Roles of Cytosolic Hsp70 and Hsp40 Molecular Chaperones in Post-translational Translocation of Presecretory Proteins PDF

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 9, Issue of February 28, pp. 7034 –7042, 2003 © 2003 by The American Society for Biochemistry and Molecular Biology, Inc....

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 9, Issue of February 28, pp. 7034 –7042, 2003 © 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Roles of Cytosolic Hsp70 and Hsp40 Molecular Chaperones in Post-translational Translocation of Presecretory Proteins into the Endoplasmic Reticulum* Received for publication, October 15, 2002 Published, JBC Papers in Press, December 19, 2002, DOI 10.1074/jbc.M210544200 Jantra Ngosuwan, Nancy M. Wang, Katie L. Fung, and William J. Chirico‡ From the Department of Anatomy and Cell Biology, State University of New York Downstate Medical Center, Brooklyn, New York 11203 Hsp70 molecular chaperones and their co-chaperones bound form, peptide substrates undergo cycles of rapid binding Downloaded from http://www.jbc.org/ at INDIAN INST OF TECHNOLOGY BOMBAY on August 31, 2019 work together in various cellular compartments to and release from Hsp70 (3). In the ADP-bound form, the inter- guide the folding of proteins and to aid the translocation actions are slower resulting in higher affinity. Hsp70s stimu- of proteins across membranes. Hsp70s stimulate protein late protein folding by binding to exposed hydrophobic se- folding by binding exposed hydrophobic sequences quences (4 – 6) and preventing their irreversible aggregation thereby preventing irreversible aggregation. Hsp40s (7). The activity of Hsp70s is regulated by Hsp40s and other stimulate the ATPase activity of Hsp70s and target un- co-chaperones. Hsp40s stimulate the ATPase activity of folded proteins to Hsp70s. Genetic and biochemical evi- Hsp70s (8) and can target unfolded proteins to them (7). dence supports a role for cytosolic Hsp70s and Hsp40s in Hsp40s preferentially bind hydrophobic polypeptides (9) and the post-translational translocation of precursor pro- can prevent the aggregation of some unfolded proteins (7, 10). teins into endoplasmic reticulum and mitochondria. To Genetic and biochemical evidence supports a role for cytoso- gain mechanistic insight, we measured the effects of Saccharomyces cerevisiae Ssa1p (Hsp70) and Ydj1p lic Hsp70s and Hsp40s in the post-translational translocation (Hsp40) on the translocation of histidine-tagged prepro- of precursor proteins into the endoplasmic reticulum and mi- ␣-factor (pp␣F6H) into microsomes. Radiolabeled tochondria (reviewed in Ref. 11). Deshaies et al. (12) reported pp␣F6H was affinity purified from wheat germ transla- that presecretory proteins and mitochondrial precursors accu- tion reactions (or Escherichia coli) to remove endoge- mulated in the yeast cytosol when the concentration of cytosolic nous chaperones. We demonstrated that either Ssa1p or Hsp70s was lowered. A temperature-sensitive mutant of the Ydj1p stimulates post-translational translocation by yeast cytosolic Hsp70 Ssa1p rapidly accumulated presecretory preventing pp␣F6H aggregation. The binding and/or hy- proteins at the nonpermissive temperature (13). Chirico et al. drolysis of ATP by Ssa1p were required to maintain the (14) showed that yeast cytosolic Hsp70s stimulated the in vitro translocation competence of pp␣F6H. To clarify the con- translocation of prepro-␣-factor (pp␣F) into yeast microsomes. tributions of membrane-bound and cytosolic Ydj1p, we Complexes containing Hsp70s and presecretory and mitochon- compared the efficiency of chaperone-dependent trans- drial precursor proteins have been identified in wheat germ location into wild-type and Ydj1p-deficient microsomes. extracts and reticulocyte lysates (15–17). The ability to stimu- Neither soluble nor membrane-bound Ydj1p was essen- late post-translational translocation is not shared by all tial for post-translational protein translocation. The Hsp70s or by other stress protein families. Neither Hsp60 (18), ability of Ssa1p, Ydj1p, or both chaperones to restore the Kar2p (19), nor Hsp90 (18) stimulates translocation. However, translocation competence of aggregated pp␣F6H was negligible. DnaK can partially substitute for yeast cytosolic Hsp70s in vitro (19, 20). Several laboratories have explored the role of DnaJ homologs Hsp701 molecular chaperones and their co-chaperones work in protein translocation. Caplan et al. (21) reported that a together in a variety of cellular compartments to guide the temperature-sensitive mutant of YDJ1 was defective for trans- folding of proteins and to aid the translocation of proteins location at the nonpermissive temperature. However, Atencio across membranes (reviewed in Ref. 1). The binding and hy- and Yaffe (22) and Becker et al. (13) showed that pp␣F is drolysis of ATP regulate the action of Hsp70s (2). In the ATP- translocated normally in deletion mutants of YDJ1. Transloca- tion of pp␣F was defective in a strain expressing a mutant form of Ydj1p that cannot be farnesylated indicating that the lipid- * This work was supported by National Science Foundation Grant modified version of Ydj1p plays a role in protein translocation MCB-9905988 and a grant from the American Heart Association (to (21). Hendrick et al. (23) showed that Escherichia coli DnaJ 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 completely inhibited post-translational translocation of pp␣F marked “advertisement” in accordance with 18 U.S.C. Section 1734 in vitro. DnaK and GrpE together relieved the DnaJ-dependent solely to indicate this fact. inhibition, but neither alone had any effect. Export of certain ‡ To whom correspondence should be addressed. Tel.: 718-270-1308; precursor proteins was defective in some dnaK and dnaJ mu- Fax: 718-270-3732; E-mail: [email protected]. 