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The Plant Cell, Vol. 1, 1223-1230, December 1989 O 1989 American Society of Plant Physiologists Severa1 Proteins lmported into Chloroplasts Form Stable Complexes with the GroEL-Related Chloroplast Molecular Chaperone Thomas H. Lubben, Gail K. Donaldson, Paul V. Viitanen, and Anthony A. Gatenby' Molecular Biology Division, Central Research and Development Department, Experimental Station, E.I. DuPont de Nemours & Co., Wilmington, Delaware 19880-0402 Nine different proteins were imported into isolated pea chloroplasts in vitro. For seven of these [the large and small subunits of ribulose-l,5bisphosphate carboxylase/oxygenase (Rubisco), B-subunit of ATP synthase, glutamine synthetase, the light-harvesting chlorophyll a / b binding protein, chloramphenicol acetyltransferase, and pre-8lactamase], a fraction was found to migrate as a stable high-molecular-weight complex during nondenaturing gel electrophoresis. This complex contained the mature forms of the imported proteins and the groEL-related chloroplast chaperonin 60 (previously known as Rubisco subonit binding protein). Thus, the stable association of imported proteins with this molecular chaperone is widespread and not necessarily restricted to Rubisco subunits or to chloroplast proteins. With two of the imported proteins (ferredoxin and superoxide dismutase), such complexes were not observed. It seems likely that, in addition to its proposed role in assembly of Rubisco, the chloroplast chaperonin 60 is involved in the assembly or folding of a wide range of proteins in chloroplasts. INTRODUCTION An abundant chloroplast stromal protein has been implicated in the assembly of the photosynthetic enzyme ribulose-1,.Ç-bisphosphate carboxylase/oxygenase (Rubisco) (Barraclough and Ellis, 1980; Roy et al., 1982; Cannon, Wang, and Roy, 1986; Gatenby et al., 1988), and has been referred to as either the large subunit binding protein (Barraclough and Ellis, 1980), Rubisco subunit binding protein (Ellis and van der Vies, 1988), or the chloroplast chaperonin (Hemmingsen, et al., 1988). Chloroplast chaperonin is related to the groEL protein of Escherichia coli (Hemmingsen et al., 1988), a heat-shock protein that is essential for cell growth (Fayet, Ziegelhoffer, and Georgopoulos, 1989) and bacteriophage assembly (Georgopoulos et al., 1973; Sternberg, 1973; Zweig and Cummings, 1973; Kochan and Murialdo, 1983). In addition, groEL is required for assembly of prokaryotic Rubisco synthesized in E. coli (Goloubinoff, Gatenby, and Lorimer, 1989), and may also be involved in protein secretion (Bochkareva, Lissin, and Girshovich, 1988; Lecker et al., 1989). To establish a uniform nomenclature for this ubiquitous class of proteins found in organelles and bacteria, we shall refer to these polypeptides of an approximate molecular weight of 60,000 as chaperonin 60 (cpn60). It has been proposed that heat-shock proteins of two distinct classes, the hsp7O-immunoglobulin heavy chain ' To whom correspondence should be addressed. binding protein family (Pelham, 1986, 1988; Chirico, Waters, and Blobel, 1988; Deshaies et al., 1988; Zimmermann et al., 1988; Flynn, Chappell, and Rothman, 1989), and the cpn60 family (Bochkareva et al., 1988; Hemmingsen et al., 1988; Cheng et al., 1989; Goloubinoff et al., 1989; Lecker et al., 1989; Ostermann et al., 1989), assist other polypeptides to maintain, or assume, a conformation required for their correct assembly or localization. How this is achieved has not been resolved, but a feature of proteins in both families is that they hydrolyze ATP (Pelham, 1988) and interact with incorrectly or partially folded proteins (Gething, McCammon, and Sambrook, 1986; Bochkareva et al., 1988; Kassenbrock et al., 1988; Lecker et al., 1989). It has been demonstrated that mitochondria contain a protein, hsp60, that is related to bacterial cpn60 (McMullin and Hallberg, 1988), and that assembly and intraorganellar targeting of some proteins in mitochondria involve this chaperonin (Cheng et al., 1989; Ostermann et al., 1989). To determine whether chloroplast cpn60 may be involved in interactionswith proteins other than Rubisco, we imported nine different proteins