Roles Of Cytosolic Hsp70 And Hsp40 Molecular Chaperones In Post-translational Translocation PDF

Roles of Cytosolic Hsp70 and Hsp40 Molecular Chaperones in Post-translational Translocation of Presecretory Proteins into the Endoplasmic Reticulum* Hsp701 molecular chaperones and their co-chaperones work together in a variety of cellular compartments to guide the folding of protein...

Roles of Cytosolic Hsp70 and Hsp40 Molecular Chaperones in Post-translational Translocation of Presecretory Proteins into the Endoplasmic Reticulum* Hsp701 molecular chaperones and their co-chaperones work together in a variety of cellular compartments to guide the folding of proteins and to aid the translocation of proteins across membranes (reviewed in Ref. 1). The binding and hy drolysis of ATP regulate the action of Hsp70s (2). In the ATP * This work was supported by National Science Foundation Grant MCB-9905988 and a grant from the American Heart Association (to W.J.C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡ To whom correspondence should be addressed. Tel.: 718-270-1308; Fax: 718-270-3732; E-mail: [email protected]. 1 The abbreviations used are: Hsp70, 70-kDa heat shock protein; AP-ppF6H, affinity purified, histidine- tagged prepro--factor; H, hex okinase; Hsc70, 70-kDa heat shock cognate protein; NEM, N-ethylma leimide; NEM-Ssa1p, N-ethylmaleimide-modified Ssa1p; Nt, nondena tured ppF6H; PRS, postribosomal supernatant; pF, pro--factor; pF6H, histidine-tagged pro--factor; ppF, prepro--factor; ppF6H, histidine-tagged prepro--factor; S, Ssa1p; Y, Ydj1p; Ni2-NTA, nickel nitrilotriacetic acid. bound form, peptide substrates undergo cycles of rapid binding and release from Hsp70 (3). In the ADP-bound form, the inter actions are slower resulting in higher affinity. Hsp70s stimu late protein folding by binding to exposed hydrophobic se quences (4–6) and preventing their irreversible aggregation (7). The activity of Hsp70s is regulated by Hsp40s and other co-chaperones. Hsp40s stimulate the ATPase activity of Hsp70s (8) and can target unfolded proteins to them (7). Hsp40s preferentially bind hydrophobic polypeptides (9) and can prevent the aggregation of some unfolded proteins (7, 10). Genetic and biochemical evidence supports a role for cytoso lic Hsp70s and Hsp40s in the post- translational translocation of precursor proteins into the endoplasmic reticulum and mi tochondria (reviewed in Ref. 11). Deshaies et al. (12) reported that presecretory proteins and mitochondrial precursors accu mulated in the yeast cytosol when the concentration of cytosolic Hsp70s was lowered. A temperature-sensitive mutant of the yeast cytosolic Hsp70 Ssa1p rapidly accumulated presecretory proteins at the nonpermissive temperature (13). Chirico et al. (14) showed that yeast cytosolic Hsp70s stimulated the in vitro translocation of prepro--factor (ppF) into yeast microsomes. Complexes containing Hsp70s and presecretory and mitochon drial precursor proteins have been identified in wheat germ extracts and reticulocyte lysates (15–17). The ability to stimu late post-translational translocation is not shared by all Hsp70s or by other stress protein families. Neither Hsp60 (18), Kar2p (19), nor Hsp90 (18) stimulates translocation. However, DnaK can partially substitute for yeast cytosolic Hsp70s in vitro (19, 20). Several laboratories have explored the role of DnaJ homologs in protein translocation. Caplan et al. (21) reported that a temperature-sensitive mutant of YDJ1 was defective for trans location at the nonpermissive temperature. However, Atencio and Yaffe (22) and Becker et al. (13) showed that ppF is translocated normally in deletion mutants of YDJ1. Transloca tion of ppFwasdefective in a strain expressing a mutant form of Ydj1p that cannot be farnesylated indicating that the lipid modified version of Ydj1p plays a role in protein translocation (21). Hendrick et al. (23) showed that Escherichia coli DnaJ completely inhibited post-translational translocation of ppF in vitro. DnaK and GrpEtogether relieved the DnaJ-dependent inhibition, but neither alone had any effect. Export of certain precursor proteins was defective in some dnaK and dnaJ mu tant strains of E. coli (24). Functional interactions between yeast Hsp70s and DnaJ homologs have been studied in vitro and in vivo (13, 25–27). Ydj1p stimulates the ATPase activity of Ssa1p (25) and to gether they constitute a protein folding machinery capable of refolding denatured luciferase (26). Becker et al. (13) explored the in vivo interactions of SSA1 and YDJ1 in protein translo 7034 Downloaded from http://www.jbc.org/ at INDIAN INST OF TECHNOLOGY BOMBAY on August 31, 2019 This paper is available on line at http://www.jbc.org Roles of Chaperones in Post-translational Translocation 7035 cation into the endoplasmic reticulum and mitochondria. A temperature-sensitive SSA1 mutant was synthetically lethal with a YDJ1 deletion mutant suggesting that SSA1 and YDJ1 genetically interact (13). Pulse-chase experiments showed that ppF, which had accumulated at the nonpermissive tempera ture, could not be translocated. These results suggested that the chaperones maintain translocation competence by binding to ppF co-translationally, but they cannot rescue aggregated ppF. Strains containing mutant versions of Ssa1p with defec tive ATP-binding pockets fail to interact productively with Ydj1p and