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. ChiricoDagger

From the Department of Anatomy and Cell Biology, State University of New York Downstate Medical Center, Brooklyn, New York 11203

Received for publication, October 15, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Hsp70 molecular chaperones and their co-chaperones work together in various cellular compartments to guide the folding of proteins and to aid the translocation of proteins across membranes. Hsp70s stimulate protein folding by binding exposed hydrophobic sequences thereby preventing irreversible aggregation. Hsp40s stimulate the ATPase activity of Hsp70s and target unfolded proteins to Hsp70s. Genetic and biochemical evidence supports a role for cytosolic Hsp70s and Hsp40s in the post-translational translocation of precursor proteins into endoplasmic reticulum and mitochondria. To gain mechanistic insight, we measured the effects of Saccharomyces cerevisiae Ssa1p (Hsp70) and Ydj1p (Hsp40) on the translocation of histidine-tagged prepro-alpha -factor (ppalpha F6H) into microsomes. Radiolabeled ppalpha F6H was affinity purified from wheat germ translation reactions (or Escherichia coli) to remove endogenous chaperones. We demonstrated that either Ssa1p or Ydj1p stimulates post-translational translocation by preventing ppalpha F6H aggregation. The binding and/or hydrolysis of ATP by Ssa1p were required to maintain the translocation competence of ppalpha F6H. To clarify the contributions of membrane-bound and cytosolic Ydj1p, we compared the efficiency of chaperone-dependent translocation into wild-type and Ydj1p-deficient microsomes. Neither soluble nor membrane-bound Ydj1p was essential for post-translational protein translocation. The ability of Ssa1p, Ydj1p, or both chaperones to restore the translocation competence of aggregated ppalpha F6H was negligible.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 hydrolysis of ATP regulate the action of Hsp70s (2). In the ATP-bound form, peptide substrates undergo cycles of rapid binding and release from Hsp70 (3). In the ADP-bound form, the interactions are slower resulting in higher affinity. Hsp70s stimulate protein folding by binding to exposed hydrophobic sequences (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 cytosolic Hsp70s and Hsp40s in the post-translational translocation of precursor proteins into the endoplasmic reticulum and mitochondria (reviewed in Ref. 11). Deshaies et al. (12) reported that presecretory proteins and mitochondrial precursors accumulated 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-alpha -factor (ppalpha F) into yeast microsomes. Complexes containing Hsp70s and presecretory and mitochondrial precursor proteins have been identified in wheat germ extracts and reticulocyte lysates (15-17). The ability to stimulate 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 translocation at the nonpermissive temperature. However, Atencio and Yaffe (22) and Becker et al. (13) showed that ppalpha F is translocated normally in deletion mutants of YDJ1. Translocation of ppalpha F was defective 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 ppalpha F in vitro. DnaK and GrpE together relieved the DnaJ-dependent inhibition, but neither alone had any effect. Export of certain precursor proteins was defective in some dnaK and dnaJ mutant 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 together 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 translocation 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 ppalpha F, which had accumulated at the nonpermissive temperature, could not be translocated. These results suggested that the chaperones maintain translocation competence by binding to ppalpha F co-translationally, but they cannot rescue aggregated ppalpha F. Strains containing mutant versions of Ssa1p with defective ATP-binding pockets fail to interact productively with Ydj1p and accumulate ppalpha F (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 preparations of ppalpha F, which was synthesized in wheat germ extracts or reticulocyte lysates, we used a histidine-tagged, affinity purified version of ppalpha F (AP-ppalpha F6H) in translocation assays. We clarified the contributions of membrane-bound and cytosolic Ydj1p, by comparing the translocation efficiencies of the chaperones using wild-type and Ydj1p-deficient microsomal membranes. We demonstrate that Ssa1p stimulates translocation by preventing the aggregation of AP-ppalpha F6H. 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-translational translocation by preventing AP-ppalpha F6H aggregation.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

Plasmids-- pKWC1, which can express ppalpha F6H in E. coli, was constructed by amplifying the cDNA for ppalpha F 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'-GTGTGCATGCGATTTCCTTCAATTTTTACTG-3' and 5'-ATATAGATCTGTACATTGGTTGGCCGGGT-3', respectively. pREP4 was obtained from Qiagen. pNWC1, which was used to generate mRNA coding for ppalpha F6H, was constructed by amplifying the cDNA for ppalpha F6H 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'-GTGTGCATGCGATTTCCTTCAATTTTTACTG-3' and 5'-ATATGAGCTCGGATCTATCAACAGGAGTCC-3', respectively. The sequences of the constructs were confirmed by automated DNA sequencing.