into isolated chloroplasts in vitro. This selection of proteins includes severa1 polypeptides that are subunits of oligomeric structures, one monomeric chloroplast protein and one monomeric secretory protein. For seven of these proteins, a fraction of the imported protein was found in a high-molecular-weight 1224 The Plant Cell complex. This complex contained the importedprotein and cpn6O. The complex was stable during electrophoresis on nondenaturing gels but was disruptedeither,by illumination of chloroplasts or by addition of ATP to soluble extracts. One monomeric protein (ferredoxin) and one polypeptide that forms a dimer (superoxide dismutase) did not form such stable complexes with cpn60. RESULTS lmport of Proteins into Chloroplasts The proteins chosen for this study were the bacterial proteins pre-P-lactamase (preBLA, monomeric) and chloramphenicol acetyltransferase (CAT, trimeric), and the plant enzymes Silene pratensis ferredoxin (FD, monomeric), pea (Pisum sativum) superoxide dismutase (SOD, homodimeric), and alfalfa (Medicago sativa) glutamine synthetase (GS, octomeric). In addition, the P-subunit of the thylakoid ATP synthase from maize (Zea mays) chloroplasts (CF,-p) and the light-harvesting chlorophyll a/b protein (LHCP) from Petunia hybrida were imported; the Anacystis nidulans large subunit (LS) and pea small subunit (SS) of Rubisco (hexadecameric) were imported as controls. Plasmids were constructed that fused the appropriate genes to a soybean (Glycine max) chloroplast Rubisco SS transit peptide (TP) coding sequence, except for ferredoxin, superoxide dismutase, and preLHCP, which were imported using their naturally occurring transit peptides. Precursor proteins, shown in Figure 1, were producedas radiolabeled polypeptides by in vitro transcription and translation, and were post-translationallyimported into isolated pea chloroplasts. Figure 2 shows radiolabeled proteins from translation reactions and from import reactions after analysis by electrophoresis on SDS-polyacrylamide gels and fluorography. It should be noted that, although the size of preLHCP is given as 28 kD (predictedfrom DNA sequence) in Figure 1, on SDS-polyacrylamide gels (Figure 2) it migrates as a protein slightly larger than the 30-kD molecular mass marker (Viitanen, Doran, and Dunsmuir, 1988). Import reactions were conducted in subdued light (less than 1 pEinstein/cm*) to preserve the cpn6O-imported protein complexes (see below), and externa1ATP providedenergy for import. As shown in Figure 2, all precursor proteins, except for TPpreBLA, were imported and processed to the size expected for proteins from which the transit peptide had been removed. Following import of TPpreBLA, three polypeptides were found inside chloroplasts (Figure 2). These were the correct size for TPpreBLA, preBLA, and BLA. This suggested that (1) the import of some TPpreBLA occurred unaccompanied by cleavage of the chloroplast transit peptide and (2) the bacterial presequence may be recognized and cleaved in the chloroplast stroma. Regardless, all proteins examined were imported well enough to undertake the binding studies described below. This required that we be able to determine whether 17'0 of the imported protein were bound to cpn60. Binding of lmported Proteins to cpn6O Chloroplast cpn60, like E. coli cpn60 (groEL), can form oligomers composed of 14 subunits, each of apparent molecular weight (M,) of 60,000 to 61,000 (Hemmingsen et al., 1988). These cpn6O oligomers, together with associated proteins, can be resolved on nondenaturing gels as a sharp band, which migrates more slowly than Rubisco holoenzyme. Both the cpn6O and Rubisco bands are clearly visible upon staining (Barracloughand Ellis, 1980; Roy et al., 1982; Gatenby et al., 1988). Furthermore, cpn60 oligomers dissociate under certain conditions (Bloom, Milos, and Roy, 1983; Hemmingsen and Ellis, 1986; Roy, Hubbs, and Cannon, 1988). To determine whether imported proteins were bound to cpn60, import reactions were analyzed by electrophoresis on low-percentage polyacrylamide gels at 4°C in the absence of denaturants. As shown in Table 1, neither imported SOD nor FD associated with cpn6O. Consistent with an earlier study (Gatenby et