accumulate ppF (27). We undertook the following study to gain insight into the mechanism by which Ssa1p and Ydj1p contribute to the post translational translocation of presecretory proteins into the endoplasmic reticulum. To avoid the effects of endogenous chaperones and other proteins contaminating earlier prepara tions of ppF, which wassynthesized in wheat germ extracts or reticulocyte lysates, we used a histidine-tagged, affinity puri fied version of ppF (AP-ppF6H) in translocation assays. We clarified the contributions of membrane-bound and cytosolic Ydj1p, by comparing the translocation efficiencies of the chap erones using wild-type and Ydj1p-deficient microsomal mem branes. We demonstrate that Ssa1p stimulates translocation by preventing the aggregation of AP-ppF6H. Although Ydj1p stimulates the ATPase activity of Ssa1p (25) and is essential for Ssa1p-dependent protein folding of luciferase in vitro (26), we show that it is not essential for post- translational protein translocation. However, Ydj1p alone stimulates post-transla tional translocation by preventing AP-ppF6H aggregation. EXPERIMENTAL PROCEDURES Plasmids—pKWC1, which can express ppF6H in E. coli, was con structed by amplifying the cDNA for ppF contained in pDJ100 (28), cutting the full-length product with BglII and SphI, and then ligating it into the corresponding sites in pQE70 (Qiagen, Inc.). The forward and reverse primers in the polymerase chain reaction were 5- GTGTGCAT GCGATTTCCTTCAATTTTTACTG-3 and 5-ATATAGATCTGTACAT TGGTTGGCCGGGT-3, respectively. pREP4 was obtained from Qia gen. pNWC1, which was used to generate mRNA coding for ppF6H, was constructed by amplifying the cDNA for ppF6H contained in pKWC1, digesting the resulting product with SphI and SacI, and then ligating it into pGEMEX-1 behind the SP6 promoter. The forward and reverse primers in the amplification reaction were 5-GTGTGCATGC GATTTCCTTCAATTTTTACTG-3 and 5-ATATGAGCTCGGATCTAT CAACAGGAGTCC-3, respectively. The sequences of the constructs were confirmed by automated DNA sequencing. Metabolic Labeling of PpF6H—E. coli IQ85 (F araD139 (argF lac)U169 rpsL150 relA1 flbB5301 deoC1 ptsF25 secYts) was trans formed sequentially with pREP4 and pKWC1. The resulting strain IQ85[pREP4, pKWC1] was grown overnight at 25 °Cin2ml ofLB media containing 2% glucose, 25 g/ml kanamycin, 25 g/ml tetracy cline, and 100 g/ml ampicillin. The cells were collected by centrifuga tion (15,700 previously (19). The concentration of Ssa1p was determined as de scribed previously (31). Ydj1p was overexpressed and purified from E. coli as described previously (25). The concentration of Ydj1p was deter mined using its extinction coefficient at 280 nm (20370 M 1 cm1). This preparation of Ydj1p stimulated the ATPase activity of Ssa1p 10-fold and was required for efficient luciferase refolding in the presence of Ssa1p (data not shown) (26). ATPase and luciferase refolding assays were performed as described previously (26, 33). Postribosomal super natants (PRS) were prepared from S. cerevisiae SKQ2N as described previously (14). Hexokinase from S. cerevisiae was obtained from Sigma. [35S]PpF6H was purified from either wheat germ translation reactions programmed with ppF6H mRNA or metabolically labeled IQ85[pREP4, pKWC1] as follows. Proteins in translation reactions (230 l) were precipitated with 460 l of ethanol and the resulting mixture was incubated on ice for 5 min. The mixture was centrifuged at 15,700 g for 5 min and then the pellet was resuspended in 1 ml of buffer B (6 M guanidine hydrochloride, 100 mM sodium phosphate, and 10 mM Tris-HCl, pH 8) and rotated for 1hat room temperature. Insoluble proteins were removed by centrifugation at 15,700 g for 5 min. Ni2-NTA resin (60 l of 50% slurry, Qiagen) was added to the supernatant and the mixture was rotated for 1 at room temperature. Unbound proteins were removed by washing the resin 5 times with 250 l of buffer C (8 M urea, 100 mM sodium phosphate, and 10 mM Tris HCl, pH8.0). [35S]PpF6H was eluted with 40 l of buffer C adjusted to pH 4.6. After the pH of eluate was neutralized with 7.5 l of 2M HEPES, pH 8, it was frozen in liquid nitrogen and stored at 70 °C. [35S]PpF6H was purified from metabolically labeled IQ85[pREP4, pKWC1] (see above) by resuspending the cells from a 10-ml culture in 500 l of buffer B. After mixing the cells for1hatroom temperature, insoluble proteins were removed by centrifugation at 15,700 g for 5 min. The supernatant was mixed with 50 lofNi2-NTA resin (50% slurry) for1hatroom temperature. Unbound proteins were removed from the resin and then [35S]ppF6H was eluted as described above. PF6Hwasproducedin either IQ85[pREP4, pKWC1] or DH5 [pREP4, pKWC1] grown and induced at 37 °C. It was purified in the same manner as ppF6H. Protein concentrations were determined using the Bradford assay (34) and bovine- globulin was used as the standard. Protein Sequencing—A 1.75-g sample of purified pF6H was deri vatized with phenylisothiocyanate and then automated Edman degra dation was performed using an Applied Biosystems 475A Protein Se quencer (Applied Biosystems, Inc.) in the Protein Sequencing Center at SUNY Downstate Medical Center. Yeast Microsomal Membranes—Microsomes were isolated from S. cerevisiae SKQ2N (MATa/ ade1/ /ade2 /his1), MYY290 (MATa leu2 his3 ura3) (22), and MYY406(MAT leu2his3mas5::URA3)(22)as previously described (30). The