in a lipid accessible compartment, but rather maintains its interaction with membranes primarily through protein-protein interactions and its resistance to urea extraction stems from the nature of its interactions with other integral membrane components (Eichler et al., 1997). There are several interesting correlations between SecA and ClpC. SecA, like ClpC. is found in both soluble and membrane associated forms and does not contain obvious membrane-spanning helixes or highly hydrophobic regions (Chapter 4; Moore and Keegstra, 1993; Cabelli at al., 1991). In addition. both ClpC and SecA are found in association with protein translocation complexes 165 (Nielsen at al., 1997; Akita et al., 1997; Wickner and Leonard. 1996). These observations imply that these two proteins may share some functional homology. Interestingly. ClpC family members have been identified as important in genetic competence in Bacillus subtilis (Msadek at al., 1994) and secretion of hemolysin in Serpulina hyodysenteriae (ter Huume et al., 1994). While the roles of ClpC homologues in these two systems have not been elucidated, in both cases potential roles of the ClpC proteins could involve translocation of macromolecules across membranes. Both SecA and ClpC contain two unrelated ATPase domains (den Blaauwen and Driessen. 1996; Schirrner et al., 1996) and the overall organization of the two proteins are similar (Figure 4.7). Although the presence of two nucleotide-binding sites has been reviewed for both the Hsp100 and SecA protein families (den Blaauwen and Driessen. 1996; Schirrner et al., 1996). comparison of these two protein families has not been previously described. The presence of two ATPase domains in a single protein is unusual, and a search of protein sequence databases revealed only one other class of proteins, the ABC transporters. containing two ATPase domains. However, unlike both the SecA and Hsp100 protein families. whose two ATPase domains are unrelated. the ATPase domains of the ABC transporter protein family are related and are thought to be the result of a duplication event (Squires and Squires, 1992). Finally. although ClpC and SecA do not display a high degree of sequence similarity, after sequences were lined up based on the location of the 166 Figure 4.7. Structural features of Hsp100 and SecA proteins. Regions of higher conservation. and the two nucleotide-binding domains (NBD) are indicated by thicker boxes. Each NBD contains Walker-type nucleotide- binding (Walker et al., 1982). The Walker A consensus region is believed to form the base of the nucleotide-binding pocket. and the Walker 8 consensus region is though to form a loop that positions Mg2+ ions. The first NBD in both Hsp100 and SecA protein families is unique in having two 8 motifs, but it is unclear If both are used in these domains. In Hsp100s. several regions of higher conservation have been identified and these are indicated in this protein by thicker boxes labeled with Roman numerals (II. III. IV, and V). At present no function has been assigned to these regions. In SecA proteins, the first NBD has been determined to have higher affinity for ATP than the second NBD, whether this is the case in Hsp1003 is still unclear. Additionally, preprotein binding. and SecB binding domains are indicated by thicker boxes. Regions determined to associate with membranes are indicated by underlying bars. 167 Hsp100 N-term ATP-1 Middle ATP-2 C-term I A1 81.1 81.2 II III A2 82 IV V SecA ATP-1 Preprotein ATP-2 SecB (High cross-linking (Low affinity) binding affinity) region A1 81.1 81.2 A2 82 I I E: membrane domain membrane domain 168 two ATPase domains, sequences similar to several conserved sequence motifs previously identified in Hsp100s (Schirmer et al., 1996) were detected in the SecA sequences (Figure 4.8). In conclusion, in this chapter we have further examined ClpC and its role in chloroplastic protein import. ClpC interacts with multiple precursors which utilize the chloroplastic protein import apparatus. and interacts with precursors in an ATP-dependent manner. Finally, a portion of ClpC is permanently associated with chloroplastic membranes and could not be removed by carbonate extraction conditions capable of removing peripherally-associated proteins. 169 Figure 4.8. Conserved motifs similar to those found in Hsp100s can be identified in corresponding regions of SecA proteins. Protein sequences from different organisms were aligned using the CLUSTAL algorithm of the DNAstar sequence analysis program (DNAstar, Madison. WI, USA). and a SecA consensus sequence was generated. Sequences similar to previously identified nucleotide-binding motifs and consensus sequences in the Hsp100 protein family (Schirmer et al., 1996) were then identified by visual inspection of corresponding regions of the SecA consensus sequence. x=any amino acid, h=hydrophobic amino acids. 170 ATP-binding 1: walker consensus sequences Classical: A 81 82 0):,ch Rx.-,h,D Rx.