al., 1988), a small but significant portion of imported SS (about 5%) formed a stable complex with cpn60. This was also observed for LHCP, regardless of whether it was Precursor Proteins 2kíLml 20 62 59 31 38 52 17 24 28 31 Figure 1. Precursor Proteins. Precursor proteins were made by in vitro transcription and translation of plasmids that contained promoters for SP6 or T2 RNA polymerase upstream of the coding regions (Lubben and Keegstra, 1986). mRNA from the transcription reactions was translated in vitro in a system derived from either reticulocytelysate or wheat germ. The resulting proteins are shown diagramatically. Chloroplast Chaperone Protein Complexes A B A B A B A B LTPLS /PpreBLA -praBLA A 30 B A B A B i A B A sented stable complexes of cpn60 with imported proteins, or merely artifacts of co-migration during electrophoresis. To help make this distinction, we treated Chloroplasts that contained imported proteins and stromal extracts from these Chloroplasts as described below. Results from these studies are shown in Figure 3. Similar results were obtained for all the proteins that comigrated with cpn60 oligomers (Figure 3). When either buffer, a glucose/hexokinase mixture or MgCI2 alone (Figure 3), or the sulfhydryl-reducing agent dithiothreitol (data not shown) were added to chloroplast extracts, no change was observed in the radiolabeled band that co-migrated with cpn60. When intact Chloroplasts that contained imported proteins were incubated in light, either in the absence (Figure 3, lane A) or presence (Figure 3, lane B) of chloramphenicol to inhibit intraorganellar protein synthesis, the radiolabeled band decreased to about one-third its control level (Figure 3, lane C). The stained protein band of cpn60 decreased similarly (data not shown). As anticipated, cpnGO oligomers dissociated when MgCI2 and ATP were added to extracts made from Chloroplasts that contained imported proteins (Figure 3, lane F). Following this treatment, some of the radiolabeled protein migrated near the top of nondenaturing gels as a high-/Wr complex (Figure 3, lane F). In contrast, no dissociation was observed when the nonhydrolyzable ATP analog /i.-y-methyleneadenosine 5'-triphosphate was used instead of ATP (Figure 3, lane G). From these studies, we concluded that (1) the observed band was indeed cpn60 and (2) those proteins that comigrated with the cpn60 band were associated with cpn60. I A -p>FD 1225 B prSOD B Figure 2. SDS-Polyacrylamide Gel Electrophoresis of Proteins Imported into Chloroplasts. Precursor proteins were incubated with intact pea Chloroplasts. After import, Chloroplasts were treated with protease (thermolysin) to remove external proteins, and intact Chloroplasts were reisolated, lysed, and analyzed by electrophoresis on SDS-polyacrylamide gels. Radiolabeled bands were then visualized by fluorography. Lanes A, translation products; lanes B, imported, protease-protected proteins. The identity of the precursor protein is indicated beside each panel; the migration of 14C-labeled molecular mass standards is shown: myosin, 200 kD; phosphorylase b, 92 kD; bovine serum albumin, 68 kD; ovalbumin, 46 kD; carbonic anhydrase, 30 kD; lysozyme, 14 kD. imported using its own transit peptide (as preLHCP) or the transit peptide of TPSS (as TPLHCP) (Table 1). In contrast, significantly larger amounts of some of the radiolabeled imported proteins (BLA, CAT, SS, LS, CF,-,cf, and GS) were found to co-migrate with the cpn60 band. For these proteins, further studies were done to determine the nature of their interaction with cpn60. Cpn60 oligomers dissociate in the presence of ATP in a reaction that requires ATP hydrolysis (Bloom et al., 1983; Hemmingsen and Ellis, 1986; Roy et al., 1988). It is also known that partial dissociation of cpn60 oligomers and associated LS occurs when intact Chloroplasts are incubated in light (Roy et al., 1988). These characteristics provided a convenient way to determine whether the radioactive bands observed in nondenaturing gels repre- Table 1. Association of Imported Proteins with Chloropiast cpn60 Precursor Protein % in cpn60 ± so TPCF,-/} TPLS TPGS TPCAT 38 ±5 34 12 24 11 17 2.8 6 2.6 5 0.6 3 0.6 3 ±0.6 TPpreBLA TPSS TPLHCP preLHCP prFD prSOD a ND" NO ND, not detectable. The limit of detection was 1%. Bands that contained cpn60 were excised from nondenaturing gels similar to those shown in Figure 3 and counted using liquid scintillation methods. Similarly, bands that contained imported proteins were also excised from SDS-polyacrylamide gels such as those in Figure 2. The amount of protein in the cpn60 band was compared with that found in the band from the SDS gel. These percentages represent the fraction of total imported protein that was found associated with cpn60. Values are the average of at least three experiments. 