post-translational translocation activi ties of microsomes isolated from wild-type strains SKQ2N and MYY290 were essentially identical. One equivalent (eq) of microsomes was de g), resuspended in 10 ml of minimal A medium (29) containing 1 mM MgSO4 , 0.2% glycerol, 0.0005% thiamine-HCl, 0.075% CSM- MET(QBIOgene, Inc.) and the above antibiotics. The culture was incubated at 25 °C until 0.5 A600. The temperature was raised to 42 °C and the culture was incubated for 2.5 h. Isopropyl-1-thio--D-galacto pyranoside (2 mM final concentration) and Tran35S-label (ICN Biomedi cals, Inc.) were added and the culture was incubated for an additional 30 min at 42 °C. The culture was placed on ice, the cells were collected by centrifugation at 4 °C, and [35S]ppF6H was purified as described below. In Vitro Transcription and Translation—Messenger RNA coding for ppForppF6H was synthesized using a RiboMAX kit (Promega). Before transcription, pDJ100 and pNWC1 were linearized with XbaI and SacI, respectively. The 3 overhangs resulting from SacI digestion of pNWC1 were converted to blunt ends using Klenow. Radiolabeled ppFandppF6Hweresynthesized in wheat germ extracts (Promega) according to the manufacturer’s instructions. Alternatively, wheat germ extracts were prepared as previously described (30). Translation grade [35S]methionine was obtained from PerkinElmer Biosciences. Protein Purification—Ssa1p was overexpressed in Saccharomyces cerevisiae strain MW141 in YPGal medium and purified as described fined as 50 A280 units/ml (35). The optical density of microsomes was measured in 1% SDS. The final concentration of the membrane was adjusted to 4 eq/ l in buffer A (20 mM HEPES-KOH, pH 7.5, 100 mM potassium acetate, 2 mM magnesium acetate, and 2 mM dithiothreitol) containing 14% glycerol. Post-translational Translocation—The post- translational transloca tion assay was described previously (14). In brief, it contained 2 lof yMix (27.25 mM HEPES, pH 7.5, 863 mM potassium acetate, 17.3 mM magnesium acetate, 15.25 mM dithiothreitol, and 12.5 mM cyclohexi mide), 1.37 l of an energy source (9.1 mM ATP, 456.2 mM creatine phosphate, and 0.365 mM GDP-mannose), 0.63 l of 8 mg/ml creatine kinase, 2 l of yeast microsomes (4 eq/ l), 1 l of water, 16 l of buffer A (PRS or chaperones to be tested), and 2 l ( 15,000 dpm) of wheat germ-translated [35S]ppF o r [35S]ppF6H. When denatured [35S]ppF6H (or [35S]ppF) was used as the substrate, it was prepared by diluting the translation reaction 4-fold into 8 M urea. The post translational translocation assay was compensated with 1.6 or 1.7 lof wheat germ translation compensation buffer (43 mM HEPES-KOH, pH 7.5, 112 mM potassium acetate, 2.1 mM magnesium acetate, 3 mM dithiothreitol) before adding 0.4 l of denatured [35S]ppF6H (or [35S]ppF) or 0.3 l of affinity purified [35S]ppF6H, respectively. De natured substrate ( 15,000 dpm) was added last and then reactions were mixed immediately. All pipette tips used to transfer denatured ppF were pretreated with SIGMACOTE (Sigma). The final volume was 25 l and the reactions were incubated at 20 °C for the indicated times. Reactions were terminated by adding an equal volume of 2 SDS-PAGEsamplebuffer andthenboiling the mixture for 2 min. In the maintenance of translocation experiments, AP- [35S]ppF6H was added to translocation assays lacking microsomal membranes and incubated at 20 °C for 30 min. Translocation was initiated by the addition of 2 l Downloaded from http://www.jbc.org/ at INDIAN INST OF TECHNOLOGY BOMBAY on August 31, 2019 Roles of Chaperones in Post-translational Translocation 7036 of microsomal membranes. In the restoration of translocation compe tence experiments, AP-[35S]ppF6H was added to translocation assays lacking microsomal membranes and chaperones and then incubated at 20 °C for 30 min. Translocation was initiated by the addition of 2 lof microsomal membranes and the appropriate amount of chaperones. Proteins in samples were separated on SDS-PAGE gels as described previously (31). The electrophoresis protein standards were phospho rylase b (97.4 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21.5 kDa), and lysozyme (14 kDa). The amount of ppF6H translocated was quantified using a PhosphorImager (Amersham Biosciences). Percent translocation was calculated as previously described (14). Protease Protection—After import, 5 l of8mM CaCl2 and 5 l of either water or 8% (w/v) Triton X-100 were added to a 25-l transloca tion reaction (36). After adding 5 lof800 g/ml trypsin, the reactions were incubated for 30 min on ice. The digestion was stopped with 5 l of 50 mM phenylmethanesulfonyl fluoride in dimethyl sulfoxide. Aggregation of ppF— AP-[35S]ppF6H (0.3 l) was diluted into buffer A (16 l) lacking chaperones or containing Ssa1p (0.68 M), Ydj1p (0.68 M), or both (each 0.68 M). After 30 min on ice, the reactions were centrifuged at 15,700 g for 15 min at 4 °C and the percent of [35S]ppF6H remaining in the supernatant was determined. An equivalent amount of supernatant was also added to post-transla tional translocation assays containing wild-type microsomes (MYY290) andtheamountof[35S]ppF6Htranslocated in 20minwasdetermined. N-Ethylmaleimide (NEM) Modification of Ssa1p—Ssa1p was stripped of bound nucleotides and then treated with NEM or water as described previously (31). Residual NEM was inactivated with dithiothreitol and then