-,h.D Hspl 0 0 consensus : Gx,GKT R/Kx.h,D RXGAIDLhD SecA consensus : Gx2GKT Rx,.,h,D Rx7WIVD Middle: Signature sequences Consensus II: Hsp100 consensus: RxxDxxxAxELRxxxIP SecA consensus: KTxExxxIYKLGxxxIP Consensus III: Hsp100 consensus: xWTGIPVth SecA consensus: xxxGIPHxVL ATP-binding 2: 'Walker consensus sequences Classical: A B Gx,GKT Rx,_,h,D Hsp100 consensus: Gx,GKT Rx,h,D SecA consensus: stKT Rx.h,GD C-tezminus Signature sequences: Consensus IV: Hsp100 consensus: FRPEFLNRLDEIIVFxxL SecA consensus: FRVEEDXPIxxxhhxxxL Consensus V: Hsp100 consensus: YGARPLRRxI SecA consensus: YGxRTthhh 171 REFERENCES Akita, M., Nielsen, E. and Keegstra, K. (1997) Identification of protein transport complexes in the chloroplastic envelope membranes via chemical cross-linking. J. Cell Biol., 136, 983-994. Arkowltz, R.A., Joly, J.C. and Wickner, W. (1993) Translocation can drive the unfolding of a preprotein domain. EMBO J., 12, 243-253. Bauerle, C., Dorl, J., and Keegstra, K. (1991) Kinetic analysis of the transport of thylakoid lumenal proteins in experiments using intact chloroplasts. J. Biol. Chem, 266, 5884-5890. Blom, J., Ktibrich, M., Rassow, J., Voos, W., Dekker, P.J.T., Maarse, A.C., Meijer, M. and Pfanner, N. (1993) The Essential Yeast Protein MIM44 (encoded by MPI1) Is Involved in an Early Step of Preprotein translocation across the Mitochondrial Inner Membrane. Mol. Cell. Biol., 13, Brodsky, J.L. and Schekman, R. (1993) A Sec63p-8iP complex from yeast is required for protein translocation in a reconstituted proteoliposome. J. Cell Biol., 123, 1355-1363. Bruce,B., Perry,S., Froehlich,J. and Keegstra,K. (1994) In vitro import of proteins into chloroplasts. Plant Molecular Biology Manual J1. Kluwer Academic Publishers, Belgium. pp. 1-15. Cabelli, R.J., Dolan, K.M., Qian, L. and Oliver, DB. (1991) Characterization of the membrane-associated and soluble states of SecA protein from wild-type and SecA51 (TS) mutant strains of Escherichia coli. J. Biol. Chem, 266, 24420- 24427. Chernoff, Y.O., Lindquist, S.L., Ono, 8., Inge-Vechtomov, S.G. and Liebman, S.W. (1995) Role of the chaperone Hsp104 in propagation of the yeast prion- Iike factor [psi+]. Science, 268, 880-884. den Blaauwen, T. and Driessen. A.J.M. (1996) Sec-dependent preprotein translocation in bacteria. Arch. Microbiol, 165, 1-8. Economou, A. and Wickner, W. (1994) SecA promotes preprotein translocation by undergoing ATP-driven cycles of membrane insertion and deinsertion. Cell, 78, 835-843. 172 Eichler, J., Brunner, J. and Vllickner, W. (1997) The protease-protected 30 kDa domain of SecA is largely inaccessible to the membrane lipid phase. EMBO J., 16, 2188-2196. Gottesmann, s.. Squires, C., Plchersky, E., Carrington, M., Hobbs, M., Mattick, J.S., Dalrymple, B., Kuramitsu, H., Shiroza, T., Foster, T., Clark, W.P., Ross, 8. and Maurizi. MR. (1990) Conservation of the regulatory subunit for the Clp ATP-dependent protease in prokaryotes and eukaryotes. Proc. Natl. Acad. Sci. USA, 87, 3513-3517. Hwang, B.J., Park, W.J., Chung, C.H. and Goldberg, A.L. (1987) Escherichia coli contains a soluble ATP-dependent protease (T i) distinct from protease La. Proc. Natl. Acad. Sci. USA, 84, 5550-5554. Hwang, B.J., Woo, K.M., Goldberg, A.L. and Chung, C.H. (1988) Protease Ti, a new ATP-dependent protease in Escherichia coli, contains protein-activated ATPase and proteolytic functions in distinct subunits. J. Biol. Chem, 263, 8727- 8734. Keegstra, K. and Yousef. A.E. (1986) Isolation and characterization of chloroplast envelope membranes. Method. Enzymol., 118, 316-325. K0, K., Doung, C. and Ko, Z.W. (1994) Nucleotide sequence of a Brassica napus Clp homolog. Plant Physiol,, 104, 1087-1089. Kronidou, N.G., Oppliger, W., Bolliger, L., Hannavy, K., Glick, 8.8., Schatz, G. and Horst, M. (1994) Dynamic interaction between lsp45 and mitochondrial hsp70 in the protein import system of the yeast mitochondrial inner membrane. Proc. Natl. Acad. Sci. USA, 91, 12818-12822. Kruger, E., Volker, U. and Hacker, M. (1994) Stress induction of prC in Bacillus subtilis and its involvement in stress tolerance. J. Bacteriol, 176, 3360- 3367. Laemmli,U.K. (1970) Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature, 227, 680-685. Li, H-m., Moore, T., and Keegstra, K. (1991) Targeting of proteins to the outer envelope membrane uses a different pathway than transport into chloroplasts. Plant Cell, 3, 709-717. Maurizi,M.R., Clark,W.P., Katayama,Y., Rudikoff,S., Pumphrey,J., Bowers,B. and Gottesman,S. (1990) Sequence and structure of Clp P. the proteolytic component of the ATP-dependent Clp protease of Escherichia coli. J. Biol. Chem, 265. 12536-12545. 173 Moore, T. and Keegstra, K. (1993) Characterization of a cDNA clone encoding a chloroplast-targeted Clp homologue. Plant Mol. Biol., 21, 525-537. Msadek, T., Kunst, F. and Rapoport, G. (1994) MecB of Bacillus subtilis. a member of the ClpC ATPase family. is a pleiotropic regulator controlling competence gene expression and growth at high temperature. Proc. Natl. Acad. Sci. USA, 91, 5788-5792. Nielsen, E., Akita, M., Dalea-Aponte, J. and Keegstra, K. (1997) Stable association of chloroplastic precursors with protein-translocation complexes that contain proteins from both envelope membranes, and a stromal Hsp100 molecular chaperone. EMBO J., 16, 935-946. Olsen, L.J., Theg, S.M., Selman, B.R., and Keegstra, K. (1989) ATP is required for the binding of precursor proteins to chloroplasts. J. Biol. Chem, 264, 6724-6729. OIsen,L.J. and Keegstra,K. (1992) The binding of precursor proteins to chloroplasts requires nucleoside triphosphates in the intermembrane space. J. Biol. Chem, 267, 