1226 The Plant Cell TPpreBLA A B C D E F G till!*} TPCAT A B C D E F G -cpn601 RubisCO TPSS A B C D E F G TPLS A B C D E F G — cpn60 — > RubisCO'1 TPCF-P TPGS A B C D E F G A B C D E F G To determine whether the imported proteins in the cpn60 complex contained a transit peptide or only mature, processed proteins, these complexes were examined further. Portions of nondenaturing gels that contained cpn60 and imported proteins were excised from nondenaturing gels, equilibrated in SDS sample buffer, and analyzed by SDS gel electrophoresis. For technical reasons, only proteins that showed a high level of association with cpn60 were analyzed. Only mature proteins, without transit peptides, were found (data not shown). We anticipated this result because the SDS gels of total imported proteins (Figure 2) showed that, except for imported TPpreBLA, only processed, mature proteins were present. To quantitate the interaction of the various imported proteins with cpn60, the radiolabeled cpnGO oligomer bands were excised from the nondenaturing gels and counted using liquid scintillation methods. Similarly, bands that contained imported proteins were also excised from SDS-polyacrylamide gels (Figure 2). We preferred this method over densitometry because stained protein bands significantly quench any associated label during fluorography but not during liquid scintillation counting of excised bands. The amount of radiolabeled protein in the cpnGO bands was compared with that found in the bands from the SDS gels. These values are shown in Table 1 and represent the fraction of total imported protein that was found associated with cpn60. These ranged from 38 ± 5% for imported CF,-0 to undetectable levels (less than 1%) for imported SOD and FD. DISCUSSION Figure 3. Effect of Treatments on Association of cpnGO with Imported Proteins. Intact chloroplasts from the reactions in Figure 2 were subjected to various treatments, followed by analysis by nondenaturing polyacrylamide gel electrophoresis and fluorography. Conditions for electrophoresis were essentially as described (Gatenby et al., 1988), except that electrophoresis was done at 4°C. The migration positions of the chloroplast chaperonin (cpn60) and Rubisco holoenzyme (RubisCO) were determined by lightly staining with bromphenol blue. This staining quenched the radioactive fluorescence of these bands so that the association of imported LS with Rubisco holoenzyme is not visible. However, radioactivity in Rubisco was readily determined by the use of liquid scintillation methods to count excised bands. The precursor proteins used in each reaction are indicated above each panel. For lanes A and B, intact chloroplasts were incubated in light at 25°C for 10 min. The reaction for lane B contained 100 Mg/mL chloamphenicol to inhibit intraorganellar protein synthesis. For lanes C to G, chloroplasts were lysed hypotonically in 10 mM HEPES-KOH, pH 7.5, and centrifuged to remove membranes. Various additions were made to fractions of the supernatant, and these reaction were incubated for 10 min at 25°C. The additions resulted in these final concentrations in the various reactions: lane C, 10 mM HEPES-KOH, pH 7.5 (control); lane D, 0.08 units/mL hexokinase, 1 mM glucose; lane E, 10 mM MgCI2; lane F, 10 mM MgCI2/5 mM ATP; lane G, 10 mM MgCI2/5 mM /3,7-methyleneadenosine5'-triphosphate. In this report we show that a wide variety of imported proteins are found associated with cpn60. Although binding of intraorganellar-synthesized LS (Barraclough and Ellis, 1980; Roy et al., 1982) and imported LS (Gatenby et al., 1988) and SS (Ellis and van der Vies, 