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INTRODUCTION |
Molecular chaperones such as members of the 70-kDa class (Hsp70s)
bind to nonnative conformations of proteins, thus facilitating their
folding and translocation across membranes (1, 2). The C-terminal
28-kDa region of Hsp70s binds unfolded polypeptides, whereas the highly
conserved N-terminal 44-kDa domain regulates that binding through its
interaction with adenine nucleotides (3). It is thought that Hsp70
proteins, like many GTPases, have a two-state conformation. When an ADP
molecule is bound in the nucleotide-binding site, the Hsp70 exhibits
relatively stable polypeptide substrate binding; when ATP is bound,
binding of substrate is relatively unstable. The 44-kDa domain has a
low intrinsic ATPase activity; therefore, ATP hydrolysis converts Hsp70
to a form having a relatively stable interaction with unfolded
proteins. Exchange of ADP for ATP results in destabilization of the
interaction. It is thought that a polypeptide first interacts with a
Hsp70 in the ATP-bound state, and then hydrolysis of ATP to ADP
stabilizes this interaction.
This cycle of interaction is facilitated by cochaperones. Procaryotes
and mitochondria contain Hsp40-type cochaperones as well as nucleotide
release factors such as GrpE of Escherichia coli and Mge1 of
Saccharomyces cerevisiae (4, 5). In the simplest scenario,
nucleotide release factors are thought to destabilize the interaction
of unfolded proteins with Hsp70, as release of ADP from a DnaK·ADP
complex can be increased up to 5000-fold by GrpE action (4). However,
the action of Hsp40s such as DnaJ of E. coli is less
well understood. Hsp40s stimulate the ATPase activity of Hsp70s, which
is thought to facilitate their binding to unfolded polypeptide
substrates (6). Hsp40s contain a canonical J domain that interacts with
the ATPase domain of Hsp70s (7). There may also be a site of
interaction of Hsp40s with the C-terminal domain of Hsp70s, since
mutant Hsp70s that show a defect in interaction with peptide substrates
also show a defect in interaction with Hsp40s (8, 9).
In addition to interacting with Hsp70s, many Hsp40s, including E. coli DnaJ, bind unfolded or partially folded polypeptides, preventing their aggregation (10). Based on a variety of in vitro analyses, a model of how Hsp40s and Hsp70s cooperate in protein folding has evolved (2, 3). Polypeptide substrates first bind
Hsp40. Then, the ATPase domain of Hsp70 interacts with Hsp40 via its J
domain. This interaction not only brings the substrate in close
proximity to Hsp70, but it also stimulates hydrolysis of ATP, trapping
the substrate. Subsequent release of nucleotide and rebinding of ATP
destabilize the Hsp70-substrate complex, resulting in its release.
In eucaryotes, all major cellular compartments contain at least one
Hsp70 and one Hsp40. Ssc1, the major Hsp70 of the mitochondrial matrix,
is involved in the translocation of proteins from the cytosol across
the mitochondrial inner membrane to the matrix and their subsequent
folding. Two temperature-sensitive mutants, ssc1-2 and
ssc1-3, have been used extensively in the analysis of the
physiological roles of Ssc1 (11-15). The ssc1-3 mutation causes an amino acid substitution in the ATPase domain that results in
severe defects in translocation. The ssc1-2 mutation, which changes a single amino acid in the peptide binding domain (P442S) (12),
has less severe effects and is therefore more useful in dissecting the
roles of Ssc1 in both translocation and folding. Intragenic suppressors
of ssc1-2, which cause an additional single amino acid
alteration in the peptide binding domain of Ssc1-2, have been
isolated. These suppressor mutations allow robust growth at the
intermediate temperature of 34 °C but do not completely suppress the
growth defect of ssc1-2 above that temperature. Two of
these, Ssc1-201 and Ssc1-202 (D519E and V524I, respectively) have
been analyzed (15). Both suppress the defects in protein translocation
as well as the interaction with the membrane tether Tim44. However,
initial results suggested that the folding defect is not suppressed
(15).