433-439. Rassow, J., Maarse, A.C., Krainer, E., Kubrich, M., Muller, H., Meijer, M., Craig, EA. and Pfanner, N. (1994) Mitochondrial Protein Import: Biochemical and Genetic Evidence for Interaction of Matrix hsp70 and the Inner Membrane Protein MIM44. J. CellBioI., 127, 1547-1556. Sanchez, Y. and Lindquist, S.L. (1990) Hsp104 is required for thennotolerance. Science, 248, 1112-1 1 15. Schatz,G. and Dobberstein,B. (1996) Common principles of protein translocation across membranes. Science, 271 , 1519-1526. Schin'ner, E.C., Glover, J.R., Singer, MA. and Lindquist, S. (1996) HSP100IClp proteins: a common mechanism explains diverse functions. Trends Biochem. Sci, 21 , 289-296. SchmitLM., Neupert.W. and Langer,T. (1995) Hsp78, a Clp homologue within mitochondria, can substitute for chaperone functions of mt-hsp70. EMBO J., 14, 3434-3444. Schneider, H.-C., Berthold, J., Bauer, M.F., Dietmeier, K., Guiard, B., Brunner, M. and Neupert, W. (1994) Mitochondrial Hsp70lMlM44 complex facilitates protein import. Nature. 371, 768-774. Shanklin,J., DertLN.D. and Flanagan,J.M. (1995) The stroma of higher plant plastids contain ClpP and ClpC, functional homologs of Escherichia coli ClpP 174 and ClpA: An archetypal two-component ATP-dependent protease. Plant Cell, 7, 1713-1722. Squires,C. and Squires,C.L. (1992) The Clp proteins: proteolysis regulators or molecular chaperones?. J. Bacteriol. 174, 1081-1085. ter Huume, A.A.H. (1994) Characterization of three putative Serpulina Hyodysenteriae hemolysins. Microb. Path09., 16, 269-282. Towbin, H. and Gordon, J. (1984) lmmunoblotting and dot immunoblotting— current status and outlook. J. Immunol. Methods. 72, 313-340. Tranel, P.J., Froehlich, J., Goyal, A. and Keegstra, K. (1995) A component of the chloroplastic protein import apparatus is targeted to the outer envelope membrane via a novel pathway. EMBO J., 14, 2436-2446. Ulbrandt, N.D., London, E. and Oliver, D.B. (1992) Deep penetration of a portion of Escherichia coli SecA protein into model membranes is promoted by anionic phospholipids. J. Biol. Chem, 267, 15184-15192. von Ahsen,O., Voos,W., Henninger,H. and Pfanner,N. (1995) The mitochondrial protein import machinery - role of ATP in dissociation of the Hsp70.Mim44 complex. J. Biol. Chem, 270, 29848-29853. Wawrzynow, A., Wojtkowiak, D., Marszalek, J., Banecki, B., Jonsen, M., Graves, B., Georgopoulos, C. and Zylicz, M. (1995) The Cle heat-shock protein of Escherichia coli. the ATP-dependent substrate specificity component of the ClpP-Cle protease, is a novel molecular chaperone. EMBO J., 14, 1867-1877. Wickner, S., Gottesman, S., Skowyra, D., Hoskins, J., McKenney, K. and Maurizi, MR. (1994) A molecular chaperone, ClpA. functions like DnaK and DnaJ. Proc. Natl. Acad. Sci. USA, 91, 12218-12222. Wickner, W. and Leonard, MR. (1996) Escherichia coli preprotein translocase. J. Biol. Chem, 271, 29514-29516. Wilson, S.A., Williams, R.J., Pearl, L.H. and Drew, RE. (1995) Identification of two new genes in the Pseudomonas aerugenosa amidase operon, encoding an ATPase (AmiB) and a putative integral membrane protein (AmiS). J. Biol. Chem, 270, 18818-18824. Woo, K.R., Kim, K.I., Goldberg, A.L., Ha, 0.8. and Chung, C.H. (1992) The heat-shock protein CIpB in Escherichia coli is a protein-activated ATPase. J. Biol. Chem, 267, 20429-20434. Chapter 5 CONCLUSIONS 175 176 Recognition of precursor proteins and their subsequent translocation into chloroplasts is thought to be mediated by proteinaceous components located in the envelope membranes (Kouranov and Schnell. 1996). At present a major focus of the research into chloroplastic protein import is the identification of these protein translocation components. Several proteins of both the outer and inner envelope membranes have been implicated as components of the protein translocation apparatus (for review see; Gray and Row, 1995; Schnell, 1995). A working model of the general chloroplastic protein import apparatus is presented in Figure 5.1. While a number of the proteins presented in Figure 5.1 have now been cloned, little is understood about their roles in the protein import process as the majority of these proteins display little or no homology with other proteins having defined functions. My research began as an attempt to determine whether molecular chaperones played a role in chloroplastic protein import. I focused first on a specific class of molecular chaperones, the Hsp70 protein family. The rationale was that members of this chaperone family had been identified as essential members of two other protein translocation systems. namely those in the mitochondrion and the endoplasmic reticulum (Kang at al., 1990; Sanders et al., 1992). In both these protein translocation systems these Hsp70$ were thought to allow for the unidirectional movement of precursors through the translocation channel (Schatz and Dobberstein, 1995). 