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INTRODUCTION |
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-
-factor (pp
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 pp
F is
translocated normally in deletion mutants of YDJ1.
Translocation of pp
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
pp
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 pp
F, which had accumulated at the nonpermissive
temperature, could not be translocated. These results suggested that
the chaperones maintain translocation competence by binding to pp
F
co-translationally, but they cannot rescue aggregated pp
F. Strains
containing mutant versions of Ssa1p with defective ATP-binding pockets
fail to interact productively with Ydj1p and accumulate pp
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 pp
F, which was synthesized in
wheat germ extracts or reticulocyte lysates, we used a
histidine-tagged, affinity purified version of pp
F (AP-pp
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-pp
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-pp
F6H aggregation.
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EXPERIMENTAL PROCEDURES |
Plasmids--
pKWC1, which can express pp
F6H in E. coli, was constructed by amplifying the cDNA for pp
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
pp
F6H, was constructed by amplifying the cDNA for pp
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 Pp
F6H--
E. coli IQ85 (F'
araD139
(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-
-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]pp
F6H was purified as described below.
In Vitro Transcription and Translation--
Messenger RNA coding
for pp
F or pp
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 pp
F and pp
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]Pp
F6H was purified from either wheat germ
translation reactions programmed with pp
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]Pp
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]Pp
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]pp
F6H was eluted as described above. 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 manner as
pp
F6H. Protein concentrations were determined using the Bradford
assay (34) and bovine
-globulin was used as the standard.
Protein Sequencing--
A 1.75-µg sample of purified p
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/
ade1/+ +/ade2 +/his1), MYY290
(MATa leu2 his3 ura3) (22), and MYY406
(MAT
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]pp
F or
[35S]pp
F6H. When denatured [35S]pp
F6H
(or [35S]pp
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]pp
F6H (or [35S]pp
F) or 0.3 µl
of affinity purified [35S]pp
F6H, respectively.
Denatured substrate (~15,000 dpm) was added last and then reactions
were mixed immediately. All pipette tips used to transfer denatured
pp
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]pp
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]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 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 pp
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 pp
F--
AP-[35S]pp
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]pp
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]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]pp
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 |
Purification of Pp
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-pp
F6H as the substrate. Pp
F used previously contained
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 altered to avoid the
possibility of compromising the signal sequence at the N terminus.
Pp
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-
-factor (p
F6H) was formed (data not shown) indicating that
the signal sequence cleavage site of pp
F6H was recognized by leader
peptidase as previously reported for authentic pp
F (37). Sequencing
the N terminus of p
F6H revealed that it was identical to that of
authentic p
F (data not shown) (38). A mixture of purified pp
F6H
and p
F6H was also electrophoresed (Fig. 1, lane 4).
Pp
F6H, like authentic pp
F (38), migrated faster than p
F6H on
SDS-PAGE gels.

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Fig. 1.
Purification of
pp F6H. Proteins from
isopropyl-1-thio- -D-galactopyranoside-induced
(lane 2) and uninduced (lane 1) E. coli IQ85 (SecYts) harboring a pp F6H
expression plasmid were separated on a SDS-PAGE gel and then stained
with Coomassie Blue. Pp F6H was affinity purified using
Ni2+-NTA affinity chromatography under denaturing
conditions (lane 3). Lane 4 contains an equal
amount of purified p F6H (upper band) and pp F6H
(lower band). Numbers on the left
represent the molecular mass (kDa) of protein standards.
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Characterization of the Translocation Competence of
Pp
F6H--
We next compared the translocation competence of pp
F
and pp
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 pp
F was removed and p
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 pp
F6H
was synthesized in wheat germ translation reactions and then added to
post-translational translocation assays four radiolabeled species
migrating more slowly than pp
F6H were detected (Fig. 2, lane
6). By analogy to reactions containing authentic pp
F, the new
bands correspond to p
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 pp
F6H and glycosylation of
p
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 pp
F and pp
F6H were essentially identical. The
translocation competence of pp
F6H described here is comparable with
that of another histidine-tagged pp
F reported previously (39).

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Fig. 2.
Post-translational translocation of
pp F6H into yeast microsomes in the presence of
PRS. [35S]Pp F and [35S]pp 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 pp F6H (22 kDa) and p F6H (23 kDa)
that contained one (26 kDa), two (28 kDa), or three (34 kDa) core
oligosaccharides.
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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 microsomes, 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) 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 pp
F and
pp
F6H are essentially identical. They also support the notion that
urea denaturation of pp
F (or pp
F6H) mimics the stimulation of
post-translational translocation by the chaperones in the PRS.