To better understand the role of Ssc1 in protein folding within the
mitochondrial matrix, we have undertaken a characterization of the
biochemical properties of Ssc1, Ssc1-2, and Ssc1-201. We found that
neither the ATPase activity of Ssc1-2 nor of the suppressor protein
Ssc1-201 was stimulated by Hsp40s. In addition, both show significant
defects in their ability to facilitate the refolding of luciferase
in vitro. These results make evident the importance of
Hsp40s in facilitating protein folding in the mitochondrial matrix
in vivo and provide insight into the complex interactions between Hsp40s and Hsp70s.
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EXPERIMENTAL PROCEDURES |
Plasmids and Strains
pRS314-SSC1(His tag) was constructed from pRS314-SSC1 (16) by
inserting six codons encoding histidine at the 3' end of the SSC1 gene using polymerase chain reaction. The segments of
ssc1-2, ssc1-201, and ssc1-202 encoding
mutations were subcloned into this plasmid.
An expression plasmid
(pGEM-Su9-DHFR*)1 that
contained a mutant version of DHFR (C7S, S42C, and D49C), DHFR*, for
use in import and folding assays was constructed. The Su9 and DHFR*
portions were polymerase chain reaction-amplified from pGEM-Su9-DHFR
(15) and DHFR* (17), respectively, using Pfu DNA polymerase
(Stratagene, La Jolla, CA). A PstI/BstXI fragment
of the final polymerase chain reaction product was inserted into
pGEM-Su9-DHFR to replace the wild-type (WT) portion of DHFR. The
plasmid was confirmed by DNA sequencing and restriction enzyme digests.
The yeast strain used for Ssc1 purification was obtained by crossing
WY11 (his3-11, 15 leu2-3, 112 ura3-52
trp1-
1 pep4::HIS3) with
PJ53-52C (trp1-1 ura3-1 leu2-3, 112 his3-11, 15 ade2-1 can1-100 GAL2+ met2-
1 lys2-
2
ssc1
ClaI::LEU2 pRS316-SSC1) followed by sporulation and dissection to generate a strain of the genotype pep4::HIS3 trp1-1
ssc1
ClaI::LEU2 pRS316-SSC1
(QL1). After transforming QL1 with
pRS314-SSC1(His tag) containing mutant or WT versions of
SSC1, cells lacking pRS316-SSC1 were selected on
5-fluoroorotic acid plates. The resulting yeast strains QL2
(ssc1-2), QL3 (ssc1-201), QL4 (ssc1-202), and QL5 (WT
SSC1) have a His-tag version of SSC1 as the only
copy of SSC1 and were used to purify Ssc1 wild-type and
mutant proteins.
Mitochondrial Assays
Mitochondria were isolated from wild-type and ssc1
mutant (ssc1-2 and ssc1-201) yeast strains
grown at 25 °C in YPGlycerol media (1% yeast extract, 2% peptone,
3% glycerol, and 2% ethanol) as described previously (12, 15).
Precursor proteins Su9-DHFR and Su9-DHFR* were synthesized in rabbit
reticulocyte lysate in the presence of [35S]methionine.
Before import, the precursor proteins were denatured by 7 M
urea, and mitochondria were incubated at 37 °C for 15 min to induce
the temperature-sensitive phenotype. The import reactions and
subsequent immunoprecipitations using Ssc1 antibodies were carried out
as described (15). Briefly, after import for 2 min at 25 °C, the
import reaction was stopped by the addition of valinomycin. Mitochondria were further incubated at 25 °C for various times then
lysed with Triton X-100 buffer (100 mM NaCl, 10 mM Tris-HCl, pH 7.5, 5 mM EDTA, 0.1% Triton
X-100). After a clarifying spin, the extract was incubated with Ssc1
antibody cross-linked to protein A-Sepharose beads at 4 °C for
1 h. The imported proteins bound to the protein A-Sepharose beads
were analyzed by SDS-polyacrylamide gel electrophoresis and digital autoradiography.
To assess folding of imported proteins, a 2-min import reaction was
carried out. Mitochondria were then disrupted with 0.6% Triton X-100
and treated with proteinase K (15 µg/ml) (Roche Molecular Biochemicals) for 15 min on ice. After the digestion was stopped by the
addition of phenylmethylsulfonyl fluoride, proteins were precipitated
with 5% trichloroacetic acid and analyzed by SDS-polyacrylamide gel
electrophoresis and digital autoradiography.