Ssa1p was dialyzed into buffer A. The concentration of NEM modified Ssa1p (NEM-Ssa1p) and water-treated Ssa1p in AP-[35S] ppF6H aggregation and post- translational translocation assays was 0.68 M. The ATPase activity of NEM-Ssa1p was only 16% that of the water-treated Ssa1p (data not shown). RESULTS Purification of PpF6H—To more clearly understand the roles of Hsp70 and Hsp40 molecular chaperones in post-trans lational translocation we modified an in vitro assay (14) by using AP-ppF6H as the substrate. PpF used previously con tained endogenous chaperones of the wheat germ translation system. We constructed a version of ppF (ppF6H) that has 6 histidine residues at its C terminus. The C terminus was al tered to avoid the possibility of compromising the signal se quence at the N terminus. PpF6H was induced (Fig. 1, com pare lanes 1 and 2) and purified (lane 3) from an E. coli strain harboring a temperature-sensitive export mutation. At the per missive temperature, histidine-tagged pro--factor (pF6H) was formed (data not shown) indicating that the signal se quence cleavage site of ppF6H was recognized by leader pep tidase as previously reported for authentic ppF (37). Sequenc ing the N terminus of pF6H revealed that it was identical to that of authentic pF (data not shown) (38). A mixture of purified ppF6H and pF6H was also electrophoresed (Fig. 1, lane 4). PpF6H, like authentic ppF (38), migrated faster than pF6H on SDS- PAGE gels. Characterization of the Translocation Competence of PpF6H—We next compared the translocation competence of ppF and ppF6H (Fig. 2). Post-translational translocation assays contained cycloheximide, an energy generating system, and yeast microsomes (30). Precursors were radiolabeled with [35S]methionine either metabolically in E. coli or during trans lation in wheat germ extracts. Changes in molecular mass because of signal sequence cleavage of precursors and glycosy lation of translocated products were monitored using SDS PAGE gels. In the presence of PRS, which contain Hsc70 mo lecular chaperones and other factors necessary for efficient post-translational translocation, the signal sequence of ppF was removed and pF was core glycosylated up to three times (Fig. 2, lane 2) as previously reported (30). In the absence of microsomes, translocation was not detected (Fig. 2, lane 1). Similarly, when ppF6Hwassynthesized in wheat germtrans lation reactions and then added to post-translational translo FIG.1.Purification of ppF6H. Proteins from isopropyl-1-thio-- D- galactopyranoside-induced (lane 2) and uninduced (lane 1) E. coli IQ85 (SecYts) harboring a ppF6H expression plasmid were separated on a SDS-PAGE gel and then stained with Coomassie Blue. PpF6H was affinity purified using Ni2-NTA affinity chromatography under denaturing conditions (lane 3). Lane 4 contains an equal amount of purified pF6H (upper band) and ppF6H (lower band). Numbers on the left represent the molecular mass (kDa) of protein standards. FIG.2.Post-translational translocation of ppF6H into yeast microsomes in the presence of PRS. [35S]PpF and [35S]ppF6H were synthesized in wheat germ extracts (WG) and then added to post-translational translocation reactions that lacked or contained wild-type microsomes as indicated. Protease protection experiments using trypsin were done in the presence or absence of Triton X-100 as indicated. Each reaction was supplemented with PRS. The numbers at the right indicate the Mr of ppF6H (22 kDa) and pF6H (23 kDa) that contained one (26 kDa), two (28 kDa), or three (34 kDa) core oligosaccharides. cation assays four radiolabeled species migrating more slowly than ppF6H were detected (Fig. 2, lane 6). By analogy to reactions containing authentic ppF, the new bands corre spond to pF6H (23 kDa) containing either one (26 kDa), two (28 kDa), or three (34 kDa) core oligosaccharides. Each histi dine-tagged species migrated more slowly than its authentic counterpart reflecting the contribution of the six histidine res idues to the molecular mass. Translocation of ppF6H and glycosylation of pF6H were also dependent on microsomes (Fig. 2, compare lanes 5 and 6). In contrast to the precursors, the translocated and glycosylated products were protected from exogenously added trypsin (Fig. 2, lanes 3 and 7). Treating the membranes with a nonionic detergent rendered the products Downloaded from http://www.jbc.org/ at INDIAN INST OF TECHNOLOGY BOMBAY on August 31, 2019 Roles of Chaperones in Post-translational Translocation 7037 FIG.3. Unfolded ppF6H is effi ciently translocated in the absence of chaperones. Wheat germ-synthesized (N)ppF(bars1and2)andppF6H(bars 4 and 5) were translocated into wild-type microsomes in the presence (bars 2 and 5) or absence (bars 1 and 4) of PRS. Wheat germ-synthesized, urea-denatured (D) ppF(bar 3) and ppF6H (bar 6) were translocated into microsomes in the ab sence of PRS. Urea-denatured, affinity purified ppF6H (AP) was also translo cated in the absence of PRS (bar 7). Each bar represents the mean S.D. of the percentage translocation during 20 min of three experiments. susceptible to proteolysis (Fig. 2, lanes 4 and 8). These results indicate that the translocation and glycosylation of ppF and ppF6H were essentially identical. The translocation compe tence of ppF6H described here is comparable with that of another histidine- tagged ppF reported previously (39). The