Metabolic Labeling of Ppalpha F6H-- E. coli IQ85 (F' araD139 Delta (argF-lac)U169 rpsL150 relA1 flbB5301 deoC1 ptsF25 secYts) was transformed sequentially with pREP4 and pKWC1. The resulting strain IQ85[pREP4, pKWC1] was grown overnight at 25 °C in 2 ml of LB media containing 2% glucose, 25 µg/ml kanamycin, 25 µg/ml tetracycline, and 100 µg/ml ampicillin. The cells were collected by centrifugation (15,700 × 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-beta -D-galactopyranoside (2 mM final concentration) and Tran35S-label (ICN Biomedicals, 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]ppalpha F6H was purified as described below.

In Vitro Transcription and Translation-- Messenger RNA coding for ppalpha F or ppalpha F6H 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 ppalpha F and ppalpha F6H were synthesized 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 previously (19). The concentration of Ssa1p was determined as described previously (31). Ydj1p was overexpressed and purified from E. coli as described previously (25). The concentration of Ydj1p was determined using its extinction coefficient at 280 nm (20370 M-1 cm-1). 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 supernatants (PRS) were prepared from S. cerevisiae SKQ2N as described previously (14). Hexokinase from S. cerevisiae was obtained from Sigma. [35S]Ppalpha F6H was purified from either wheat germ translation reactions programmed with ppalpha F6H mRNA or metabolically labeled IQ85[pREP4, pKWC1] as follows. Proteins in translation reactions (230 µl) were precipitated with 460 µl 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 1 h at room temperature. Insoluble proteins were removed by centrifugation at 15,700 × g for 5 min. Ni2+-NTA resin (60 µl 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 µl of buffer C (8 M urea, 100 mM sodium phosphate, and 10 mM Tris-HCl, pH 8.0). [35S]Ppalpha F6H was eluted with 40 µl of buffer C adjusted to pH 4.6. After the pH of eluate was neutralized with 7.5 µl of 2 M HEPES, pH 8, it was frozen in liquid nitrogen and stored at -70 °C. [35S]Ppalpha F6H was purified from metabolically labeled IQ85[pREP4, pKWC1] (see above) by resuspending the cells from a 10-ml culture in 500 µl of buffer B. After mixing the cells for 1 h at room temperature, insoluble proteins were removed by centrifugation at 15,700 × g for 5 min. The supernatant was mixed with 50 µl of Ni2+-NTA resin (50% slurry) for 1 h at room temperature. Unbound proteins were removed from the resin and then [35S]ppalpha F6H was eluted as described above. Palpha F6H was produced in either IQ85[pREP4, pKWC1] or DH5alpha [pREP4, pKWC1] grown and induced at 37 °C. It was purified in the same manner as ppalpha F6H. Protein concentrations were determined using the Bradford assay (34) and bovine gamma -globulin was used as the standard.

Protein Sequencing-- A 1.75-µg sample of purified palpha F6H was derivatized with phenylisothiocyanate and then automated Edman degradation was performed using an Applied Biosystems 475A Protein Sequencer (Applied Biosystems, Inc.) in the Protein Sequencing Center at SUNY Downstate Medical Center.