Protein Purification
The yeast strains QL2, QL3,
QL4, and QL5 were grown at 25 °C in YPGlycerol
media. Cells were harvested at A600 = 1.0-1.5, and mitochondria were isolated as described previously with some modifications (12). Briefly, after treatment with zymolyase (0.45 mg/ml) (ICN Biomedicals, Costa Mesa, CA), the cells were homogenized in
homogenization buffer (0.6 M sorbitol, 10 mM
Tris-HCl, pH 7.5, and 1 mM phenylmethylsulfonyl fluoride).
Mitochondria were separated from unbroken cells and nuclei by
2,300 × g centrifugation for 5 min and pelleted from
other cytosolic components by 20,000 × g
centrifugation for 12 min. The mitochondria pellet was resuspended in
0.5% Triton X-100 in IMAC buffer (20 mM Hepes-KOH, pH 7.4, 150 mM KCl, 2.5 mM magnesium acetate, 20 mM imidazole, 10% glycerol) with 1 mM
phenylmethylsulfonyl fluoride. The extract was loaded onto a nickel
column (Novagen, Madison, WI) equilibrated with IMAC buffer. After
washing with IMAC buffer including 1 mM ATP, 1 M KCl, the column was eluted with an imidazole gradient
(20-240 mM in IMAC buffer). The fractions containing Ssc1
protein were pooled and loaded onto a Q-Sepharose column (Amersham
Pharmacia Biotech) equilibrated with low salt buffer (20 mM
Hepes-KOH, pH 7.4, 50 mM KCl, 10% glycerol). After washing
with low salt buffer, Ssc1 was eluted with high salt buffer (20 mM Hepes-KOH, pH 7.4, 240 mM KCl, 10%
glycerol). The fractions containing Ssc1 protein were pooled, frozen in
liquid nitrogen, and stored at
75 °C. Mge1, Mdj1, and DnaJ
were purified as described before (9, 16, 18-20).
Fluorescence Anisotropy Peptide Binding Assay
Kd Measurement--
Peptide P5 (CALLLSAPRR) was labeled
with fluorescein to generate F-P5 as described (21). Various
concentrations of Ssc1 proteins were incubated with F-P5 (10 nM) at 25 °C in buffer A (25 mM Hepes-KOH,
pH 7.4, 100 mM KCl, 11 mM magnesium acetate, 10% glycerol). After binding reached equilibrium, anisotropy
measurements were made with the Beacon 2000 fluorescence polarization
system (Panvera, Madison, WI) at 25 °C with excitation at 490 nm and emission at 535 nm. The data were fitted to a quadratic single site
binding equation by using Microsoft EXCEL to calculate the Kd.
In experiments utilizing ADP, Ssc1 was incubated with 500 µM nucleotide for 15 min in buffer A. Similar results
were obtained with Ssc1 proteins with and without a 37 °C
preincubation and in the absence of ADP. For the binding in the
presence of ATP, Ssc1 proteins were incubated with 2 mM ATP
at 25 °C for 30 s in buffer A, then F-P5 was added. By 2 min,
before significant hydrolysis occurred, binding equilibrium was
reached, and anisotropy readings were taken.
koff Measurement--
F-P5 (10 nM) was
incubated with Ssc1 proteins at a concentration of ~5
µM. At this Ssc1 concentration, >80% F-P5 was bound to
wild-type and mutant Ssc1 proteins at equilibrium. After the addition
of a 1000-fold excess of unlabeled P5 to the reaction, anisotropy
measurements were taken every 10 s to monitor the release of F-P5.
The release rate koff was calculated by fitting
the release curve to one phase exponential decay using Prism 2.0 (GraphPad, San Diego, CA).
Single-turnover ATPase Assays
Ssc1·ATP complexes were isolated as described (16), with
several modifications. 25 µg of Ssc1 protein was incubated with 100 µCi of [
-32P]ATP (DuPont, 3000 Ci/mmol) in buffer A
containing 25 µM ATP in a 100-µl final volume on ice
for 4 min. The complex was isolated immediately on a NICK column
(Amersham Pharmacia Biotech). Single-turnover ATPase assays were
performed as described (16) at 25 °C in buffer A in the presence of
different proteins (Mge1, Mdj1, or DnaJ) and peptide P5 at various
concentrations. At the indicated times, the reaction was stopped, and
the percent conversion of ATP to ADP was determined. The rate of ATP
hydrolysis was calculated by fitting the data to a first-order rate
equation by nonlinear regression analysis using Prism 2.0.