rate of translocation of unfolded ppF was previously shown to be greater than that of native ppF (14). To test whether the conformation of ppF6H affects its translocation competence, we denatured ppF6H with urea and then diluted it into translocation reactions (Fig. 3). Denatured ppF and ppF6Hweretranslocated, respectively, 3.3- and 3.1-fold more efficiently than their corresponding nondenatured controls (Fig. 3, compare bars 1 and 3 and 4 and 6). Denatured, affinity purified ppF6H (AP-ppF6H) was translocated 4.8-fold more efficiently than its native control (compare bars 4 and 7). Like ppF and ppF6H, AP-ppF6H was translocated into micro somes, its signal sequence was removed, and the resulting product was glycosylated (data not shown). The efficiency of translocation of the denatured ppFs (bars 3, 6, and 7) ap proached that of the corresponding nondenatured forms in the presence of PRS (bars 2 and 5). Together, these results indicate that translocation efficiencies of the denatured forms of ppF and ppF6H are essentially identical. They also support the notion that urea denaturation of ppF (or ppF6H) mimics the stimulation of post-translational translocation by the chaper ones in the PRS. Upon dilution from urea, ppF (or ppF6H) likely folds rap idly and loses translocation competence. To test this hypothesis we measured the amount of ppF6H translocated at various times after dilution. Translocation assays were restricted to 2 min to minimize the contribution of background translocation during longer incubations. The translocation competence of AP-ppF6Hwasessentially lost by 15 min and its half-life was 2.2 min (Fig. 4). Kinetics of Translocation in the Presence of Ssa1p and Ydj1p—The availability of AP-ppF6H allowed us to explore the role of yeast cytosolic Hsp70 and Hsp40 molecular chaper ones in post-translational translocation without interference from chaperones in wheat germ translation systems. We deter mined the time course of translocation of AP-ppF6H (Fig. 5). For reference, the time course of translocation of wheat germ FIG.4.Loss of translocation competence. AP-[35S]ppF6H was diluted into post-translational translocation reactions lacking micro somes. At the indicated times, wild-type microsomes were added and the amount of ppF6H translocated during a 2-min incubation was determined. Each point represents the mean of the percentage trans location of two experiments. synthesized, nondenatured ppF6H is also shown (Fig. 5, line Nt). The initial rate of translocation ( 20% translocation) of AP-ppF6H(line None) was indistinguishable from that in the presence of Ssa1p (line S), Ydj1p (line Y), the control protein hexokinase (line H), both Ssa1p and Ydj1p (line S Y), or both Ssa1p and hexokinase (Fig. 5, line S H). However, the final relative yields of translocation were S, S Y, S H None,Y, H Nt. Hexokinase was used as a control because it is a cytosolic protein whose crystal structure resembles that of the ATP-binding domain of Hsc70 (40). Together, these results suggest that immediately after dilution, translocation of some Downloaded from http://www.jbc.org/ at INDIAN INST OF TECHNOLOGY BOMBAY on August 31, 2019 Roles of Chaperones in Post-translational Translocation 7038 FIG.5.Time course of translocation of ppF6H in the presence of yeast cytosolic chaperones. AP-[35S]ppF6H was diluted into post-translational translocation reactions containing wild-type microsomes in the absence of chaperones (line None) or in the presence of Ssa1p (line S), Ydj1p (line Y), hexokinase (line H), both Ssa1p and Ydj1p (line S Y), or both Ssa1p and hexokinase (line S H). At the indicated times the amount of [35S]ppF6H translocated was determined. The concentration of each protein was 0.68 M. For comparison, the time course of wheat germ- synthesized, nondenatured [35S]ppF6H translocation in the absence of chaperones is shown (line Nt). Each point represents the mean S.D. of the percentage translocation of three experiments. Inset, the time course of translocation of AP-[35S]ppF6H in the presence of Ssa1p was replotted as log (translocated fraction (TF)) versus time. AP-ppF6H was rapid and independent of exogenously added chaperones. At later times, however, Ssa1p played an increas ingly important role in maintaining or restoring precursor translocation competence. Ydj1p did not markedly stimulate translocation of AP- ppF6H in the presence or absence of Ssa1p. Nevertheless, Ydj1p stimulated the ATPase activity of Ssa1p 10-fold and was required for efficient luciferase refolding in the presence of Ssa1p (data not shown) (26, 33). The results also suggest that during a 60-min translocation assay AP ppF6Hexists in at least two states: one that is rapidly trans located and the other that is translocated more slowly. A replot of the Ssa1p time course data (log translocated fraction versus time) is biphasic supporting the idea that AP-ppF6H exists in at least two states (Fig. 5, inset). The rapidly translocated form may be unfolded, whereas the more slowly translocated form may be either bound to chaperones, folded, or aggregated. Chaperones Differentially Affect Translocation—During the course of post-translational translocation, chaperones may in teract at different stages of precursor folding. We postulate that AP- ppF6H has at least three fates upon dilution from denaturant into translocation assays. It may 1) enter