177 Figure 5.1 Working model of the general chloroplastic protein import apparatus. T0086 and Toc75 interact directly with the transit peptide of precursors (Perry and Keegstra, 1994; Ma et al., 1996). At present. Toc86 is thought to act as a receptor protein for transit peptides (Schnell et al., 1994). and Toc75 is thought to form all or part of a translocation channel (T ranel at al., 1995). Toc34 associates with these two components in the outer membrane, but has not been found in direct association with precursors (Schnell at al., 1994). Upon addition of low levels of ATP to chloroplasts, precursors are inserted across the outer membrane and can interact with inner membrane translocation components (Ma et al., 1996; Nielsen at al., 1997; Akita at al., 1997). Tic110 and ClpC are members of precursor-containing complexes at this stage of import (Ltlbeck at al., 1997; Nielsen at al., 1997; Akita at al., 1997). As discussed in Chapters 2 - and 4, ClpC interacts with translocation complexes in the absence of precursors. and is tightly-associated with membranes. Import may require GTP as well as ATP. although the role of GTP is unclear (Olsen and Keegstra, 1992; Kessler et al., 1994). Some components of the import apparatus remain to be identified (represented by component ‘X’). 178 179 If Hsp708 were involved in the protein import system in chloroplasts this would provide not only an additional component of the import apparatus, but additionally it would provide a component whose activity was already known in other protein import systems. This parallel might then give insight into the mechanism by which the chloroplastic import components interacted with the Hsp70 in the import process. Initial experiments to determine whether the stromal Hsp70. S78. played a role in chloroplastic protein import were either negative or ambiguous, so I therefore expanded my investigations to determine if perhaps stromal molecular chaperones of other families were involved. This conclusions chapter summarizes the development of detergent solubilization techniques to characterize chloroplastic protein translocation complexes (Chapter2), the evaluation of three classes of stromal molecular chaperones. Hsp100. Hsp70, and Hsp60, for roles in chloroplastic protein import (Chapter 3), the observations which led to the formation of the hypothesis that the stromal Hsp100, ClpC. plays a role in protein import into chloroplasts (Chapters 3 and 4), and suggestions on future research to address unanswered questions arising from these investigations and regarding ClpC’s function in the chloroplastic protein translocation apparatus. In order to examine the role of molecular chaperones in protein translocation into chloroplasts it was first necessary to generate and detect stable protein translocation complexes. Because precursors become stably 180 associated with chloroplastic envelope membranes in the presence of low ATP concentrations. and this presumably reflected a stable association with translocation components, this stage of protein import was chosen as a starting point in the search for stable translocation complexes. The characterization of membrane-associated complexes solubilized with non-denaturing detergents is presented in Chapter 2. The results of these experiments led to three important observations. First, precursors associated with translocation complexes containing translocation components of both the outer and inner envelope membranes as well as a putative stromal component. Second, these complexes could be observed even in the absence of added precursors. Finally, the stromal Hsp100 chaperone. ClpC was associated with these complexes. Further evaluation of whether stromal members of three different molecular chaperone families. Hsp100s (ClpC). Hsp70s (S78). and Hsp60s play roles in chloroplastic protein translocation is presented in Chapter 3. Stromal Hsp60 could be found in association with newly-imported proteins in the stroma, but was not found associated with precursors in chloroplastic membranes. These results are consistent with stromal Hsp60 playing a role in folding or assembly of newly imported proteins rather than having a direct role in the translocation of precursors through the envelope membrane. Both ClpC and S78 could be found associated with complexes containing precursors in chloroplastic membranes. The presence of precursors in complexes precipitated with anti-ClpC or anti-S78 antibodies disappeared as import progressed indicating that these precursors were on the import pathway. 181 However. ClpC-containing translocation complexes were efficiently solubilized and ClpC always co-migrated with precursor and translocation complexes. Fractions containing both S78 and precursors were not efficiently solubilized, and S78 did not co-migrate with precursors and translocation components upon further manipulation of these fractions. The possibility that S78 interacts in a relevant manner with precursors cannot be eliminated, but since ClpC was observed in precursor-containing complexes under conditions where S78 cannot, I favor ClpC over S78 as a chloroplastic translocation component. Further evidence for ClpC’s role as a protein translocation component is presented in Chapter 4. Additionally. preliminary evidence of the manner in which ClpC interacts with the chloroplastic protein translocation apparatus is discussed. Finally, Chapter 4 highlights several interesting correlations between the Hsp100 molecular chaperone family and the bacterial protein secretion component SecA. The data presented in this dissertation provide a detailed analysis of the role of stromal molecular chaperones in chloroplastic protein translocation. Several unanswered questions remain regarding the manner in which chloroplastic protein translocation components associate to form translocation complexes, and as to the function of ClpC in protein import in chloroplasts. The remainder of this chapter addresses some of these unanswered questions and potential avenues for future research. 