1 The abbreviations used are: Hsp70, 70-kDa heat shock protein; tant strains of E. coli (24). AP-pp␣F6H, affinity purified, histidine-tagged prepro-␣-factor; H, hex- Functional interactions between yeast Hsp70s and DnaJ okinase; Hsc70, 70-kDa heat shock cognate protein; NEM, N-ethylma- homologs have been studied in vitro and in vivo (13, 25–27). leimide; NEM-Ssa1p, N-ethylmaleimide-modified Ssa1p; Nt, nondena- Ydj1p stimulates the ATPase activity of Ssa1p (25) and to- tured pp␣F6H; PRS, postribosomal supernatant; p␣F, pro-␣-factor; p␣F6H, histidine-tagged pro-␣-factor; pp␣F, prepro-␣-factor; pp␣F6H, gether they constitute a protein folding machinery capable of histidine-tagged prepro-␣-factor; S, Ssa1p; Y, Ydj1p; Ni2⫹-NTA, nickel- refolding denatured luciferase (26). Becker et al. (13) explored nitrilotriacetic acid. the in vivo interactions of SSA1 and YDJ1 in protein translo- 7034 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 previously (19). The concentration of Ssa1p was determined as de- temperature-sensitive SSA1 mutant was synthetically lethal scribed previously (31). Ydj1p was overexpressed and purified from E. coli as described previously (25). The concentration of Ydj1p was deter- with a YDJ1 deletion mutant suggesting that SSA1 and YDJ1 mined using its extinction coefficient at 280 nm (20370 M⫺1 cm⫺1). This genetically interact (13). Pulse-chase experiments showed that preparation of Ydj1p stimulated the ATPase activity of Ssa1p 10-fold pp␣F, which had accumulated at the nonpermissive tempera- and was required for efficient luciferase refolding in the presence of ture, could not be translocated. These results suggested that Ssa1p (data not shown) (26). ATPase and luciferase refolding assays the chaperones maintain translocation competence by binding were performed as described previously (26, 33). Postribosomal super- to pp␣F co-translationally, but they cannot rescue aggregated natants (PRS) were prepared from S. cerevisiae SKQ2N as described previously (14). Hexokinase from S. cerevisiae was obtained from pp␣F. Strains containing mutant versions of Ssa1p with defec- Sigma. [35S]Pp␣F6H was purified from either wheat germ translation tive ATP-binding pockets fail to interact productively with reactions programmed with pp␣F6H mRNA or metabolically labeled Ydj1p and accumulate pp␣F (27). IQ85[pREP4, pKWC1] as follows. Proteins in translation reactions (230 We undertook the following study to gain insight into the ␮l) were precipitated with 460 ␮l of ethanol and the resulting mixture mechanism by which Ssa1p and Ydj1p contribute to the post- was incubated on ice for 5 min. The mixture was centrifuged at translational translocation of presecretory proteins into the 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 endoplasmic reticulum. To avoid the effects of endogenous 10 mM Tris-HCl, pH 8) and rotated for 1 h at room temperature. chaperones and other proteins contaminating earlier prepara- Insoluble proteins were removed by centrifugation at 15,700 ⫻ g for 5 Downloaded from http://www.jbc.org/ at INDIAN INST OF TECHNOLOGY BOMBAY on August 31, 2019 tions of pp␣F, which was synthesized in wheat germ extracts or min. Ni2⫹-NTA resin (60 ␮l of 50% slurry, Qiagen) was added to the reticulocyte lysates, we used a histidine-tagged, affinity puri- supernatant and the mixture was rotated for 1 at room temperature. fied version of pp␣F (AP-pp␣F6H) in translocation assays. We Unbound proteins were removed by washing the resin 5 times with 250 clarified the contributions of membrane-bound and cytosolic ␮l of buffer C (8 M urea, 100 mM sodium phosphate, and 10 mM Tris- HCl, pH 8.0). [35S]Pp␣F6H was eluted with 40 ␮l of buffer C adjusted to Ydj1p, by comparing the translocation efficiencies of the chap- pH 4.6. After the pH of eluate was neutralized with 7.5 ␮l of 2 M erones using wild-type and Ydj1p-deficient microsomal mem- HEPES, pH 8, it was frozen in liquid nitrogen and stored at ⫺70 °C. branes. We demonstrate that Ssa1p stimulates translocation [35S]Pp␣F6H was purified from metabolically labeled IQ85[pREP4, by preventing the aggregation of AP-pp␣F6H. Although Ydj1p pKWC1] (see above) by resuspending the cells from a 10-ml culture in stimulates the ATPase activity of Ssa1p (25) and is essential for 500 ␮l of buffer B. After mixing the cells for 1 h at room temperature, Ssa1p-dependent protein folding of luciferase in vitro (26), we insoluble proteins were removed by centrifugation at 15,700 ⫻ g for 5 min. The supernatant was mixed with 50 ␮l of Ni2⫹-NTA resin (50% show that it is not essential for post-translational protein slurry) for 1 h at room temperature. Unbound proteins were removed translocation. However, Ydj1p alone stimulates post-transla- from the resin and then [35S]pp␣F6H was eluted as described above. tional translocation by preventing AP-pp␣F6H aggregation. P␣F6H was produced in either IQ85[pREP4, pKWC1] or DH5␣[pREP4, pKWC1] grown and induced at 37 °C. It was purified in the same EXPERIMENTAL PROCEDURES manner as pp␣F6H. Protein concentrations were determined using the Plasmids—pKWC1, which can express pp␣F6H in E. coli, was con- Bradford assay (34) and bovine ␥-globulin was used as the standard. structed by amplifying the cDNA for pp␣F contained in pDJ100 (28), Protein Sequencing—A 1.75-␮g sample of purified p␣F6H was deri- cutting the full-length product with BglII and SphI, and then ligating it vatized with phenylisothiocyanate and then automated Edman degra- into the corresponding sites in pQE70 (Qiagen, Inc.). The forward and dation was performed using an Applied Biosystems 475A Protein Se- reverse primers in the polymerase chain reaction were 5⬘-GTGTGCAT- quencer (Applied Biosystems, Inc.) in the Protein Sequencing Center at GCGATTTCCTTCAATTTTTACTG-3⬘ and 5⬘-ATATAGATCTGTACAT- SUNY Downstate Medical