micro somes in an unfolded conformation directly, 2) interact with chaperones that maintain its translocation competence, or 3) fold/aggregate. To elucidate the contributions of Ssa1p and Ydj1p we performed translocation assays under three condi tions. In the first case, we mimicked in vivo translocation by diluting AP-ppF6H into reactions containing microsomes and different combinations of chaperones. Second, to investigate the ability of the chaperones to maintain translocation compe tence, we preincubated AP-ppF6H with different combina tions of chaperones before adding microsomes. Third, to test restoration of translocation competence activity we allowed AP-ppF6H to fold/aggregate before adding chaperones and microsomes. We refer to these assay conditions as complete (C), maintenance (M), and restoration (R), respectively. Upon dilution into a complete translocation reaction, about Downloaded from http://www.jbc.org/ at INDIAN INST OF TECHNOLOGY BOMBAY on August 31, 2019 Roles of Chaperones in Post-translational Translocation 7039 FIG.6. Ssa1p and Yjd1p can maintain, but not restore, translocation competence of ppF6H. In “complete” reactions (line C), AP-[35S]ppF6H was diluted into post-translational translocation reactions containing wild-type (MYY290, panels a, c, and e) or Ydj1p-deficient microsomes (MYY406, panels b, d, and f) and different concentrations of Ssa1p (panel a and b), Ydj1p (panel c and d), or both (panel e and f). “Maintenance” reactions (line M) were identical to “complete” reactions, except that microsomes were added 30 min after AP-[35S]ppF6H was diluted into reactions. “Restoration” reactions (line R) were identical to complete reactions, except that microsomes and chaperones were added 30 min after AP-[35S]ppF6H was diluted into reactions. The percent of [35S]ppF6H translocated during a 20-min incubation is shown. Each point represents the mean of the percentage translocation of three experiments. 48% of AP-ppF6H was rapidly translocated into wild-type microsomes in the absence of chaperones (Fig. 6a, c, and e, line C). In the presence of 0.68 M Ssa1p, translocation peaked at 62% (Fig. 6a, line C). Higher concentrations of Ssa1p inhibited translocation. When the addition of microsomes was delayed by 30 min, the amount of AP-ppF6Htranslocated was only 8% in the absence of chaperones (Fig. 6a, c, and e, line M). However, including Ssa1p in the preincubation increased translocation 2-fold suggesting that it can either maintain or restore the translocation competence of AP-ppF6H (Fig. 6a, line M). To distinguish between these two possibilities Ssa1p and micro somes were added after AP-ppF6H folded/aggregated. Less than 10% of folded/aggregated AP-ppF was translocated (Fig. 6a, line R). Together, these results indicate that Ssa1p can maintain the translocation competence of AP-ppF. However, its ability to restore translocation competence to folded/aggre gated AP-ppF is negligible. To determine whether membrane-bound Ydj1p cooperates with Ssa1p to stimulate the translocation of ppF6H, we re peated these experiments using microsomal membranes iso lated from a strain lacking Ydj1p (Fig. 6b). Wild-type microso mal membranes contain farnesylated Ydj1p (41) that might obscure the effects of exogenous Ydj1p. We found that the effects of Ssa1p on AP-ppF6Htranslocationinto wild- type and Ydj1p-deficient microsomes were essentially identical (com pare Fig. 6, a and b, lines C, M, and R). These results suggest that membrane-bound Ydj1p does not play a significant role in the translocation of AP-ppF6H into microsomes in the ab sence or presence of Ssa1p. In contrast to Ssa1p, Ydj1p slightly inhibited AP-ppF6H translocation when present during dilution (Fig. 6c, line C). At the highest Ydj1p concentration tested, translocation efficiency decreased 10%. Importantly, Ydj1p stimulated translocation when the addition of microsomes was delayed (Fig. 6c, line M). This suggests that Ydj1p can act as a molecular chaperone in post-translational translocation by maintaining translocation competence. Ydj1p did not stimulate translocation of refolded AP-ppF6H (Fig. 6c, line R). To elucidate the role of mem Downloaded from http://www.jbc.org/ at INDIAN INST OF TECHNOLOGY BOMBAY on August 31, 2019 Roles of Chaperones in Post-translational Translocation 7040 brane- bound Ydj1p, we repeated these experiments using Ydj1p-deficient microsomes (Fig. 6d). The patterns were essen tially identical to those obtained using wild-type microsomes supporting the notion that membrane-bound Ydj1p is not re quired for translocation (compare Fig. 6, c and d). To determine whether the chaperones cooperate during pro tein translocation, we monitored translocation at different con centrations of both Ssa1p and Ydj1p maintained at a 1:1 molar ratio. Reciprocal dose- response studies indicated that activity was maximal at this ratio (data not shown). The concentration dependence of Ssa1p and Ydj1p together on translocation was similar to that of Ssa1p alone (compare line C in Fig. 6, a and e). This result suggests that either exogenous Ydj1p does not cooperate with Ssa1p or that Ydj1p is not limiting in the reac tion because of its presence on wild-type membranes. Ssa1p and Ydj1p together maintained translocation competence about 2-fold better than either did alone (compare line M in Fig. 6, a, c, and e). Such an additive