1988; Gatenby et al., 1988) to cpnBO has been reported, the association of nonRubisco proteins with cpn60 in chloroplasts has not been described before. The interaction of several disparate imported proteins with cpn60 may indicate a general role for this oligomer at some point in the folding or assembly pathway of imported chloroplast proteins. The folding and assembly of proteins to their final conformation has previously been thought to be an intrinsic feature of the amino acid sequence of a protein (Anfinsen, 1973). However, in some instances, it is now known that this process requires the presence of auxiliary proteins (Cheng et al., 1989; Goloubinoff et al., 1989; Ostermann et al., 1989). One such class of proteins are chaperonins (Hemmingsen et al., 1988), which are involved in protein folding (Ostermann et al., 1989; Goloubinoff et al., 1990) and are required for the assembly of some oligomeric structures (Georgopoulos et al., 1973; Sternberg, 1973; Zweig and Cummings, 1973; Kochan Chloroplast Chaperone Protein Complexes and Murialdo, 1983; Cheng et al., 1989; Goloubinoff et al., 1989). Therefore, binding of imported proteins to cpn60, as observed in the present study, may be an early event in the folding of chloroplast proteins. It should be noted that a stable association was observed between cpn60 and the monomeric protein BLA, which obviously does not become assembled into an oligomeric structure. Apparently, monomeric polypeptides, as well as those that are components of oligomeric structures, can form complexes with cpn60 upon import. The product from the association of nascent or imported polypeptides with cpn60 might be folded monomers, or assembly-competent subunits of oligomeric proteins, instead of assembled oligomers, thus invoking a role for cpn60 in protein folding directly. However, both the recognition determinants and the exact role of these molecular chaperones in protein folding and assembly pathways remain to be determined before a functional model can be established. If binding of these imported proteins represents an initial event in protein folding, then the interaction of such a diverse group of imported proteins with cpn60 indicates that a common recognition motif may be present in these proteins. Because chaperonins are not considered to be part of the final structure (Hemmingsen et al., 1988) and apparently do not bind to completely folded proteins (Goloubinoff et al., 1990), this motif may be accessible only in polypeptides entering the chloroplast in an incompletely folded state. This ability to recognize common sequential or transiently exposed structural features of the partially folded imported protein would account for the observed broad specificity of cpn6O. This type of protein interaction could also explain the association of cpn60 with Rubisco LS, synthesized within the chloroplast (Barraclough and Ellis, 1980; Roy et al., 1982). Elongating polypeptide chains, emerging from ribosomes, may present features during translationor after release in an unfolded or partially folded form that are available for interaction with cpn60 in a manner analogous to the interaction with polypeptides entering the organelle. Two proteins, prSOD and prFD, did not form stable complexes with cpn60. These proteins were imported using their own transit peptides rather than the transit peptide of TPSS. This might lead one to conclude that the interaction of proteins with cpn6O was mediated by the transit peptide of TPSS. However, we think this is unlikely for severa1reasons. First, the Same low level of association of LHCP with cpn60 was observed whether LHCP import was directed via its own transit peptide (as preLHCP) or via the transit peptide of TPSS (as TPLHCP). Second, when cpn60-imported protein complexes from nondenaturing gels were analyzed by SDS gel electrophoresis, only mature proteins without transit peptides were found. Finally, a number of proteins without transit peptides have been shown to associate with cpn60 or groEL. For example, chloroplast-synthesized LS, which contains no transit peptide, strongly binds to cpn60 oligomers (Barraclough and Ellis, 1980, Roy et al., 1982, 1988), and two 1227 bacterial proteins, preBLA