Yeast Microsomal Membranes-- Microsomes were isolated from S. cerevisiae SKQ2N (MATa/alpha ade1/+ +/ade2 +/his1), MYY290 (MATa leu2 his3 ura3) (22), and MYY406 (MATalpha leu2 his3 mas5::URA3) (22) as previously described (30). The post-translational translocation activities of microsomes isolated from wild-type strains SKQ2N and MYY290 were essentially identical. One equivalent (eq) of microsomes was defined 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/µl 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 translocation assay was described previously (14). In brief, it contained 2 µl of 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 cycloheximide), 1.37 µl of an energy source (9.1 mM ATP, 456.2 mM creatine phosphate, and 0.365 mM GDP-mannose), 0.63 µl of 8 mg/ml creatine kinase, 2 µl of yeast microsomes (4 eq/µl), 1 µl of water, 16 µl of buffer A (PRS or chaperones to be tested), and 2 µl (~15,000 dpm) of wheat germ-translated [35S]ppalpha F or [35S]ppalpha F6H. When denatured [35S]ppalpha F6H (or [35S]ppalpha F) 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 µl of 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 µl of denatured [35S]ppalpha F6H (or [35S]ppalpha F) or 0.3 µl of affinity purified [35S]ppalpha F6H, respectively. Denatured substrate (~15,000 dpm) was added last and then reactions were mixed immediately. All pipette tips used to transfer denatured ppalpha F were pretreated with SIGMACOTE (Sigma). The final volume was 25 µl and the reactions were incubated at 20 °C for the indicated times. Reactions were terminated by adding an equal volume of 2× SDS-PAGE sample buffer and then boiling the mixture for 2 min. In the maintenance of translocation experiments, AP-[35S]ppalpha F6H was added to translocation assays lacking microsomal membranes and incubated at 20 °C for 30 min. Translocation was initiated by the addition of 2 µl of microsomal membranes. In the restoration of translocation competence experiments, AP-[35S]ppalpha 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 µl 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 phosphorylase 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 ppalpha F6H translocated was quantified using a PhosphorImager (Amersham Biosciences). Percent translocation was calculated as previously described (14).

Protease Protection-- After import, 5 µl of 8 mM CaCl2 and 5 µl of either water or 8% (w/v) Triton X-100 were added to a 25-µl translocation reaction (36). After adding 5 µl of 800 µg/ml trypsin, the reactions were incubated for 30 min on ice. The digestion was stopped with 5 µl of 50 mM phenylmethanesulfonyl fluoride in dimethyl sulfoxide.

Aggregation of ppalpha F-- AP-[35S]ppalpha F6H (0.3 µl) was diluted into buffer A (16 µl) lacking chaperones or containing Ssa1p (0.68 µM), Ydj1p (0.68 µM), or both (each 0.68 µM). After 30 min on ice, the reactions were centrifuged at 15,700 × g for 15 min at 4 °C and the percent of [35S]ppalpha F6H remaining in the supernatant was determined. An equivalent amount of supernatant was also added to post-translational translocation assays containing wild-type microsomes (MYY290) and the amount of [35S]ppalpha 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]ppalpha F6H aggregation and post-translational translocation assays was 0.68 µM. The ATPase activity of NEM-Ssa1p was only 16% that of the water-treated Ssa1p (data not shown).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Purification of Ppalpha F6H-- To more clearly understand the roles of Hsp70 and Hsp40 molecular chaperones in post-translational translocation we modified an in vitro assay (14) by using AP-ppalpha F6H as the substrate. Ppalpha F used previously contained endogenous chaperones of the wheat germ translation system. We constructed a version of ppalpha F (ppalpha F6H) that has 6 histidine residues at its C terminus. The C terminus was altered to avoid the possibility of compromising the signal sequence at the N terminus. Ppalpha F6H was induced (Fig. 1, compare lanes 1 and 2) and purified (lane 3) from an E. coli strain harboring a temperature-sensitive export mutation. At the permissive temperature, histidine-tagged pro-alpha -factor (palpha F6H) was formed (data not shown) indicating that the signal sequence cleavage site of ppalpha F6H was recognized by leader peptidase as previously reported for authentic ppalpha F (37). Sequencing the N terminus of palpha F6H revealed that it was identical to that of authentic palpha F (data not shown) (38). A mixture of purified ppalpha F6H and palpha F6H was also electrophoresed (Fig. 1, lane 4). Ppalpha F6H, like authentic ppalpha F (38), migrated faster than palpha F6H on SDS-PAGE gels.