182 THE EFFECT OF NUCLEOTIDES ON PROTEIN IMPORT COMPLEXES In addition to the identification of novel chloroplastic protein translocation components it is imperative to begin to understand how these components interact to form translocation complexes and cooperate to accomplish the movement of precursors through the envelope membranes. In mitochondria association of inner and outer membrane translocation complexes is dependent upon the presence of precursors (Horst at al., 1995), and inclusion of inner membrane translocation complexes depends on the presence of a A‘P across the inner membrane (Schatz. 1996). In other systems. binding of nucleotides to translocation components also plays an important role in protein translocation complex formation and stability. In mitochondria. association of the matrix-Hsp70 with the inner membrane translocation component, TIM44, is ATP-dependent (Schneider at al., 1994). In the endoplasmic reticulum. the association of SRP and SR, and subsequent delivery of this complex to the Sec61 translocation channel is regulated by a series of GTP-binding proteins (Walter and Johnson, 1995), and the association of the lumenal Hsp70, BiP. with the translocation component, Se063p. is regulated by ATP. Chloroplasts do not require a membrane potential across the envelope membranes to import proteins (T heg et al., 1989), and translocation complexes containing both outer and inner envelope components in the absence of added precursors have been observed (Chapter 2). However, several of the protein 183 translocation components identified in chloroplasts bind nucleotides. These include two outer envelope membrane components. T0086 and Toc34, which bind GTP (Kessler at al., 1994; Hirsch et al., 1994). Additionally. ClpC, which has two ATP-binding sites, has been identified as a member of the chloroplastic protein import complex. The presence of nucleotide-binding sites in several of the protein translocation components raises the possibility that the association of these components into protein translocation complexes may be regulated by the presence or absence of nucleotides in their nucleotide-binding domains. It is likely that nucleotides are involved in several different aspects of chloroplastic protein import complex foImation. Several questions could be addressed relatively easily by extending the immunoprecipitation and immunoblotting techniques developed in this thesis to determine the composition of protein translocation complexes in the presence of various nucleotides. Are chloroplastic protein translocation complexes permanent structures, or is their formation regulated by nucleotides? Earlier models of chloroplastic protein translocation envisioned precursors at this stage of import interacting exclusively, or at least primarily. with outer envelope membrane translocation components (for reviews see; Schnell, 1995; Gray and Row. 1995). These models were based on the observations that, when precursors associated with chloroplasts in limiting ATP concentrations, they remained susceptible to 184 exogenous protease. and the stromally-localized processing protease did not have access to the precursor's transit peptide. As described in Chapter 2. the ATP-dependent binding of precursors not only involves outer membrane translocation components but translocation complexes at this stage also contain components of the inner envelope membrane and stromal compartment (Ma et al., 1996; Nielsen at al., 1997; Akita at al., 1997). Indeed. it appears that the transit peptides of precursors halted at the ATP-dependent binding stage of import have already been inserted across the outer envelope membrane and interact with inner envelope or stromal translocation components (Ma et al., 1996). The observation that, even in the absence of added precursors. translocation components from both the inner and outer envelope membranes were associated implied one of two possibilities. The first of these was that these components associated into permanent structures. However, only a portion of the various translocation components were observed in complexes containing both outer and inner membrane translocation components (Figure 2.3). Additionally, translocation complexes containing only outer membrane translocation components have been identified (Schnell at al., 1994; Waegemann and Soll, 1992). and a translocation intermediate localized in the inter-membrane space was detected during protein import into chloroplasts under hypertonic conditions (Scott and Theg, 1996). These results along with the observation that outer and inner membrane components readily segregated to their respective membrane fractions upon separation of the outer and inner 185 envelope membranes are not consistent with the notion that translocation complexes containing both outer and inner translocation components are permanent structures. The second possibility is that the association of inner and outer membrane translocation components is regulated by the presence of nucleotides added to the in vitro