Center. TGGTTGGCCGGGT-3⬘, respectively. pREP4 was obtained from Qia- Yeast Microsomal Membranes—Microsomes were isolated from S. gen. pNWC1, which was used to generate mRNA coding for pp␣F6H, cerevisiae SKQ2N (MATa/␣ ade1/⫹ ⫹/ade2 ⫹/his1), MYY290 (MATa was constructed by amplifying the cDNA for pp␣F6H contained in leu2 his3 ura3) (22), and MYY406 (MAT␣ leu2 his3 mas5::URA3) (22) as pKWC1, digesting the resulting product with SphI and SacI, and then previously described (30). The post-translational translocation activi- ligating it into pGEMEX-1 behind the SP6 promoter. The forward and ties of microsomes isolated from wild-type strains SKQ2N and MYY290 reverse primers in the amplification reaction were 5⬘-GTGTGCATGC- were essentially identical. One equivalent (eq) of microsomes was de- GATTTCCTTCAATTTTTACTG-3⬘ and 5⬘-ATATGAGCTCGGATCTAT- fined as 50 A280 units/ml (35). The optical density of microsomes was CAACAGGAGTCC-3⬘, respectively. The sequences of the constructs measured in 1% SDS. The final concentration of the membrane was were confirmed by automated DNA sequencing. adjusted to 4 eq/␮l in buffer A (20 mM HEPES-KOH, pH 7.5, 100 mM Metabolic Labeling of Pp␣F6H—E. coli IQ85 (F⬘ araD139 ⌬(argF- potassium acetate, 2 mM magnesium acetate, and 2 mM dithiothreitol) lac)U169 rpsL150 relA1 flbB5301 deoC1 ptsF25 secYts) was trans- containing 14% glycerol. formed sequentially with pREP4 and pKWC1. The resulting strain Post-translational Translocation—The post-translational transloca- IQ85[pREP4, pKWC1] was grown overnight at 25 °C in 2 ml of LB tion assay was described previously (14). In brief, it contained 2 ␮l of media containing 2% glucose, 25 ␮g/ml kanamycin, 25 ␮g/ml tetracy- yMix (27.25 mM HEPES, pH 7.5, 863 mM potassium acetate, 17.3 mM cline, and 100 ␮g/ml ampicillin. The cells were collected by centrifuga- magnesium acetate, 15.25 mM dithiothreitol, and 12.5 mM cyclohexi- tion (15,700 ⫻ g), resuspended in 10 ml of minimal A medium (29) mide), 1.37 ␮l of an energy source (9.1 mM ATP, 456.2 mM creatine containing 1 mM MgSO4, 0.2% glycerol, 0.0005% thiamine-HCl, 0.075% phosphate, and 0.365 mM GDP-mannose), 0.63 ␮l of 8 mg/ml creatine CSM-MET (QBIOgene, Inc.) and the above antibiotics. The culture was kinase, 2 ␮l of yeast microsomes (4 eq/␮l), 1 ␮l of water, 16 ␮l of buffer incubated at 25 °C until 0.5 A600. The temperature was raised to 42 °C A (PRS or chaperones to be tested), and 2 ␮l (⬃15,000 dpm) of wheat and the culture was incubated for 2.5 h. Isopropyl-1-thio-␤-D-galacto- germ-translated [35S]pp␣F or [35S]pp␣F6H. When denatured pyranoside (2 mM final concentration) and Tran35S-label (ICN Biomedi- [35S]pp␣F6H (or [35S]pp␣F) was used as the substrate, it was prepared cals, Inc.) were added and the culture was incubated for an additional by diluting the translation reaction 4-fold into 8 M urea. The post- 30 min at 42 °C. The culture was placed on ice, the cells were collected translational translocation assay was compensated with 1.6 or 1.7 ␮l of by centrifugation at 4 °C, and [35S]pp␣F6H was purified as described wheat germ translation compensation buffer (43 mM HEPES-KOH, pH below. 7.5, 112 mM potassium acetate, 2.1 mM magnesium acetate, 3 mM In Vitro Transcription and Translation—Messenger RNA coding for dithiothreitol) before adding 0.4 ␮l of denatured [35S]pp␣F6H (or pp␣F or pp␣F6H was synthesized using a RiboMAX kit (Promega). [35S]pp␣F) or 0.3 ␮l of affinity purified [35S]pp␣F6H, respectively. De- Before transcription, pDJ100 and pNWC1 were linearized with XbaI natured substrate (⬃15,000 dpm) was added last and then reactions and SacI, respectively. The 3⬘ overhangs resulting from SacI digestion were mixed immediately. All pipette tips used to transfer denatured of pNWC1 were converted to blunt ends using Klenow. Radiolabeled pp␣F were pretreated with SIGMACOTE (Sigma). The final volume pp␣F and pp␣F6H were synthesized in wheat germ extracts (Promega) was 25 ␮l and the reactions were incubated at 20 °C for the indicated according to the manufacturer’s instructions. Alternatively, wheat times. Reactions were terminated by adding an equal volume of 2⫻ germ extracts were prepared as previously described (30). Translation SDS-PAGE sample buffer and then boiling the mixture for 2 min. In the grade [35S]methionine was obtained from PerkinElmer Biosciences. maintenance of translocation experiments, AP-[35S]pp␣F6H was added Protein Purification—Ssa1p was overexpressed in Saccharomyces to translocation assays lacking microsomal membranes and incubated cerevisiae strain MW141 in YPGal medium and purified as described at 20 °C for 30 min. Translocation was initiated by the addition of 2 ␮l 7036 Roles of Chaperones in Post-translational Translocation of microsomal membranes. In the restoration of translocation compe- tence experiments, AP-[35S]pp␣F6H 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 ␮l of 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 pp␣F6H translocated was quantified using a PhosphorImager (Amersham Biosciences). Percent translocation was calculated as previously described (14). Protease Protection—After import, 5 ␮l of 8 mM 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 ␮l of 800 ␮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 pp␣F—AP-[35S]pp␣F6H (0.3 ␮l) was diluted into buffer A (16 ␮l) lacking chaperones or containing Ssa1p (0.68 ␮M), Downloaded from http://www.jbc.org/ at INDIAN INST OF TECHNOLOGY BOMBAY on August 31, 2019 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]pp␣F6H 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) and the amount of [35S]pp␣F6H translocated in 20 min was determined. 