and CAT, which contain no chloroplast transit peptide, associate transiently with the heat-shock groEL protein in E. coli S30 extracts (Bochkareva et al., 1988). However, regardless of what determines whether a protein interacts with cpn60, the observation that a variety of proteins form stable complexes with cpn6O provides a tool with which to examine further the role of cpn60 in protein folding and import. Because cpn6O proteins have been found in a wide variety of organisms (McMullin and Hallberg, 1988) and share a high level of amino acid sequence homology (Hemmingsen et al., 1988), it is likely that they also exhibit a broad specificity with regard to the proteins with which they interact in the folding or assembly process. Indeed, the data shown here, which demonstrate a stable association between pea chloroplast cpn60 and a variety of heterologous proteins, support this. Thus, the chloroplast chaperonin interacts with the prokaryotic BLA, CAT, and LS proteins, in addition to binding to plant proteins from different species (maize CF,-0, alfalfa GS, petunia LHCP). Furthermore, it is noteworthy that a number of different temperature-sensitive mutations can be suppressed in vivo by overproduction of the E. coli groE proteins in bacterial hosts (Van Dyk, Gatenby, and LaRossa, 1989). Other heatshock proteins also exhibit broad specificity. In vitro interactions betweena large number of short synthetic peptides and another heat-shock protein (hsc70) have been demonstrated (Flynn et al., 1989). Our results suggest that chloroplast cpn6O may be crucial in mediating the posttranslational folding and assembly of many imported or organelle-encoded proteins. Indeed, a similar role has been suggested recently for mitochondrial cpn60, which is involved in the assembly and intraorganellar targeting of mitochondrial proteins (Cheng et al., 1989, Ostermann et al., 1989). METHODS Enzymes and Reagents Most biochemicals were from Sigma unless indicated otherwise. Radioisotopes, EN3HANCE,and Econofluor were from DuPontNew England Nuclear; SP6 RNA polymerase and restriction enzymes were from Bethesda Research Laboratories; NCS tissue solubilizerwas from Amersham; RNasin,and lysate for translation, made from either rabbit reticulocyte or wheat germ, was from Promega; acrylamide, bisacrylamide, and N,N,N',N'-tetramethylethylenediamine were from Bio-Rad. Plasmid Construction Plasmids that encode TPSS, TPLS, and TPCF-P (Lubben and Keegstra,1986; Gatenby et al., 1988) have been described before. Plasmids pSPFD22 (Smeekens, van Binsbergen, and Weisbeek, 1228 The Plant Cell 1985), which encodes prFD, pSPA2 (Scioli and Zalinskas, 1988), which encodes prSOD, and M13mpl8/preLHCP (Viitanen et al., 1988), which encodes preLHCP, have also been described before. To make pTPBLA, which encodes TPpreBLA, pGMTP was cut with Pstl, treated with T4 DNA polymerase, and cut with Ball. This fragment encodes the transit peptide of TPSS, but with a Ball site at the 5’ end. This was ligated into pTG2 (Kadonaga et al., 1984), which had been cut with EcoRl and treated with the Klenow fragment of DNA polymerase. pGMTP was made by ligation of the small Hindlll/EcoRIfragment of pGM22/3 (Gatenby et al., 1988), which encodes the same transit peptide found in TPSS, into Hindlll/EcoRI-cut pGEMl /BAL. pGEMl/BAL was made by cutting pGEM1 (Promega) with Sphl, treating with T4 DNA polymerase, and inserting Ball linkers (Bethesda Research Laboratories, 5’-GTGGCCAC-3’). To make pTPCAT, which encodes TPCAT, a Bglll/partial Hincll fragment encoding CAT was subcloned from pB3CA81 (French and Ahlquist, 1987) via a pUC18 polylinker into pGM22/3 cut with Sphl/Pstl. To make pTPGS, which encodes TPGS, pGSl O0 (DasSarma, Tischer, and Goodman, 1986) was cut with Bglll, filled in with Klenow, and cut with Stul. This 1.2-kb glutamine synthetase coding region was ligated into pGM22/3, which had been cut with Sal1 and repaired with Klenow. Plasmid pTPLHCP, which encodes TPLHCP (a chimeric protein in which the transit protein of TPSS has been fused to mature LHCP), was constructed from a modified version of M13mpl8/preLHCP (Viitanen et al., 1988). Briefly, the region encoding the first methionine of mature LHCP ( C