binding or import assays. Whether various nucleotides effect the association of translocation components of the outer and inner membranes into translocation complexes can be examined using the techniques developed during the course of this thesis. It would be relatively easy to monitor the association, or dissociation of translocation components in the presence of various nucleotides using immunoprecipitation and immunoblotting techniques. What role does GTP play in chloroplastic protein import? GTP is able to partially replace the ATP requirement for stable association of precursors with chloroplasts (Olsen et al., 1989). Additionally. the stable association of precursors with chloroplasts is blocked by GTPyS, even in the presence of ATP (Olsen and Keegstra, 1992; Kessler at al., 1994; personal observation). Precursors can be cross-linked with the putative receptor protein. T0086. even in the absence added nucleotides (Perry and Keegstra, 1994). or in the presence of GTPyS (Ma at al., 1996). This complex contains T0075 and T0034, and migrates with purified outer envelope membranes (Perry and Keegstra, 1994; Ma et al., 1996). Since both T0086 and Toc34 are GTP-binding proteins (Kessler at al., 1994; Seedorf at al., 1994; Hirsch et al., 1994), GTP probably plays a role in 186 the formation of these complexes. However. the role of GTP at this step is still unknown. If the association of T0086 or T0034 with other translocation components is affected by GTP or GDP, some insight as to the functions of these proteins in chloroplastic protein import might be gained by performing immunoprecipitation and immunoblotting techniques in the presence of these nucleotides. These results could then be combined with an analysis the effect of these nucleotides on association and import of precursors during in vitro import assays with isolated. intact chloroplasts. What effect does ATP have on the association of ClpC with translocation complexes? Evidence was presented in Chapter 4 that the association of ClpC with precursor-containing complexes was disrupted by the presence of ATP. In mitochondrial and endoplasmic reticulum protein translocation systems molecular chaperones utilize ATP hydrolysis to drive successive binding and release from precursors emerging from the translocation channel into the interior of the organelle (Schatz and Dobberstein, 1996). In both these systems the association of the molecular chaperone with translocation channels is disrupted by ATP (Schneider at al., 1994; Brodsky and Schekman, 1993). In bacterial secretion. SecA mediates the movement of precursors through the translocation channel (Arkowitz et al., 1993). In this system however, SecA does not dissociate from the translocation channel. but rather undergoes a conformational 187 change that pushes a portion of the precursor through the translocation channel. where it is then released (Economou and Wickner, 1994). As discussed in Chapter 4, at present it is not known which of these possibilities best describes the ATP-dependent dissociation of ClpC and precursor-containing complexes. Further analysis of the composition of the ClpC-containing complexes for other translocation components may give some insight into how ATP causes this dissociation. These experiments are presently ongoing in the laboratory. and based on preliminary results it appears that ClpC does not dissociate from either Ti0110, or Toc75. in the presence of ATP. These results support a model in which ClpC does not dissociate from translocation complexes in the presence of ATP (Figure 4.6A). Although Toc75 is maintained in ClpC immunoprecipitated complexes. the extent to which other outer envelope components remain in association with these complexes is still unclear. DOES CLPC INTERACT DIRECTLY WITH PRECURSORS DURING PROTEIN IMPORT? In this thesis ClpC has been observed in association with chloroplastic protein translocation complexes in a manner consistent with its having a role in the import process (Chapters 2. 3. and 4). In order to understand the details of its role, the manner in which ClpC interacts with chloroplastic protein translocation components must be addressed. Because ClpC is a member of the Hsp100 molecular chaperone family, one could speculate that this protein 188 might drive the unidirectional movement of precursors into chloroplasts. as previously observed for molecular chaperones in other protein import systems (Kang et al., 1990; Brodsky and Schekman. 1993). However. evidence of such an interaction is still lacking in chloroplasts. Chemical cross-linking techniques have been used in both the mitochondrial and endoplasmic reticulum protein translocation systems to determine the direct interaction of molecular chaperones with translocating precursors (Scherer et al., 1991; Vogel at al., 1990). Chemical cross-linking techniques have also been utilized in chloroplasts for the identification of protein translocation components that interact directly with translocating precursors. Perry and Keegstra, 1994. utilized a chemical cross- linking strategy to identify both T0086 and Toc75 as chloroplastic translocation components, and an identical strategy was utilized later by Ma et al., 1996. to identify the inner membrane translocation component, Ti025. ClpC was not detected in either of