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Fig. 1.   Purification of ppalpha F6H. Proteins from isopropyl-1-thio-beta -D-galactopyranoside-induced (lane 2) and uninduced (lane 1) E. coli IQ85 (SecYts) harboring a ppalpha F6H expression plasmid were separated on a SDS-PAGE gel and then stained with Coomassie Blue. Ppalpha F6H was affinity purified using Ni2+-NTA affinity chromatography under denaturing conditions (lane 3). Lane 4 contains an equal amount of purified palpha F6H (upper band) and ppalpha F6H (lower band). Numbers on the left represent the molecular mass (kDa) of protein standards.

Characterization of the Translocation Competence of Ppalpha F6H-- We next compared the translocation competence of ppalpha F and ppalpha F6H (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 translation in wheat germ extracts. Changes in molecular mass because of signal sequence cleavage of precursors and glycosylation of translocated products were monitored using SDS-PAGE gels. In the presence of PRS, which contain Hsc70 molecular chaperones and other factors necessary for efficient post-translational translocation, the signal sequence of ppalpha F was removed and palpha F 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 ppalpha F6H was synthesized in wheat germ translation reactions and then added to post-translational translocation assays four radiolabeled species migrating more slowly than ppalpha F6H were detected (Fig. 2, lane 6). By analogy to reactions containing authentic ppalpha F, the new bands correspond to palpha F6H (23 kDa) containing either one (26 kDa), two (28 kDa), or three (34 kDa) core oligosaccharides. Each histidine-tagged species migrated more slowly than its authentic counterpart reflecting the contribution of the six histidine residues to the molecular mass. Translocation of ppalpha F6H and glycosylation of palpha F6H 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 susceptible to proteolysis (Fig. 2, lanes 4 and 8). These results indicate that the translocation and glycosylation of ppalpha F and ppalpha F6H were essentially identical. The translocation competence of ppalpha F6H described here is comparable with that of another histidine-tagged ppalpha F reported previously (39).


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Fig. 2.   Post-translational translocation of ppalpha F6H into yeast microsomes in the presence of PRS. [35S]Ppalpha F and [35S]ppalpha F6H 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 ppalpha F6H (22 kDa) and palpha F6H (23 kDa) that contained one (26 kDa), two (28 kDa), or three (34 kDa) core oligosaccharides.

The rate of translocation of unfolded ppalpha F was previously shown to be greater than that of native ppalpha F (14). To test whether the conformation of ppalpha F6H affects its translocation competence, we denatured ppalpha F6H with urea and then diluted it into translocation reactions (Fig. 3). Denatured ppalpha F and ppalpha 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 ppalpha F6H (AP-ppalpha F6H) was translocated 4.8-fold more efficiently than its native control (compare bars 4 and 7). Like ppalpha F and ppalpha F6H, AP-ppalpha F6H was translocated into microsomes, its signal sequence was removed, and the resulting product was glycosylated (data not shown). The efficiency of translocation of the denatured ppalpha Fs (bars 3, 6, and 7) approached 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 ppalpha F and ppalpha F6H are essentially identical. They also support the notion that urea denaturation of ppalpha F (or ppalpha F6H) mimics the stimulation of post-translational translocation by the chaperones in the PRS.


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Fig. 3.   Unfolded ppalpha F6H is efficiently translocated in the absence of chaperones. Wheat germ-synthesized (N) ppalpha F (bars 1 and 2) and ppalpha 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) ppalpha F (bar 3) and ppalpha F6H (bar 6) were translocated into microsomes in the absence of PRS. Urea-denatured, affinity purified ppalpha F6H (AP) was also translocated in the absence of PRS (bar 7). Each bar represents the mean ± S.D. of the percentage translocation during 20 min of three experiments.

Upon dilution from urea, ppalpha F (or ppalpha F6H) likely folds rapidly and loses translocation competence. To test this hypothesis we measured the amount of ppalpha F6H 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-ppalpha F6H was essentially lost by 15 min and its half-life was 2.2 min (Fig. 4).