C E A ) was converted to an Sphl site ( G C E C ) , using oligonucleotide-directed mutagenesis as previously described (Viitanen et al., 1988). A fragment that encoded mature LHCP was excised from this modified plasmid with Sphl/Xbal. This fragment was ligated into Sphl/Xbal-cut pGM22/3 (which contains the transit peptide of TPSS) to produce pTPLHCP. were subjected to seven different treatments. Each aliquot contained the equivalent of 30 pL (of a total of 220 pL) of the original import reaction. For the first two treatments,two aliquots Of intact chloroplasts were incubated in light at 25°C for 10 min. To one of these chloramphenicol was added to a final concentration of 1O0 pg/mL to inhibit intraorganellar protein synthesis. After this incubation, chloroplasts were collectedby centrifugationand lysed hypotonically in 10 mM HEPES-KOH, pH 7.5, and centrifuged to remove membranes. For the other five treatments, soluble extracts were prepared from chloroplasts that had been lysed hypotonically as described above. Various additions were made to these extracts (as described in the legend to Figure 3), and these were incubated for 10 min at 25°C. After incubation, portions of these reactions and the extracts from the two light-treated chloroplasts were analyzed by SDS-PAGE (Laemmli, 1970) and by nondenaturing gel electrophoresis through 6% polyacrylamide gels as described (Laemmli, 1970) except that neither SDS nor 8mercaptoethanol was present (Gatenby et al., 1988). Radiolabeled bands were visualized by fluorography after treatment of gels with EN3HANCE.Quantitation of the number of imported molecules was calculated from the number of leucine or methionine residues per imported protein after liquid scintillationcounting of gel slices that had been sdubilized in NCS tissue solubilizer (Walter, Ibrahimi, and Blobel, 1981). ACKNOWLEDGMENTS We would like to thank Roy French, Howard M. Goodman, Jeremy R. Knowles, Janet Scioli, and Sjef Smeekens for providing plasmids, and Cathy Kalbach and Karen Bacot for excellent technical assistance. In Vitro Transcription and Translation and Protein lmport Transcription of linearized plasmid DNA templates to produce mRNA was performed as described previously (Lubben and Keegstra, 1986). Translation of this mRNA, in lysate from wheat germ or reticulocyte lysate, was done according to the manufacturer’s (Promega) protocol, using either 3H-le~~ine or 35S-methionine. Protein import reactions were done as described previously (Lubben and Keegstra, 1986), except that reactions were done in subdued light (less than 1 pEinsteinlcm2)with 45 pL of translation per reaction, and contained 10 mM ATP to provide energy for import. After a 30-min incubationat room temperature, reactions (220 pL) were centrifuged and chloroplasts were resuspended in 0.1 mg/mL thermolysin and incubated for 30 min to remove externally bound proteins (Cline et al., 1985). lntact chloroplasts were repurifiedby centrifugationthrough 40% Percoll (Cline et al., 1985), and the chloroplastswere resuspended in 220 pL of impor? buffer (Cline et al., 1985) and divided into aliquots for further treatment. Analysis of Binding of lmported Proteins to cpn6O Oligomers For each precursor protein, protease-treatedchloroplastscontaining imported proteins or soluble extracts from these chloroplasts Received August 1O, 1989; revised October 16, 1989. REFERENCES Anfinsen, C.B. (1973). 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Several proteins imported into chloroplasts form stable complexes with the GroEL-related chloroplast molecular chaperone. T H Lubben, G K Donaldson, P V Viitanen and A A Gatenby Plant Cell 1989;1;1223-1230 DOI 10.1105/tpc.1.12.1223 This information is current as of August 3, 2017 Permissions https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298 X eTOCs Sign up for eTOCs at: http://www.plantcell.org/cgi/alerts/ctmain CiteTrack Alerts Sign up for CiteTrack Alerts at: http://www.plantcell.org/cgi/alerts/ctmain Subscription Information Subscription Information for The Plant Cell and Plant Physiology is available at: http://www.aspb.org/publications/subscriptions.cfm © American Society of Plant Biologists ADVANCING THE SCIENCE OF PLANT BIOLOGY