these two studies, even though similar ATP concentrations to were used form the translocation complexes in both studies. There are several possibilities for the lack of detection of ClpC in these two studies. First, both these studies utilized a heterobifunctional cross-linker that attached to the precursors through cysteine residues. As a result. the cysteine residue in precursor transit peptide must be in a suitable position for the attached cross-linker to be able to interact with the ClpC molecule. If this were the case re-examination with precursor constructs where the cross-linkers can be 189 attached in different positions might reveal ClpC. and/or other new translocation components. Second. ClpC might have been cross-linked but as it migrates immediately above T0086 on SDS-PAGE (personal observation) it might not have been identified. If ClpC was overlooked for these reasons it would be relatively simple to repeat these experiments and use antibodies specific to ClpC to immunoprecipitate ClpC and determine if it any was cross-linked to precursor associated with chloroplasts in docking conditions. Finally. ClpC may be present in translocation complexes under ATP- dependent docking conditions, but may not associate directly with precursors until later stages of import. At present, attempts to create stable import intermediates halted after the docking step of import are ongoing in the laboratory. If ClpC interacts with precursors at these later stages of import. detection of such an interaction will have to wait until techniques to create these late import intermediates have been developed. In the event that cross-linking strategies are not successful in identifying direct interaction between ClpC and translocating precursors a second, complementary strategy might provide some information on the possibility that ClpC is capable of interacting directly with precursor proteins. Although two different members of the Hsp100 protein family (ClpA. and Cle of Escherichia colr) have been shown to possess molecular chaperone activity (Wickner at al., 1994; Wawrzynow at al., 1995), ClpC, and its particular subgroup of the Hsp100 family. the C-type Hsp1003, have not yet been demonstrated to have molecular 190 chaperone activity. Therefore it is necessary to determine if ClpC does in fact associate with proteins as a molecular chaperone. This might be done by measuring if ClpC has a peptide-stimulated ATPase activity. Such experiments have been utilized to determine chaperone activity of several molecular chaperone families (Chappell et al., 1986; Flynn et al., 1988; Rothman, 1989) including HSp‘IOOS (Wawrzynow at al., 1995). IDENTIFICATION OF INNER MEMBRANE PROTEINS THAT INTERACT WITH CLPC ClpC is found in association with protein translocation components even in the absence of added precursors (Chapter 2). In other protein translocation systems soluble molecular chaperones in the interior of the organelle interact with integral membrane protein translocation components (For review see; Schatz and Dobberstein, 1996). ClpC most likely also interacts specifically with one or more inner envelope membrane translocation components. To address which inner envelope membrane translocation components interact with ClpC, one could extend the detergent solubilization experiments to analyze protein complexes of purified inner membranes. In the event that ClpC-containing complexes of the inner envelope membrane contain a large number of different proteins complementary chemical-cross-linking strategies could be attempted to determine which of these interacted directly with ClpC. 191 One possible complication is that, at present. inner envelope membranes are always contaminated by outer envelope membranes after fractionation protocols. Identification of translocation complexes containing only inner envelope membrane components might be difficult if complexes containing outer envelope membrane components were also detected. If this is the case. inner envelope membrane fractions could be further purified from contaminating outer envelope vesicles by immunodepletion with antibodies specific for outer envelope membrane translocation components such as Toc75. Two possible candidates for ClpC-interacting translocation components are the inner translocation components Tic110 (Labeck et al., 1996), and Ti025 (Ma et al., 1996). Antibodies are available for both these proteins, therefore it would be relatively easy to determine if either of these components could be found in association with ClpC solubilized from inner envelope membranes by coimmunoprecipitation analysis. It is likely that not all inner envelope membrane translocation components have been identified. If ClpC interacts with a previously unidentified inner membrane translocation components it may be necessary to detect interacting proteins by Coomassie or silver-staining methods. or detection of a protein modifying reagent, such as biotinylation. 