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Fig. 4.   Loss of translocation competence. AP-[35S]ppalpha F6H was diluted into post-translational translocation reactions lacking microsomes. At the indicated times, wild-type microsomes were added and the amount of ppalpha F6H translocated during a 2-min incubation was determined. Each point represents the mean of the percentage translocation of two experiments.

Kinetics of Translocation in the Presence of Ssa1p and Ydj1p-- The availability of AP-ppalpha F6H allowed us to explore the role of yeast cytosolic Hsp70 and Hsp40 molecular chaperones in post-translational translocation without interference from chaperones in wheat germ translation systems. We determined the time course of translocation of AP-ppalpha F6H (Fig. 5). For reference, the time course of translocation of wheat germ-synthesized, nondenatured ppalpha F6H is also shown (Fig. 5, line Nt). The initial rate of translocation (<20% translocation) of AP-ppalpha F6H (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 AP-ppalpha F6H was rapid and independent of exogenously added chaperones. At later times, however, Ssa1p played an increasingly important role in maintaining or restoring precursor translocation competence. Ydj1p did not markedly stimulate translocation of AP-ppalpha F6H 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-ppalpha F6H exists in at least two states: one that is rapidly translocated 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-ppalpha F6H 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.


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Fig. 5.   Time course of translocation of ppalpha F6H in the presence of yeast cytosolic chaperones. AP-[35S]ppalpha 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]ppalpha F6H translocated was determined. The concentration of each protein was 0.68 µM. For comparison, the time course of wheat germ-synthesized, nondenatured [35S]ppalpha 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]ppalpha F6H in the presence of Ssa1p was replotted as log (translocated fraction (TF)) versus time.

Chaperones Differentially Affect Translocation-- During the course of post-translational translocation, chaperones may interact at different stages of precursor folding. We postulate that AP-ppalpha F6H has at least three fates upon dilution from denaturant into translocation assays. It may 1) enter microsomes 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 conditions. In the first case, we mimicked in vivo translocation by diluting AP-ppalpha F6H into reactions containing microsomes and different combinations of chaperones. Second, to investigate the ability of the chaperones to maintain translocation competence, we preincubated AP-ppalpha F6H with different combinations of chaperones before adding microsomes. Third, to test restoration of translocation competence activity we allowed AP-ppalpha F6H 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 48% of AP-ppalpha F6H 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 µM 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-ppalpha F6H translocated 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-ppalpha F6H (Fig. 6a, line M). To distinguish between these two possibilities Ssa1p and microsomes were added after AP-ppalpha F6H folded/aggregated. Less than 10% of folded/aggregated AP-ppalpha F was translocated (Fig. 6a, line R). Together, these results indicate that Ssa1p can maintain the translocation competence of AP-ppalpha F. However, its ability to restore translocation competence to folded/aggregated AP-ppalpha F is negligible.


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Fig. 6.   Ssa1p and Yjd1p can maintain, but not restore, translocation competence of ppalpha F6H. In "complete" reactions (line C), AP-[35S]ppalpha 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]ppalpha 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]ppalpha F6H was diluted into reactions. The percent of [35S]ppalpha F6H translocated during a 20-min incubation is shown. Each point represents the mean of the percentage translocation of three experiments.

To determine whether membrane-bound Ydj1p cooperates with Ssa1p to stimulate the translocation of ppalpha F6H, we repeated these experiments using microsomal membranes isolated from a strain lacking Ydj1p (Fig. 6b). Wild-type microsomal membranes contain farnesylated Ydj1p (41) that might obscure the effects of exogenous Ydj1p. We found that the effects of Ssa1p on AP-ppalpha F6H translocation into wild-type and Ydj1p-deficient microsomes were essentially identical (compare 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-ppalpha F6H into microsomes in the absence or presence of Ssa1p.

In contrast to Ssa1p, Ydj1p slightly inhibited AP-ppalpha F6H 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-ppalpha F6H (Fig. 6c, line R). To elucidate the role of membrane-bound Ydj1p, we repeated these experiments using Ydj1p-deficient microsomes (Fig. 6d). The patterns were essentially identical to those obtained using wild-type microsomes supporting the notion that membrane-bound Ydj1p is not required for translocation (compare Fig. 6, c and d).