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] FIG. 1. Purification of pp␣F6H. Proteins from isopropyl-1-thio-␤- D-galactopyranoside-induced (lane 2) and uninduced (lane 1) E. coli pp␣F6H aggregation and post-translational translocation assays was IQ85 (SecYts) harboring a pp␣F6H expression plasmid were separated 0.68 ␮M. The ATPase activity of NEM-Ssa1p was only 16% that of the on a SDS-PAGE gel and then stained with Coomassie Blue. Pp␣F6H water-treated Ssa1p (data not shown). was affinity purified using Ni2⫹-NTA affinity chromatography under RESULTS denaturing conditions (lane 3). Lane 4 contains an equal amount of purified p␣F6H (upper band) and pp␣F6H (lower band). Numbers on Purification of Pp␣F6H—To more clearly understand the the left represent the molecular mass (kDa) of protein standards. roles of Hsp70 and Hsp40 molecular chaperones in post-trans- lational translocation we modified an in vitro assay (14) by using AP-pp␣F6H as the substrate. Pp␣F used previously con- tained endogenous chaperones of the wheat germ translation system. We constructed a version of pp␣F (pp␣F6H) 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. Pp␣F6H 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 (p␣F6H) was formed (data not shown) indicating that the signal se- quence cleavage site of pp␣F6H was recognized by leader pep- tidase as previously reported for authentic pp␣F (37). Sequenc- ing the N terminus of p␣F6H revealed that it was identical to FIG. 2. Post-translational translocation of pp␣F6H into yeast that of authentic p␣F (data not shown) (38). A mixture of microsomes in the presence of PRS. [35S]Pp␣F and [35S]pp␣F6H were synthesized in wheat germ extracts (WG) and then added to purified pp␣F6H and p␣F6H was also electrophoresed (Fig. 1, post-translational translocation reactions that lacked or contained lane 4). Pp␣F6H, like authentic pp␣F (38), migrated faster wild-type microsomes as indicated. Protease protection experiments than p␣F6H on SDS-PAGE gels. using trypsin were done in the presence or absence of Triton X-100 as Characterization of the Translocation Competence of indicated. Each reaction was supplemented with PRS. The numbers at the right indicate the Mr of pp␣F6H (22 kDa) and p␣F6H (23 kDa) that Pp␣F6H—We next compared the translocation competence of contained one (26 kDa), two (28 kDa), or three (34 kDa) core pp␣F and pp␣F6H (Fig. 2). Post-translational translocation oligosaccharides. assays contained cycloheximide, an energy generating system, and yeast microsomes (30). Precursors were radiolabeled with cation assays four radiolabeled species migrating more slowly [35S]methionine either metabolically in E. coli or during trans- than pp␣F6H were detected (Fig. 2, lane 6). By analogy to lation in wheat germ extracts. Changes in molecular mass reactions containing authentic pp␣F, the new bands corre- because of signal sequence cleavage of precursors and glycosy- spond to p␣F6H (23 kDa) containing either one (26 kDa), two lation of translocated products were monitored using SDS- (28 kDa), or three (34 kDa) core oligosaccharides. Each histi- PAGE gels. In the presence of PRS, which contain Hsc70 mo- dine-tagged species migrated more slowly than its authentic lecular chaperones and other factors necessary for efficient counterpart reflecting the contribution of the six histidine res- post-translational translocation, the signal sequence of pp␣F idues to the molecular mass. Translocation of pp␣F6H and was removed and p␣F was core glycosylated up to three times glycosylation of p␣F6H were also dependent on microsomes (Fig. 2, lane 2) as previously reported (30). In the absence of (Fig. 2, compare lanes 5 and 6). In contrast to the precursors, microsomes, translocation was not detected (Fig. 2, lane 1). the translocated and glycosylated products were protected from Similarly, when pp␣F6H was synthesized in wheat germ trans- exogenously added trypsin (Fig. 2, lanes 3 and 7). Treating the lation reactions and then added to post-translational translo- membranes with a nonionic detergent rendered the products  Roles of Chaperones in Post-translational Translocation 7037 FIG. 3. Unfolded pp␣F6H is effi- ciently translocated in the absence of chaperones. Wheat germ-synthesized (N) pp␣F (bars 1 and 2) and pp␣F6H (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) pp␣F (bar 3) and pp␣F6H (bar 6) were translocated into microsomes in the ab- sence of PRS. Urea-denatured, affinity purified pp␣F6H (AP) was also translo- Downloaded from http://www.jbc.org/ at INDIAN INST OF TECHNOLOGY BOMBAY on August 31, 2019 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 pp␣F and pp␣F6H were essentially identical. The translocation compe- tence of pp␣F6H described here is comparable with that of another histidine-tagged pp␣F reported previously (39). The rate of translocation of unfolded pp␣F was previously shown to be greater than that of native pp␣F (14). To test whether the conformation of pp␣F6H affects its translocation competence, we denatured pp␣F6H with urea and then diluted it into translocation reactions (Fig. 3). Denatured pp␣F and pp␣F6H were translocated, 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 pp␣F6H (AP-pp␣F6H) was translocated 4.8-fold more efficiently than its native control (compare bars 4 and 7). Like pp␣F and pp␣F6H, AP-pp␣F6H 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 pp␣Fs (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 pp␣F and pp␣F6H are essentially identical. They also support the notion that urea denaturation of pp␣F (or pp␣F6H) mimics the FIG. 4. Loss of translocation competence. AP-[35S]pp␣F6H was stimulation of post-translational translocation by the chaper- diluted into post-translational translocation reactions lacking micro- ones in the PRS. somes. At the indicated times, wild-type microsomes were added and Upon dilution from urea, pp␣F (or pp␣F6H) likely folds rap- the amount of pp␣F6H translocated during a 2-min incubation was idly and loses translocation competence. To test this