192 REFERENCES Akita, M., Nielsen, E. and Keegstra, K. (1997) Identification of protein transport complexes in the chloroplastic envelope membranes via chemical cross-linking. J. CellBioI., 136, 983-994. Brodsky, J.L. and Schekman, R. (1993) A Sec63p-BiP complex from yeast is required for protein translocation in a reconstituted proteoliposome. J. Cell Biol., 123, 1355-1363. Chappell, T.G., Welch, W.J., Schlossman, D.M., Palter, K.8., Schlessinger, M.J and Rothman, J.E. (1986) Uncoating ATPase is a member of the 70 kilodalton family of stress proteins. Cell, 45, 3-13. Flynn, G.C., Chappell, T.G. and Rothman, J.E. (1989) Peptide binding and release of proteins implicated as catalysts of protein assembly. Science, 245, 385-390. Gray,J.C. and Row,P.E. (1995) Protein translocation across chloroplast envelope membranes. Trends Cell Biol., 5, 243-247. Hirsch, S., Muckel, E., Heemeyer, F., von Heijne, G. and Soll, J. (1994) A receptor component of the chloroplast protein translocation machinery. Science, 266, 1989-1992. Horst, M., Hilfiker-Rothenfluh, S., Oppliger, W. and Schatz, G. (1995) Dynamic interaction of the protein translocation 'systems in the inner and outer membranes of yeast mitochondria. EMBO J., 14, 2293-2297. Kang, P.-J., Ostermann, J., Shilling, J., Neupert, W., Craig, EA. and Pfanner, N. (1990) Requirement for hsp70 in the mitochondrial matrix for translocation and folding of precursor proteins. Nature, 348, 137-143. Kessler, F., Blobel, G., Patel, HA. and Schnell, D.J. (1994) Identification of two GTP-binding proteins in the chloroplast protein import machinery. Science, 266, 1035-1039. Kouranov, A. and Schnell, D.J. (1996) Protein translocation at the envelope and thylakoid membranes of chloroplasts. J. Biol. Chem, 271, 31009-31012. Lilbeck, J., Soll, J., Akita, M., Nielsen, E. and Keegstra, K. (1996) Topology of IEP110, a component of the chloroplast protein import machinery present in the inner envelope membrane. EMBO J., 15, 4230-4238. Ma, Y., Kouranov, A., LaSaIa, S. and Schnell, D.J. (1996) Two components 193 of the chloroplast protein import apparatus. IAP86 and IAP75, interact with the transit sequence during the recognition and translocation of precursor proteins at the outer envelope. J. Cell Biol., 134, 315-327. Nielsen, E., Akita, M., Davila-Aponte, J. and Keegstra, K. (1997) Stable association of chloroplastic precursors with protein-translocation complexes that contain proteins from both envelope membranes, and a stromal Hsp100 molecular chaperone. EMBO J., 16, 935-946. Olsen, L.J., Theg, S.M., Selman, B.R., and Keegstra, K. (1989) ATP is required for the binding of precursor proteins to chloroplasts. J. Biol. Chem, 264, 6724-6729. OIsen,L.J. and Keegstra,K. (1992) The binding of precursor proteins to chloroplasts requires nucleoside triphosphates in the intermembrane space. J. Biol. Chem, 267, 433-439. Perry, S.E., and Keegstra, K. (1994) Envelope membrane proteins that interact with chloroplastic precursor proteins. Plant Cell. 6, 93—105. Rothman, J.E. (1989) Polypeptide chain binding proteins: catalysts of protein folding and related processes in cells. Cell, 59, 591-601. Sanders, S.L., Whitfield, K.M., Vogel, J.P., Rose, MD. and Schekman, R.W. (1992) Se061p and BiP directly facilitate polypeptide translocation into the ER. Cell, 69, 353-365. Schatz, G. (1996) The protein import system of mitochondria. J. Biol. Chem, 271, 31763-31766. Schatz,G. and Dobberstein,B. (1996) Common principles of protein translocation across membranes. Science, 271, 1519-1526. Scherer,P.E., Krieg,U.C., Hwang,S.T., Vestweber,D. and Schatz,G. (1990) A precursor protein partly translocated into yeast mitochondria is bound to a 70 kd mitochondrial stress protein. EMBO J., 9, 4315-4322. Schneider, H.-C., Berthold, J., Bauer, M.F., Dietmeier, K., Guiard, 8., Brunner, M. and Neupert, W. (1994) Mitochondrial Hsp70/MIM44 complex facilitates protein import. Nature, 371, 768-774. Schnell, D.J., Kessler, F. and Blobel, G. (1994) Isolation of components of the chloroplast protein import machinery. Science, 266, 1007-1012. Schnell, D.J. (1995) Shedding light on the chloroplastic protein import machinery. Cell. 83, 521-524. 194 Scott,S.V. and Theg,S.M. (1996) A new chloroplast protein import intermediate reveals distinct translocation machineries in the two envelope membranes: Energetics and mechanistic implications. J. Cell Biol., 132, 63-75. Seedorf, M., Waegemann, K and Soll, J. (1995) A constituent of the chloroplast import complex represents a new type of GTP-binding protein. Plant J., 7, 401-411. Theg, S.M., Baurle, C., Olsen, L.J., Selman, B.R., and Keegstra, K. (1989) lntemal ATP is the only requirement for the translocation of precursor proteins across chloroplastic membranes. J. Biol. Chem, 264. 6730-6736. Vogel, J.P., Misra, L.M. and Rose, MD. (1990) Loss of BiP/GRP78 function blocks translocation of secretory proteins in yeast. J. Cell Biol., 110, 1885-1895. Waegemann,K. and Soll,J. (1991) Characterization of the protein import apparatus in isolated outer envelopes of chloroplasts. Plant J., 1, 149-158. Wawrzynow, A., Wojtkowiak, D., Marszalek, J., Banecki, 8., Jonsen, M., Graves, 8., Georgopoulos, C. and Zylicz, M. (1995) The Cle heat-shock protein of Escherichia coli, the ATP-dependent substrate specificity component of the ClpP-CIpX protease, is a novel molecular chaperone. EMBO J., 14, 1867-1877. Wickner, s.. Gottesman, s.. Skowyra, D., Hoskins, J., McKenney, K. and Maurizi, MR. (1994) A molecular chaperone, ClpA, functions like DnaK and DnaJ. Proc. Natl. Acad. Sci. USA, 91, 12218-12222. ntcman STATE UNIV. LIBRARIES llllllllllllllllllllllllllllllllllllllll 31293015719671