To determine whether the chaperones cooperate during protein translocation, we monitored translocation at different concentrations 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 reaction 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 ppalpha F6H translocated in the presence of both Ssa1p and Ydj1p was negligible (Fig. 6e, line R). The dependence of AP-ppalpha F6H translocation into Ydj1p-deficient and wild-type membranes on the concentration 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 Ppalpha F6H-- We showed above that Ssa1p and Ydj1p can maintain the translocation competence of ppalpha F6H. To test the hypothesis that they maintain translocation competence by preventing aggregation we first diluted AP-ppalpha F6H into buffer lacking chaperones or containing Ssa1p, Ydj1p, or both. After 30 min, aggregated AP-ppalpha F6H was removed by centrifugation and the translocation competence of AP-ppalpha F6H remaining in the supernatant was determined. In the absence of chaperones, more than 90% of AP-ppalpha F6H was removed by centrifugation indicating that it had aggregated (Fig. 7A, bars Initial and N). Ssa1p and Ydj1p increased the solubility of ppalpha F6H by 14- and 4.4-fold, respectively (Fig. 7A, compare bars N, S, and Y). In the presence of both chaperones, the solubility of AP-ppalpha F6H was comparable with that in the presence of Ssa1p alone (Fig. 7A, bars S and S+Y). Together these results indicate that although both chaperones can prevent the aggregation of AP-ppalpha F6H, Ssa1p is more effective. Furthermore, Ydj1p does not increase the ability of Ssa1p to prevent aggregation under these reaction conditions. To determine whether preventing aggregation improves post-translational translocation we measured the translocation competence of AP-ppalpha F6H in the supernatants (Fig. 7B). The translocation efficiency of AP-ppalpha F6H maintained 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 stimulate translocation independently by preventing AP-ppalpha F6H aggregation.


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Fig. 7.   Ssa1p and Yjd1p stimulate translocation by preventing aggregation. A, AP-[35S]ppalpha F6H was diluted into buffer containing no 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]ppalpha F6H remaining in the supernatant was determined. A control reaction was not centrifuged (Initial). B, [35S]ppalpha F6H 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.

We and others previously showed that the post-translational translocation activity of Ssa1p is dependent on its ATPase activity (27, 31). We demonstrated that NEM inhibits the ability 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-ppalpha F6H, we measured the ability of NEM-Ssa1p to prevent the aggregation of AP-ppalpha F6H and to stimulate the translocation of the AP-ppalpha F6H remaining in solution. We found that 2 ± 0.2-fold more AP-ppalpha F6H remained soluble in the presence of NEM-Ssa1p than in the presence of water-treated Ssa1p (data not shown). However, NEM-Ssa1p was only 12 ± 0.8% as effective as water-treated Ssa1p in translocating the soluble AP-ppalpha F6H into microsomes post-translationally (data not shown). Hexokinase neither prevented aggregation nor maintained the translocation competence of AP-ppalpha F6H (data not shown). Together these results suggest that the ATP binding and/or ATPase activity of Ssa1p are not required for preventing the aggregation of AP-ppalpha F6H, but are required for ensuring the productive release and translocation of AP-ppalpha F6H.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We explored the roles of cytosolic chaperones in post-translational translocation in vitro using AP-ppalpha F6H. In the absence of chaperones, urea-denatured AP-ppalpha F6H was rapidly translocated into wild-type or Ydj1p-deficient microsomes upon dilution into reactions. In the absence of microsomes and chaperones, AP-ppalpha F6H aggregated and became translocation incompetent. Ssa1p, Ydj1p, or both chaperones prevented aggregation and maintained the translocation competence of some AP-ppalpha F6H. These results provide the first biochemical evidence supporting the notion that Ssa1p and Ydj1p stimulate post-translational translocation into the endoplasmic reticulum by preventing the aggregation of presecretory proteins. Ydj1p, however, is not essential for translocation and its farnesylated, membrane-bound version provides no substantial benefit. Although the chaperones alone or together maintained translocation competence of AP-ppalpha F6H, their ability to restore 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 reported here demonstrating that Ssa1p acts by preventing ppalpha F aggregation. Further insight was obtained using NEM-Ssa1p, which has defective nucleotide-binding and ATPase activities (31). NEM-Ssa1p prevented the aggregation of AP-ppalpha F6H, but failed to maintain its translocation competence. This is consistent with the idea that NEM-Ssa1p can bind AP-ppalpha F6H, 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-ppalpha F6H. We previously reported that cytosolic Hsc70 associates with ppalpha F in wheat germ extracts (16). Recently, others have shown that Hsc70 can be photocross-linked to ppalpha F translated in reticulocyte lysates (15). The translocation competence of mitochondrial precursor proteins is also maintained by cytosolic Hsp70 chaperones likely through direct association (44).