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Fig. 3.
Unfolded pp F6H is
efficiently 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 absence of PRS. Urea-denatured,
affinity purified pp 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.
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Upon dilution from urea, pp
F (or pp
F6H) likely folds rapidly and
loses translocation competence. To test this hypothesis 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 during longer incubations. The
translocation competence of AP-pp
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]pp F6H was diluted into post-translational
translocation reactions lacking microsomes. At the indicated times,
wild-type microsomes were added and the amount of pp F6H translocated
during a 2-min incubation was determined. Each point represents the
mean of the percentage translocation of two experiments.
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Kinetics of Translocation in the Presence of Ssa1p and
Ydj1p--
The availability of AP-pp
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-pp
F6H (Fig. 5).
For reference, the time course of translocation of wheat
germ-synthesized, nondenatured pp
F6H is also shown (Fig. 5,
line Nt). The initial rate of translocation (<20%
translocation) of AP-pp
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-pp
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-pp
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-pp
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-pp
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
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 µM. 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.
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Chaperones Differentially Affect Translocation--
During the
course of post-translational translocation, chaperones may interact at
different stages of precursor folding. We postulate that AP-pp
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-pp
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-pp
F6H with different combinations of
chaperones before adding microsomes. Third, to test restoration of
translocation competence activity we allowed AP-pp
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-pp
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-pp
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-pp
F6H (Fig. 6a, line M). To
distinguish between these two possibilities Ssa1p and microsomes were
added after AP-pp
F6H folded/aggregated. Less than 10% of
folded/aggregated AP-pp
F was translocated (Fig. 6a, line R). Together, these results indicate that
Ssa1p can maintain the translocation competence of AP-pp
F. However,
its ability to restore translocation competence to folded/aggregated
AP-pp
F is negligible.

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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.
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To determine whether membrane-bound Ydj1p cooperates with Ssa1p to
stimulate the translocation of pp
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-pp
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-pp
F6H into
microsomes in the absence or presence of Ssa1p.
In contrast to Ssa1p, Ydj1p slightly inhibited AP-pp
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-pp
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 pp
F6H translocated 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 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 Pp
F6H--
We showed
above that Ssa1p and Ydj1p can maintain the translocation competence of
pp
F6H. To test the hypothesis that they maintain translocation
competence by preventing aggregation we first diluted AP-pp
F6H into
buffer lacking chaperones or containing 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 had
aggregated (Fig. 7A, bars Initial and N). Ssa1p and Ydj1p
increased the solubility of pp
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-pp
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-pp
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-pp
F6H in the
supernatants (Fig. 7B). The translocation efficiency of
AP-pp
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-pp
F6H aggregation.

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Fig. 7.
Ssa1p and Yjd1p stimulate translocation by
preventing aggregation. A,
AP-[35S]pp 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]pp F6H remaining in the supernatant was
determined. A control reaction was not centrifuged
(Initial). B, [35S]pp 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.
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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-pp
F6H, we measured the ability of NEM-Ssa1p to prevent the
aggregation of AP-pp
F6H and to stimulate the translocation of the
AP-pp
F6H remaining in solution. We found that 2 ± 0.2-fold more AP-pp
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-pp
F6H into microsomes
post-translationally (data not shown). Hexokinase neither prevented
aggregation nor maintained the translocation competence of AP-pp
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-pp
F6H, but are required for ensuring the
productive release and translocation of AP-pp
F6H.
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DISCUSSION |
We explored the roles of cytosolic chaperones in
post-translational translocation in vitro using AP-pp
F6H.
In the absence of chaperones, urea-denatured AP-pp
F6H was rapidly
translocated into wild-type or Ydj1p-deficient microsomes upon dilution
into reactions. In the absence of microsomes and chaperones,
AP-pp
F6H aggregated and became translocation incompetent. Ssa1p,
Ydj1p, or both chaperones prevented aggregation and maintained the
translocation competence of some AP-pp
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-pp
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 pp
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-pp
F6H, but failed to
maintain its translocation competence. This is consistent with the idea
that NEM-Ssa1p can bind AP-pp
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-pp
F6H. We previously reported that cytosolic Hsc70 associates
with pp
F in wheat germ extracts (16). Recently, others have shown
that Hsc70 can be photocross-linked to pp
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-pp
F6H aggregation and
maintained its translocation competence, it, unlike Ssa1p, did not
stimulate translocation of AP-pp
F6H upon dilution into complete
reactions containing microsomes. This result suggests that Ydj1p may
prevent AP-pp
F6H aggregation less effectively than Ssa1p. Results
reported here demonstrating that AP-pp
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 pp
F is
efficiently exported from ydj1 null strains (13, 22). In such strains, however, pp
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
pp
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 pp
F into yeast
microsomes. DnaK and GrpE reversed the inhibition. In contrast, we
found that Ydj1p only partially inhibited translocation of AP-pp
F6H
even when present at high concentrations. Ydj1p may inhibit
post-translational translocation by competing with Ssa1p for binding
sites on pp
F6H or by stabilizing ADP forms of the Ssa1p·pp
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 pp
F, whereas the pp
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 pp
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 pp
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 pp
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 pp
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 pp
F aggregation, to maintain translocation
competence of pp
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 pp
F.