Luciferase Refolding Assay
Firefly luciferase (4 µM) was denatured for 3 h at 30 °C in buffer B (40 mM Tris-HCl, pH 7.4, 50 mM KCl, 1 mM dithiothreitol, 15 mM
magnesium acetate) containing 6 M urea. For refolding,
diluted luciferase (50 nM) was incubated at 25 °C for
2 h in buffer B (40 µl) supplemented with ATP (5 mM), an ATP regenerating system (10 mM
phosphocreatine, 100 µg/ml phosphocreatine kinase), 0.15 mg/ml bovine
serum albumin, and chaperone proteins as indicated in the figure
legends. The luciferase activity was determined in a Beckman
scintillation counter using the luciferase assay system E1500 (Promega,
Madison, WI).
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RESULTS |
Ssc1-2 and Ssc1-201 Are Defective in Folding in Both
Mitochondrial and in Vitro Assays--
To study protein folding within
the mitochondrial matrix, DHFR was utilized as a test substrate. A
protein, Su9-DHFR, containing a presequence that directs the protein
synthesized in the cytosol into the mitochondrial matrix fused to the
mouse DHFR was synthesized in reticulocyte lysate in the presence of
[35S]methionine. The synthesized protein was denatured
with urea and imported into mitochondria isolated from wild-type,
ssc1-2, and ssc1-201cells. In agreement with
previous results (15), this unfolded protein was imported into
ssc1-2 as efficiently as into mitochondria from wild-type
or ssc1-201 cells, as very similar amounts of radiolabeled
protein were imported into the 3 mitochondria preparations (Fig.
1A). The folding state of the imported DHFR was assessed by testing its susceptibility to digestion by protease. After import, the isolated mitochondria were disrupted by
Triton X-100, and the extracts were treated with proteinase K. 49% of
imported DHFR was proteinase K-resistant in wild-type mitochondria,
whereas only 17% was resistant in ssc1-2 mitochondria. 23% of imported DHFR was resistant in ssc1-201
mitochondria. Therefore, both ssc1-2 and
ssc1-201 mitochondria are defective in the folding of this
test substrate. However, DHFR imported into ssc1-201
mitochondria consistently showed slightly more proteinase K resistance
than that in ssc1-2 mitochondria.

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Fig. 1.
Folding of proteins imported into isolated
mitochondria. After denaturation with urea, radiolabeled
preprotein Su9-DHFR and Su9-DHFR* were imported into isolated WT,
ssc1-2, and ssc1-201 mitochondria for 2 min.
Mitochondria were lysed, and the folding state of imported DHFR and
DHFR* was accessed by the resistance of the mature protein
(m) to treatment with proteinase K.
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Since we observed a defect in folding in ssc1-2 and
ssc1-201 mitochondria, we wanted to test the ability of the
mutant proteins to function in the refolding of denatured proteins
in vitro. First we developed a purification scheme for
wild-type and mutant Ssc1 proteins. We utilized an SSC1 gene
encoding a C-terminal polyhistidine extension. These constructs rescued
the lethality of the
ssc1 mutation, and strains carrying
His-tagged mutant versions maintained the same temperature-sensitive
growth phenotype as those carrying untagged genes (data not shown).
Under the conditions used, refolding of denatured luciferase was
dependent upon the presence of Ssc1, the mitochondrial Hsp40 Mdj1, and
the nucleotide exchange factor Mge1 as well as Hsp78, a member of the
Hsp100 family of chaperones that facilitates disaggregation of protein
aggregates (22, 23). In the absence of Ssc1, less than 1% of
luciferase activity was recovered, whereas in the presence of Ssc1,
~60% of luciferase activity was found as compared with the native
luciferase (Fig. 2A). However,
Ssc1-2 was not able to significantly facilitate the refolding of
luciferase under any condition tested. In addition, no activity of
Ssc1-201 was observed at low concentrations of Ssc1, where the
refolding activity of wild-type Ssc1 is nearly maximal (1 µM). However, at increasing concentrations, some
refolding activity was observed, reaching about 45% maximal wild-type
activity at 6 µM. Therefore, although the suppressor
protein has some refolding activity, it is severely compromised
compared with wild-type protein. This result is consistent with the
slightly increased level of resistance of imported DHFR to protease in
ssc1-201 mitochondria compared with ssc1-2
mitochondria, even though the level of Ssc1 protein is similar in all
three types of mitochondria (data not shown).