hypothesis determined. Each point represents the mean of the percentage trans- location of two experiments. we measured the amount of pp␣F6H translocated at various times after dilution. Translocation assays were restricted to 2 min to minimize the contribution of background translocation synthesized, nondenatured pp␣F6H is also shown (Fig. 5, line during longer incubations. The translocation competence of Nt). The initial rate of translocation (⬍20% translocation) of AP-pp␣F6H was essentially lost by 15 min and its half-life was AP-pp␣F6H (line None) was indistinguishable from that in the 2.2 min (Fig. 4). presence of Ssa1p (line S), Ydj1p (line Y), the control protein Kinetics of Translocation in the Presence of Ssa1p and hexokinase (line H), both Ssa1p and Ydj1p (line S⫹Y), or both Ydj1p—The availability of AP-pp␣F6H allowed us to explore Ssa1p and hexokinase (Fig. 5, line S⫹H). However, the final the role of yeast cytosolic Hsp70 and Hsp40 molecular chaper- relative yields of translocation were S, S ⫹ Y, S ⫹ H ⬎ None, Y, ones in post-translational translocation without interference H ⬎ Nt. Hexokinase was used as a control because it is a from chaperones in wheat germ translation systems. We deter- cytosolic protein whose crystal structure resembles that of the mined the time course of translocation of AP-pp␣F6H (Fig. 5). ATP-binding domain of Hsc70 (40). Together, these results For reference, the time course of translocation of wheat germ- suggest that immediately after dilution, translocation of some 7038 Roles of Chaperones in Post-translational Translocation Downloaded from http://www.jbc.org/ at INDIAN INST OF TECHNOLOGY BOMBAY on August 31, 2019 FIG. 5. Time course of translocation of pp␣F6H in the presence of yeast cytosolic chaperones. AP-[35S]pp␣F6H 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]pp␣F6H translocated was determined. The concentration of each protein was 0.68 ␮M. For comparison, the time course of wheat germ-synthesized, nondenatured [35S]pp␣F6H 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]pp␣F6H in the presence of Ssa1p was replotted as log (translocated fraction (TF)) versus time. AP-pp␣F6H was rapid and independent of exogenously added teract at different stages of precursor folding. We postulate chaperones. At later times, however, Ssa1p played an increas- that AP-pp␣F6H has at least three fates upon dilution from ingly important role in maintaining or restoring precursor denaturant into translocation assays. It may 1) enter micro- translocation competence. Ydj1p did not markedly stimulate somes in an unfolded conformation directly, 2) interact with translocation of AP-pp␣F6H in the presence or absence of chaperones that maintain its translocation competence, or 3) Ssa1p. Nevertheless, Ydj1p stimulated the ATPase activity of fold/aggregate. To elucidate the contributions of Ssa1p and Ssa1p 10-fold and was required for efficient luciferase refolding Ydj1p we performed translocation assays under three condi- in the presence of Ssa1p (data not shown) (26, 33). The results tions. In the first case, we mimicked in vivo translocation by also suggest that during a 60-min translocation assay AP- diluting AP-pp␣F6H into reactions containing microsomes and pp␣F6H exists in at least two states: one that is rapidly trans- different combinations of chaperones. Second, to investigate located and the other that is translocated more slowly. A replot the ability of the chaperones to maintain translocation compe- of the Ssa1p time course data (log translocated fraction versus tence, we preincubated AP-pp␣F6H with different combina- time) is biphasic supporting the idea that AP-pp␣F6H exists in tions of chaperones before adding microsomes. Third, to test at least two states (Fig. 5, inset). The rapidly translocated form restoration of translocation competence activity we allowed may be unfolded, whereas the more slowly translocated form AP-pp␣F6H to fold/aggregate before adding chaperones and may be either bound to chaperones, folded, or aggregated. microsomes. We refer to these assay conditions as complete (C), Chaperones Differentially Affect Translocation—During the maintenance (M), and restoration (R), respectively. course of post-translational translocation, chaperones may in- Upon dilution into a complete translocation reaction, about  Roles of Chaperones in Post-translational Translocation 7039 Downloaded from http://www.jbc.org/ at INDIAN INST OF TECHNOLOGY BOMBAY on August 31, 2019 FIG. 6. Ssa1p and Yjd1p can maintain, but not restore, translocation competence of pp␣F6H. In “complete” reactions (line C), AP-[35S]pp␣F6H 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]pp␣F6H 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]pp␣F6H was diluted into reactions. The percent of [35S]pp␣F6H translocated during a 20-min incubation is shown. Each point represents the mean of the percentage translocation of three experiments. 48% of AP-pp␣F6H was rapidly translocated into wild-type peated these experiments using microsomal membranes iso- microsomes in the absence of chaperones (Fig. 6a, c, and e, line lated from a strain lacking Ydj1p (Fig. 6b). Wild-type microso- C). In the presence of 0.68 ␮M Ssa1p, translocation peaked at mal membranes contain farnesylated Ydj1p (41) that might 62% (Fig. 6a, line C). Higher concentrations of Ssa1p inhibited obscure the effects of exogenous Ydj1p. We found that the translocation. When the addition of microsomes was delayed by effects of Ssa1p on AP-pp␣F6H translocation into wild-type and 30 min, the amount of AP-pp␣F6H translocated was only 8% in Ydj1p-deficient microsomes were essentially identical (com- the absence of chaperones (Fig. 6a, c, and e, line M). However, pare Fig. 6, a and b, lines C, M, and R). These results suggest including Ssa1p in the preincubation increased translocation