Ydj1p was previously shown to stimulate the ATPase activity 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-ppalpha F6H aggregation and maintained its translocation competence, it, unlike Ssa1p, did not stimulate translocation of AP-ppalpha F6H upon dilution into complete reactions containing microsomes. This result suggests that Ydj1p may prevent AP-ppalpha F6H aggregation less effectively than Ssa1p. Results reported here demonstrating that AP-ppalpha F6H can be post-translationally 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 obtained from in vivo experiments showing that ppalpha F is efficiently exported from ydj1 null strains (13, 22). In such strains, however, ppalpha F 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 ppalpha F·Ssa1p 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-translated ppalpha F into yeast microsomes. DnaK and GrpE reversed the inhibition. In contrast, we found that Ydj1p only partially inhibited translocation of AP-ppalpha F6H even when present at high concentrations. Ydj1p may inhibit post-translational translocation by competing with Ssa1p for binding sites on ppalpha F6H or by stabilizing ADP forms of the Ssa1p·ppalpha F6H 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 ppalpha F, whereas the ppalpha F 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 together or in parallel in post-translational translocation of presecretory 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 monopolize this pathway if they recognized overlapping sites on ppalpha F. 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 temperature-sensitive mutant of Ydj1p accumulated ppalpha F at 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 ppalpha F and its glycosylated intermediates at 23 °C (13). Third, mutants of Ssa1p containing amino acid substitutions in the ATP-binding domain failed to bind unfolded polypeptides, did not stimulate post-translational translocation of ppalpha F 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 nonpermissive conditions (13, 21) or lacking the full complement of cytosolic Hsp70 chaperones at permissive temperatures (13), contain insufficient amounts of Ssa1p to support post-translational translocation. In our in vitro experiments, Ydj1p did not enhance the ability of Ssa1p to prevent ppalpha F aggregation, to maintain translocation competence of ppalpha F, or to stimulate post-translational translocation. The normally beneficial cooperation between Ssa1p and Ydj1p during protein folding (26) may be unnecessary and counterproductive in the post-translational translocation of unfolded ppalpha F.

    ACKNOWLEDGEMENTS

We thank Irina Kovatch and David Jin for technical assistance, Julie Rushbrook for amino acid sequence analysis, and Betty Craig and Don Oliver for strains.

    FOOTNOTES

* 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. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 718-270-1308; Fax: 718-270-3732; E-mail: william.chirico@downstate.edu.

Published, JBC Papers in Press, December 19, 2002, DOI 10.1074/jbc.M210544200

    ABBREVIATIONS

The abbreviations used are: Hsp70, 70-kDa heat shock protein; AP-ppalpha F6H, affinity purified, histidine-tagged prepro-alpha -factor; H, hexokinase; Hsc70, 70-kDa heat shock cognate protein; NEM, N-ethylmaleimide; NEM-Ssa1p, N-ethylmaleimide-modified Ssa1p; Nt, nondenatured ppalpha F6H; PRS, postribosomal supernatant; palpha F, pro-alpha -factor; palpha F6H, histidine-tagged pro-alpha -factor; ppalpha F, prepro-alpha -factor; ppalpha F6H, histidine-tagged prepro-alpha -factor; S, Ssa1p; Y, Ydj1p; Ni2+-NTA, nickel-nitrilotriacetic acid.

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