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Fig. 2.
Chaperone-mediated refolding of firefly
luciferase. A, refolding of chemically denatured
luciferase was examined in the presence of Hsp78 (1 µM),
Mdj1 (0.2 µM), Mge1 (0.1 µM), and different
concentrations of wild-type Ssc1 (WT), Ssc1-2, or Ssc1-201
as indicated. B, refolding of luciferase was investigated in
the presence of wild-type Ssc1 (WT) (2 µM), Ssc1-2 (2 µM), or Ssc1-201 (2 µM), Mge1 (0.1 µM), Hsp78 (1 µM), and different
concentrations of Mdj1. 100% of activity corresponds to measurements
made with untreated native luciferase.
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Ssc1-2 and Ssc1-201 Have a Prolonged Interaction with Imported
Protein--
Ssc1 interaction with imported proteins can be monitored
in mitochondria by testing for the ability of Ssc1-specific antibodies to coimmunoprecipitate radiolabeled imported protein. In wild-type mitochondria, this association is transient. Consistent with previously published results (15, 24), only a low percentage of imported protein,
about 5%, was immunoprecipitated immediately after import (Fig.
3A). With time, that amount
decreased, to 1-2% by 15-35 min after import. Significantly larger
amounts of DHFR were coimmunoprecipitated in ssc1-2 and
ssc1-201 mitochondria. Initially 12-15% was precipitated; by 35 min after import, 7-9% was precipitated.

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Fig. 3.
Interaction of Ssc1 with imported
proteins. Wild-type (DHFR) and mutant (DHFR*) versions of
Su9-DHFR, panels A and B, respectively, were
imported into WT, ssc1-2, and ssc1-201
mitochondria as described in the legend to Fig. 1. After inhibition of
further import by the addition of valinomycin, the mitochondria were
incubated at 25 °C for various times ( t). One aliquot
of the sample taken at zero time was analyzed by SDS-polyacrylamide gel
electrophoresis and autoradiography (lanes 1-3) to
determine the amount of protein imported. The samples taken at other
time points were lysed and subjected to immunoprecipitation with
anti-Ssc1 antibody (lanes 4-16). The lower panels in
A and B show the quantification of the autoradiograms.
The total amount of imported protein was set to 100% in each
case.
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Thus, ssc1-2 and ssc1-201 mitochondria showed a
defect in folding of DHFR and a prolonged association with imported
protein. Two possible explanations for the prolonged association came
to mind. First, Ssc1-2 and Ssc1-201 might have an increased affinity for unfolded proteins. Perhaps the substrates are released more slowly
once they have bound to the mutant Hsp70, causing this prolonged
association. Alternatively, the mutant Hsp70s might have normal
interaction with unfolded proteins but be defective in other
interactions, resulting in a defect in folding. Proteins would thus
remain in a partially unfolded state for a long period of time and
remain substrates for Ssc1 binding. In this case each DHFR molecule
would undergo many more cycles of interaction with mutant Ssc1
proteins, causing an apparent prolonged interaction. We proceeded to
test these ideas.
Ssc1-2, but Not Ssc1-201, Has a Lowered Affinity for Peptide
Substrate in the ADP-bound Form but Not the ATP-bound Form--
To
examine substrate binding properties, wild-type, Ssc1-2, and Ssc1-201
proteins were tested in a fluorescence anisotropy-based peptide binding
assay. This assay has been previously used to assess the interaction of
peptide substrates with DnaK (9, 21). For studies of Ssc1, a model
peptide P5 (CALLLSAPRR), having a portion of the
mitochondrial-targeting sequence of aspartate aminotransferase from
chicken, was selected. P5 was fluorescently labeled on its N-terminal
cysteine with fluorescein (F-P5). The anisotropy assay follows the
relative rotational diffusion of the fluorescein after excitation with
polarized light. Because of its small size, F-P5 should rotate in
solution rather rapidly and, thus, have a low anisotropy value. When
F-P5 is bound to Ssc1, it should rotate more slowly and, thus, display
a significantly higher anisotropy value.