that membrane-bound Ydj1p does not play a significant role in 2-fold suggesting that it can either maintain or restore the the translocation of AP-pp␣F6H into microsomes in the ab- translocation competence of AP-pp␣F6H (Fig. 6a, line M). To sence or presence of Ssa1p. distinguish between these two possibilities Ssa1p and micro- In contrast to Ssa1p, Ydj1p slightly inhibited AP-pp␣F6H somes were added after AP-pp␣F6H folded/aggregated. Less translocation when present during dilution (Fig. 6c, line C). At than 10% of folded/aggregated AP-pp␣F was translocated (Fig. the highest Ydj1p concentration tested, translocation efficiency 6a, line R). Together, these results indicate that Ssa1p can decreased 10%. Importantly, Ydj1p stimulated translocation maintain the translocation competence of AP-pp␣F. However, when the addition of microsomes was delayed (Fig. 6c, line M). its ability to restore translocation competence to folded/aggre- This suggests that Ydj1p can act as a molecular chaperone in gated AP-pp␣F is negligible. post-translational translocation by maintaining translocation To determine whether membrane-bound Ydj1p cooperates competence. Ydj1p did not stimulate translocation of refolded with Ssa1p to stimulate the translocation of pp␣F6H, we re- AP-pp␣F6H (Fig. 6c, line R). To elucidate the role of mem- 7040 Roles of Chaperones in Post-translational Translocation 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 Downloaded from http://www.jbc.org/ at INDIAN INST OF TECHNOLOGY BOMBAY on August 31, 2019 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 pp␣F6H translo- cated in the presence of both Ssa1p and Ydj1p was negligible (Fig. 6e, line R). The dependence of AP-pp␣F6H translocation into Ydj1p-deficient and wild-type membranes on the concen- tration of the Ssa1p and Ydj1p pair was similar, indicating that membrane-bound Ydj1p does not play an essential role in Ssa1p- dependent translocation (compare Fig. 6, e and f). Chaperones Prevent the Aggregation of Pp␣F6H—We showed above that Ssa1p and Ydj1p can maintain the translocation competence of pp␣F6H. To test the hypothesis that they main- tain translocation competence by preventing aggregation we first diluted AP-pp␣F6H into buffer lacking chaperones or con- taining Ssa1p, Ydj1p, or both. After 30 min, aggregated AP- pp␣F6H was removed by centrifugation and the translocation competence of AP-pp␣F6H remaining in the supernatant was determined. In the absence of chaperones, more than 90% of AP-pp␣F6H was removed by centrifugation indicating that it FIG. 7. Ssa1p and Yjd1p stimulate translocation by preventing had aggregated (Fig. 7A, bars Initial and N). Ssa1p and Ydj1p aggregation. A, AP-[35S]pp␣F6H was diluted into buffer containing no increased the solubility of pp␣F6H by 14- and 4.4-fold, respec- chaperones (N) or Ssa1p (S), Ydj1p (Y), or both (S⫹Y) as indicated. After 30 min, the reactions were centrifuged and the percent of [35S]pp␣F6H tively (Fig. 7A, compare bars N, S, and Y). In the presence of remaining in the supernatant was determined. A control reaction was both chaperones, the solubility of AP-pp␣F6H was comparable not centrifuged (Initial). B, [35S]pp␣F6H in the supernatants was also with that in the presence of Ssa1p alone (Fig. 7A, bars S and added to post-translational translocation assays containing wild-type S⫹Y). Together these results indicate that although both chap- microsomes and the amount translocated in 20 min was determined. Translocation efficiencies are expressed relative to reactions lacking erones can prevent the aggregation of AP-pp␣F6H, Ssa1p is chaperones (N). Each bar represents the mean ⫾ S.D. of three more effective. Furthermore, Ydj1p does not increase the abil- experiments. ity of Ssa1p to prevent aggregation under these reaction con- ditions. To determine whether preventing aggregation im- pp␣F6H remained soluble in the presence of NEM-Ssa1p than proves post-translational translocation we measured the in the presence of water-treated Ssa1p (data not shown). How- translocation competence of AP-pp␣F6H in the supernatants ever, NEM-Ssa1p was only 12 ⫾ 0.8% as effective as water- (Fig. 7B). The translocation efficiency of AP-pp␣F6H main- treated Ssa1p in translocating the soluble AP-pp␣F6H into tained in solution by Ssa1p and Ydj1p was, respectively, 4- and microsomes post-translationally (data not shown). Hexokinase 2-fold greater than that in reactions lacking chaperones (Fig. neither prevented aggregation nor maintained the transloca- 7B, compare bars N, S, and Y). Translocation in the presence of tion competence of AP-pp␣F6H (data not shown). Together both chaperones was comparable with that of Ssa1p alone. these results suggest that the ATP binding and/or ATPase Together these results suggest that Ssa1p and Ydj1p can stim- activity of Ssa1p are not required for preventing the aggrega- ulate translocation independently by preventing AP-pp␣F6H tion of AP-pp␣F6H, but are required for ensuring the produc- aggregation. tive release and translocation of AP-pp␣F6H. We and others previously showed that the post-translational translocation activity of Ssa1p is dependent on its ATPase DISCUSSION activity (27, 31). We demonstrated that NEM inhibits the abil- We explored the roles of cytosolic chaperones in post-trans- ity of Ssa1p to bind and hydrolyze ATP by modifying 3 cysteine lational translocation in vitro using AP-pp␣F6H. In the ab- residues in its ATP-binding domain (31). To determine whether sence of chaperones, urea-denatured AP-pp␣F6H was rapidly the ATPase activity of Ssa1p is also required for maintaining translocated into wild-type or Ydj1p-deficient microsomes upon the translocation competence of AP-pp␣F6H, we measured the dilution into reactions. In the absence of microsomes and chap- ability of NEM-Ssa1p to prevent the aggregation of