response suggests that they independently maintain translocation competence under these reaction conditions. The amount of refolded ppF6H translo cated in the presence of both Ssa1p and Ydj1p was negligible (Fig. 6e, line R). The dependence of AP-ppF6H translocation into Ydj1p-deficient and wild-type membranes on the concen tration of the Ssa1p and Ydj1p pair was similar, indicating that membrane- boundYdj1pdoesnotplayanessentialroleinSsa1p dependent translocation (compare Fig. 6, e and f). Chaperones Prevent the Aggregation of PpF6H—Weshowed above that Ssa1p and Ydj1p can maintain the translocation competence of ppF6H. To test the hypothesis that they main tain translocation competence by preventing aggregation we first diluted AP-ppF6H into buffer lacking chaperones or con taining Ssa1p, Ydj1p, or both. After 30 min, aggregated AP ppF6H was removed by centrifugation and the translocation competence of AP-ppF6H remaining in the supernatant was determined. In the absence of chaperones, more than 90% of AP-ppF6H was removed by centrifugation indicating that it had aggregated (Fig. 7A, bars Initial and N). Ssa1p and Ydj1p increased the solubility of ppF6H by 14- and 4.4-fold, respec tively (Fig. 7A, compare bars N, S, and Y). In the presence of both chaperones, the solubility of AP-ppF6H was comparable with that in the presence of Ssa1p alone (Fig. 7A, bars S and S Y).Together these results indicate that although both chap erones can prevent the aggregation of AP- ppF6H, Ssa1p is more effective. Furthermore, Ydj1p does not increase the abil ity of Ssa1p to prevent aggregation under these reaction con ditions. To determine whether preventing aggregation im proves post-translational translocation we measured the translocation competence of AP-ppF6H in the supernatants (Fig. 7B). The translocation efficiency of AP-ppF6H main tained in solution by Ssa1p and Ydj1p was, respectively, 4- and 2-fold greater than that in reactions lacking chaperones (Fig. 7B, compare bars N, S, and Y). Translocation in the presence of both chaperones was comparable with that of Ssa1p alone. Together these results suggest that Ssa1p and Ydj1p can stim ulate translocation independently by preventing AP-ppF6H aggregation. Weandothers previously showed that the post- translational translocation activity of Ssa1p is dependent on its ATPase activity (27, 31). We demonstrated that NEM inhibits the abil ity of Ssa1p to bind and hydrolyze ATP by modifying 3 cysteine residues in its ATP-binding domain (31). To determine whether the ATPase activity of Ssa1p is also required for maintaining the translocation competence of AP-ppF6H, we measured the ability of NEM- Ssa1p to prevent the aggregation of AP ppF6H and to stimulate the translocation of the AP-ppF6H remaining in solution. We found that 2 0.2-fold more AP Downloaded from http://www.jbc.org/ FIG.7.Ssa1p and Yjd1p stimulate translocation by preventing aggregation. A, AP-[35S]ppF6H was diluted into buffer containing no chaperones (N) or Ssa1p(S), Ydj1p (Y), or both (S Y)asindicated. After 30 min, the reactions were centrifuged and the percent of [35S]ppF6H remaining in the supernatant was determined. A control reaction was not centrifuged (Initial). B, [35S]ppF6H in the supernatants was also added to post-translational translocation assays containing wild-type microsomes and the amount translocated in 20 min was determined. Translocation efficiencies are expressed relative to reactions lacking chaperones (N). Each bar represents the mean S.D. of three experiments. ppF6Hremained soluble in the presence of NEM-Ssa1p than in the presence of water-treated Ssa1p (data not shown). How ever, NEM-Ssa1p was only 12 0.8% as effective as water treated Ssa1p in translocating the soluble AP-ppF6H into microsomes post-translationally (data not shown). Hexokinase neither prevented aggregation nor maintained the transloca tion competence of AP-ppF6H (data not shown). Together these results suggest that the ATP binding and/or ATPase activity of Ssa1p are not required for preventing the aggrega tion of AP-ppF6H, but are required for ensuring the produc tive release and translocation of AP-ppF6H. DISCUSSION We explored the roles of cytosolic chaperones in post-trans lational translocation in vitro using AP-ppF6H. In the ab sence of chaperones, urea- denatured AP-ppF6H was rapidly translocated into wild-type or Ydj1p-deficient microsomes upon dilution into reactions. In the absence of microsomes and chap erones, AP-ppF6H aggregated and became translocation in competent. Ssa1p, Ydj1p, or both chaperones prevented aggre gation and maintained the translocation competence of some at INDIAN INST OF TECHNOLOGY BOMBAY on August 31, 2019 Roles of Chaperones in Post-translational Translocation 7041 AP-ppF6H. These results provide the first biochemical evi dence supporting the notion that Ssa1p and Ydj1p stimulate post- translational translocation into the endoplasmic reticu lum by preventing the aggregation of presecretory proteins. Ydj1p, however, is not essential for translocation and its far nesylated, membrane-bound version provides no substantial benefit. Although the chaperones alone or together maintained translocation competence of AP-ppF6H, their ability to re store the translocation competence of aggregated precursor was negligible. Genetic (12, 13, 27) and biochemical (14, 42, 43) studies have shown that