AP- erones, AP-pp␣F6H aggregated and became translocation in- pp␣F6H and to stimulate the translocation of the AP-pp␣F6H competent. Ssa1p, Ydj1p, or both chaperones prevented aggre- remaining in solution. We found that 2 ⫾ 0.2-fold more AP- gation and maintained the translocation competence of some  Roles of Chaperones in Post-translational Translocation 7041 AP-pp␣F6H. These results provide the first biochemical evi- an affinity purified version of pp␣F, whereas the pp␣F used by dence supporting the notion that Ssa1p and Ydj1p stimulate Hendrick et al. (23) was not purified after synthesis in a crude post-translational translocation into the endoplasmic reticu- reticulocyte lysate. lum by preventing the aggregation of presecretory proteins. An important issue is whether Ssa1p and Ydj1p work to- Ydj1p, however, is not essential for translocation and its far- gether or in parallel in post-translational translocation of pre- nesylated, membrane-bound version provides no substantial secretory proteins into the endoplasmic reticulum. Our results benefit. Although the chaperones alone or together maintained support the idea that they work in parallel. However, the low translocation competence of AP-pp␣F6H, their ability to re- cellular concentration of Ydj1p and the higher maintenance of store the translocation competence of aggregated precursor translocation activity of Ssa1p suggest that Ssa1p would mo- was negligible. nopolize this pathway if they recognized overlapping sites on Genetic (12, 13, 27) and biochemical (14, 42, 43) studies have pp␣F. Three reports support the idea that Ydj1p and Ssa1p shown that cytosolic Hsp70s stimulate the post-translational cooperate in post-translational translocation of presecretory translocation of precursor proteins into endoplasmic reticulum proteins into the endoplasmic reticulum (13, 21). First, a tem- and mitochondria. Insight into the mechanism by which Ssa1p perature-sensitive mutant of Ydj1p accumulated pp␣F at the stimulates translocation is provided by the experiments re- nonpermissive temperature and the mutant protein, ydj1– ported here demonstrating that Ssa1p acts by preventing pp␣F 151p, only weakly stimulated the ATPase activity of Ssa1p Downloaded from http://www.jbc.org/ at INDIAN INST OF TECHNOLOGY BOMBAY on August 31, 2019 aggregation. Further insight was obtained using NEM-Ssa1p, (21). Second, Becker et al. (13) found a synthetically lethal which has defective nucleotide-binding and ATPase activities relationship between SSA1 and YDJ1. Furthermore, a strain (31). NEM-Ssa1p prevented the aggregation of AP-pp␣F6H, expressing Ssa1p, but lacking Ssa2p, Ssa3p, Ssa4p, and Ydj1p but failed to maintain its translocation competence. This is accumulated pp␣F and its glycosylated intermediates at 23 °C consistent with the idea that NEM-Ssa1p can bind AP- (13). Third, mutants of Ssa1p containing amino acid substitu- pp␣F6H, but its subsequent release, if any, is inadequate to tions in the ATP-binding domain failed to bind unfolded support translocation. Similarly, NEM-Ssa1p can prevent the polypeptides, did not stimulate post-translational transloca- aggregation of denatured luciferase, but cannot refold it (33). tion of pp␣F into microsomes, and their ATPase activity could Ssa1p likely prevents aggregation by directly interacting with not be stimulated by Ydj1p (27). It remains possible, however, AP-pp␣F6H. We previously reported that cytosolic Hsc70 asso- that cells expressing high levels of unfolded proteins at non- ciates with pp␣F in wheat germ extracts (16). Recently, others permissive conditions (13, 21) or lacking the full complement of have shown that Hsc70 can be photocross-linked to pp␣F trans- cytosolic Hsp70 chaperones at permissive temperatures (13), lated in reticulocyte lysates (15). The translocation competence contain insufficient amounts of Ssa1p to support post-transla- tional translocation. In our in vitro experiments, Ydj1p did not of mitochondrial precursor proteins is also maintained by cy- enhance the ability of Ssa1p to prevent pp␣F aggregation, to tosolic Hsp70 chaperones likely through direct association (44). maintain translocation competence of pp␣F, or to stimulate Ydj1p was previously shown to stimulate the ATPase activity post-translational translocation. The normally beneficial coop- of Ssa1p (25, 26), to facilitate the release of bound polypeptides eration between Ssa1p and Ydj1p during protein folding (26) from Ssa1p (25, 27), to prevent the aggregation of rhodanese may be unnecessary and counterproductive in the post-trans- (10), and to cooperate with Ssa1p in the folding of denatured lational translocation of unfolded pp␣F. luciferase (26, 45). Although we found that Ydj1p hindered AP-pp␣F6H aggregation and maintained its translocation com- Acknowledgments—We thank Irina Kovatch and David Jin for tech- petence, it, unlike Ssa1p, did not stimulate translocation of nical assistance, Julie Rushbrook for amino acid sequence analysis, and AP-pp␣F6H upon dilution into complete reactions containing Betty Craig and Don Oliver for strains. microsomes. This result suggests that Ydj1p may prevent AP- REFERENCES pp␣F6H aggregation less effectively than Ssa1p. Results re- 1. Bukau, B., and Horwich, A. L. (1998) Cell 92, 351–366 ported here demonstrating that AP-pp␣F6H can be post-trans- 2. Palleros, D. R., Reid, K. L., Shi, L., Welch, W. J., and Fink, A. L. (1993) Nature lationally translocated into microsomes isolated from ydj1 null 365, 664 – 666 3. Schmid, D., Baici, A., Gehring, H., and Christen, P. (1994) Science 263, strains indicate that membrane-bound Ydj1p is not essential 971–973 for this process. These results are consistent with those ob- 4. Flynn, G. C., Pohl, J., Flocco, M. T., and Rothman, J. E. (1991) Nature 353, 726 –730 tained from in vivo experiments showing that pp␣F is effi- 5. Blond-Elguindi, S., Cwirla, S. E., Dower, W. J., Lipshutz, R. J., Sprang, S. R., ciently exported from ydj1 null strains (13, 22). In such strains, Sambrook, J. F., and Gething, M. J. (1993) Cell 75, 717–728 6. Rudiger, S., Germeroth, L., Schneider-Mergener, J., and Bukau, B. (1997) however, pp␣F also has the option of entering the endoplasmic EMBO J. 16, 1501–1507 reticulum co-translationally via the SRP-dependent pathway. 7. Langer, T., Lu, C., Echols, H., Flanagan, J., Hayer, M. K., and Hartl, F. U. Although our results do not support the notion that membrane- (1992) Nature 356, 683– 689 8. Liberek, K., Marszalek, J., Ang, D., Georgopoulos, C., and Zylicz, M. (1991) bound Ydj1p acts as a release factor for pp␣F䡠Ssa1p complexes, Proc. Natl. Acad. Sci. U. S. A. 88, 2874 –2878 this hypothesis requires further study. 9. Rudiger, S., Schneider-Mergener, J., and Bukau, B. (2001) EMBO J. 20, 1042–1950 Using chaperones isolated from E. coli, Hendrick et al. (23, 10. Cyr, D. M. (1995) FEBS Lett. 359, 129 –132 32) reported that E. coli DnaJ completely inhibited the in vitro 11. Fewell, S. W., Travers, K. J., Weissman, J. S., and Brodsky, J. L. (2001) Annu. post-translational translocation of reticulocyte lysate-trans- Rev. Genet. 35, 149 –191 12. Deshaies, R. J., Koch, B. D., Werner-Washburne, M., Craig, E. A., and Schek- lated pp␣F into yeast microsomes. DnaK and GrpE reversed man, R. (1988) Nature 332, 800 – 805 the inhibition. In contrast, we found that Ydj1p only partially 13. Becker, J., Walter, W., Yan, W., and Craig, E. A. (1996) Mol. Cell. Biol. 16, 4378 – 4386 inhibited translocation of AP-pp␣F6H even when present at 14. Chirico, W. J., Waters, M. G., and Blobel, G. (1988) Nature 332, 805– 810 high concentrations. Ydj1p may inhibit post-translational 15. Plath, K., and Rapoport, T. A. (2000) J. Cell Biol. 151, 167–178 16. Chirico, W. J. (1992) Biochem. Biophys. Res. Commun. 189, 1150 –1156 translocation by competing with Ssa1p for binding sites on 17. Lain, B., Iriarte, A., and Martinez-Carrion, M. (1994) J. Biol. Chem. 269, pp␣F6H or by stabilizing ADP forms of the Ssa1p䡠pp␣F6H 15588 –15596 complexes. Perhaps the lack of agreement between the two 18. Wiech, H., Buchner, J., Zimmermann, M., Zimmermann, R., and Jakob, U. (1993) J. Biol. Chem. 268, 7414 –7421 studies arises from the difference in the source of reaction 19. Brodsky, J. L., Hamamoto, S., Feldheim, D., and Schekman, R. (1993) J. Cell components. All components used in our system were derived Biol. 120, 95–102 20. Waters, M. G., Chirico, W. J., Henriquez, R., and Blobel, G. (1989) in Stress- from yeast, whereas those used by Hendrick et al. (23) were induced Proteins, (Pardue, M. L., Feramisco, J. R., and Lindquist, S. L., eds) derived from three different organisms. Furthermore, we used Vol. 96, pp. 163–174, Alan R. Liss, Inc., New York 7042 Roles of Chaperones in Post-translational Translocation 21. Caplan, A. J., Cyr, D. M., and Douglas, M. G. (1992) Cell 71, 1143–1155 34. Bradford, M. M. (1976) Anal. Biochem. 72, 248 –254 22. Atencio, D. P., and Yaffe, M. P. (1992) Mol. Cell. Biol. 12, 283–291 35. Walter, P., and Blobel, G. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 7112–7116 23. Hendrick, J. P., Langer, T., Davis, T. A., Hartl, F. U., and Wiedmann, M. 36. Waters, M. G., and Blobel, G. (1986) J. Cell Biol. 102, 1543–1550 (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10216 –10220 37. Lecker, S., Meyer, D., and Wickner, W. (1989) J. Biol. Chem. 264, 1882–1886 24. Wild, J., Altman, E., Yura, T., and Gross, C. A. (1992) Genes Dev. 6, 1165–1172 38. Waters, M. G., Evans, E. A., and Blobel, G. (1988) J. Biol. Chem. 263, 25. Cyr, D. M., Lu, X., and Douglas, M. G. (1992) J. Biol. Chem. 267, 20927–20931 6209 – 6214 26. Levy, E. J., McCarty, J., Bukau, B., and Chirico, W. J. (1995) FEBS Lett. 368, 39. Bush, G. L., Tassin, A. M., Friden, H., and Meyer, D. I. (1991) J. Biol. Chem. 435– 440 266, 13811–13814 27. McClellan, A. J., and Brodsky, J. L. (2000) Genetics 156, 501–512 40. Bork, P., Sander, C., and Valencia, A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 28. Julius, D., Schekman, R., and Thorner, J. (1984) Cell 36, 309 –318 7290 –7294 29. Miller, J. H. (1972) Experiments in Molecular Genetics, Cold Spring Harbor 41. Caplan, A. J., Tsai, J., Casey, P. J., and Douglas, M. G. (1992) J. Biol. Chem. Laboratory, Cold Spring Harbor 267, 18890 –18895 30. Waters, M. G., Chirico, W. J., and Blobel, G. (1986) J. Cell Biol. 103, 42. Murakami, H., Pain, D., and Blobel, G. (1988) J. Cell Biol. 107, 2051–2057 2629 –2636 43. Zimmermann, R., Sagstetter, M., Lewis, M. J., and Pelham, H. R. (1988) 31. Liu, Q., Levy, E. J., and Chirico, W. J. (1996) J. Biol. Chem. 271, 29937–29944 EMBO J. 7, 2875–2880 32. Hendrick, J. P., and Hartl, F.-U. (1993) Annu. Rev. Biochem. 1993 62, 349 –384 44. Komiya, T., Sakaguchi, M., and Mihara, K. (1996) EMBO J. 15, 399 – 407 33. Hermawan, A., and Chirico, W. J. (1999) Arch. Biochem. Biophys 369, 157–162 45. Lu, Z., and Cyr, D. M. (1998) J. Biol. Chem. 273, 27824 –27830 Downloaded from http://www.jbc.org/ at INDIAN INST OF TECHNOLOGY BOMBAY on August 31, 2019  Roles of Cytosolic Hsp70 and Hsp40 Molecular Chaperones in Post-translational Translocation of Presecretory Proteins into the Endoplasmic Reticulum Jantra Ngosuwan, Nancy M. Wang, Katie L. Fung and William J. Chirico J. Biol. Chem. 2003, 278:7034-7042. doi: 10.1074/jbc.M210544200 originally published online December 19, 2002 Access the most updated version of this article at doi: 10.1074/jbc.M210544200 Downloaded from http://www.jbc.org/ at INDIAN INST OF TECHNOLOGY BOMBAY on August 31, 2019 Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's e-mail alerts This article cites 44 references, 25 of which can be accessed free at http://www.jbc.org/content/278/9/7034.full.html#ref-list-1