cytosolic Hsp70s stimulate the post-translational translocation of precursor proteins into endoplasmic reticulum and mitochondria. Insight into the mechanism by which Ssa1p stimulates translocation is provided by the experiments re ported here demonstrating that Ssa1p acts by preventing ppF aggregation. Further insight was obtained using NEM-Ssa1p, which has defective nucleotide-binding and ATPase activities (31). NEM-Ssa1p prevented the aggregation of AP-ppF6H, but failed to maintain its translocation competence. This is consistent with the idea that NEM-Ssa1p can bind AP ppF6H, but its subsequent release, if any, is inadequate to support translocation. Similarly, NEM-Ssa1p can prevent the aggregation of denatured luciferase, but cannot refold it (33). Ssa1p likely prevents aggregation by directly interacting with AP-ppF6H. Wepreviously reported that cytosolic Hsc70 asso ciates with ppF in wheat germ extracts (16). Recently, others have shownthatHsc70canbephotocross-linked to ppF trans lated in reticulocyte lysates (15). The translocation competence of mitochondrial precursor proteins is also maintained by cy tosolic Hsp70 chaperones likely through direct association (44). Ydj1p waspreviously showntostimulatetheATPaseactivity of Ssa1p (25, 26), to facilitate the release of bound polypeptides from Ssa1p (25, 27), to prevent the aggregation of rhodanese (10), and to cooperate with Ssa1p in the folding of denatured luciferase (26, 45). Although we found that Ydj1p hindered AP-ppF6Haggregation and maintained its translocation com petence, it, unlike Ssa1p, did not stimulate translocation of AP- ppF6H upon dilution into complete reactions containing microsomes. This result suggests that Ydj1p may prevent AP ppF6H aggregation less effectively than Ssa1p. Results re ported here demonstrating that AP-ppF6H can be post-trans lationally translocated into microsomes isolated from ydj1 null strains indicate that membrane-bound Ydj1p is not essential for this process. These results are consistent with those ob tained from in vivo experiments showing that ppF is effi ciently exported from ydj1 null strains (13, 22). In such strains, however, ppF also has the option of entering the endoplasmic reticulum co- translationally via the SRP-dependent pathway. Although our results do not support the notion that membrane bound Ydj1p acts as a release factor for ppFSsa1p complexes, this hypothesis requires further study. Using chaperones isolated from E. coli, Hendrick et al. (23, 32) reported that E. coli DnaJ completely inhibited the in vitro post-translational translocation of reticulocyte lysate-trans lated ppF into yeast microsomes. DnaK and GrpE reversed the inhibition. In contrast, we found that Ydj1p only partially inhibited translocation of AP-ppF6H even when present at high concentrations. Ydj1p may inhibit post-translational translocation by competing with Ssa1p for binding sites on ppF6H or by stabilizing ADP forms of the Ssa1pppF6H complexes. Perhaps the lack of agreement between the two studies arises from the difference in the source of reaction components. All components used in our system were derived from yeast, whereas those used by Hendrick et al. (23) were derived from three different organisms. Furthermore, we used an affinity purified version of ppF, whereas the ppF used by Hendrick et al. (23) was not purified after synthesis in a crude reticulocyte lysate. An important issue is whether Ssa1p and Ydj1p work to gether or in parallel in post-translational translocation of pre secretory proteins into the endoplasmic reticulum. Our results support the idea that they work in parallel. However, the low cellular concentration of Ydj1p and the higher maintenance of translocation activity of Ssa1p suggest that Ssa1p would mo nopolize this pathway if they recognized overlapping sites on ppF. Three reports support the idea that Ydj1p and Ssa1p cooperate in post-translational translocation of presecretory proteins into the endoplasmic reticulum (13, 21). First, a tem perature- sensitive mutant of Ydj1p accumulated ppFat the nonpermissive temperature and the mutant protein, ydj1 151p, only weakly stimulated the ATPase activity of Ssa1p (21). Second, Becker et al. (13) found a synthetically lethal relationship between SSA1 and YDJ1. Furthermore, a strain expressing Ssa1p, but lacking Ssa2p, Ssa3p, Ssa4p, and Ydj1p accumulated ppF and its glycosylated intermediates at 23 °C (13). Third, mutants of Ssa1p containing amino acid substitu tions in the ATP-binding domain failed to bind unfolded polypeptides, did not stimulate post-translational transloca tion of ppF into microsomes, and their ATPase activity could not be stimulated by Ydj1p (27). It remains possible, however, that cells expressing high levels of unfolded proteins at non permissive conditions (13, 21) or lacking the full complement of cytosolic Hsp70 chaperones at permissive temperatures (13), contain insufficient amounts of Ssa1p to support post-transla tional translocation. In our in vitro experiments, Ydj1p did not enhance the ability of Ssa1p to prevent ppF aggregation, to maintain translocation competence of ppF, or to stimulate post-translational translocation. The normally beneficial coop eration between Ssa1p and Ydj1p during protein folding (26) may be unnecessary and counterproductive in the post- trans lational translocation of unfolded ppF