Binding assays were performed using increasing concentrations of Ssc1
protein, and the increase in anisotropy was fitted to a single-site
binding model. Analysis of wild-type Ssc1 binding experiments, carried
out in the presence of ADP, yielded a dissociation constant
(Kd) of 0.22 (±0.036) µM (Fig.
4, A and B). Ssc1-2 showed about a 5-fold lower affinity for peptide, having a
Kd of 1.1 (±0.095) µM. However, the
affinity of Ssc1-201 for peptide was only slightly lower than that of
wild-type, having a Kd of 0.28 (±0.031)
µM. Therefore, the suppressor mutation appears to have
significantly reversed the defect of Ssc1-2 in binding peptide
substrate.

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Fig. 4.
Fluorescence anisotropy assay of peptide
binding and release. A, F-P5 (10 nM) was
incubated in the presence of the indicated concentrations of wild-type
Ssc1 (WT), Ssc1-2, or Ssc1-201 proteins as described under
"Experimental Procedures." Anisotropy measurements were taken at
25 °C. Raw polarization values were plotted as a function of
increasing Ssc1 concentration. B, data of F-P5 binding in
the presence of ADP from panel A were fitted to a quadratic
single-site binding equation to determine the Kd for
each Ssc1 protein. Data are expressed as the fraction of F-P5 bound to
Ssc1 protein. C, each Ssc1 protein (~5 µM)
was incubated with F-P5 (10 nM) for 2 h to achieve
binding equilibrium in the presence of 500 µM ADP. At
zero time, a 1000× excess of unlabeled P5 was added. The release of
bound F-P5 was monitored by anisotropy measurement every 10 s.
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To look at the defect of Ssc1-2 in more detail, we monitored the
displacement of the prebound F-P5 after the addition of a large excess
of unlabeled P5 by measuring the decrease in anisotropy. Upon the
addition of P5 to a wild-type Ssc1·F-P5 complex, the anisotropy
change showed a single phase with a rate constant of 0.0024 (±0.000022) s
1 (Fig. 4C).
Experiments with Ssc1-2 revealed a very similar
koff (0.0028 (±0.000059)
s
1). This small difference in
koff observed using ADP-bound Ssc1-2 cannot
account for the 5-fold difference in Kd observed. Therefore, we conclude that this difference in Kd is due to a difference in the on-rate of peptide. Similar values were
obtained when the experiments were performed in the absence of nucleotide.
We also tested the suppressor protein Ssc1-201 to determine the basis
of the restoration of the Kd to wild-type levels. The koff was decreased about 2.2-fold compared
with wild-type protein, 0.0011 (±0.0000062)
s
1 compared with 0.0024 s
1. Therefore, to obtain a
Kd similar to wild-type, the kon of Ssc1-201 would need to be 2.8-fold
slower than that of wild type. We proposed that both the
kon and koff are affected by the suppressor mutation, resulting in a nearly normal affinity.
The affinity of Hsp70 for peptide substrates is much lower in the
ATP-bound form than in the ADP-bound form (25, 26). As expected, upon
the addition of ATP, the anisotropy readings rapidly decreased,
indicating peptide release. Because of the rapidity of this reduction,
we were unable to compare the rate of release of peptide upon ATP
addition among the different proteins.
To look more carefully at the interaction of Ssc1 with peptide when it
is in the ATP-bound state, we carried out anisotropy assays in the
presence of ATP. As expected, in the presence of ATP, the affinity for
peptide of both wild-type Ssc1 and Ssc1-2 was dramatically reduced
(Fig. 4A). Because of this reduction we were unable to
approach saturation of binding. However, the affinity of wild-type Ssc1
and Ssc1-2 appears similar, based on the increase in anisotropy
observed at high concentrations of protein. We estimate that the
Kd of both wild-type and Ssc1-2 proteins in the
ATP-bound form is on the order of 20 µM, if at saturation
the anisotropy reading is similar to that of the ADP-bound form.
Therefore, there is about a 90-fold difference between the affinity of
the ATP and ADP form of wild-type protein for P5. This difference is
similar to the difference in affinity of ATP and ADP-bound DnaK for
peptides (25, 27, 28). In summary, our results indicate that wild-type
Ssc1 and Ssc1-2 have similar affinity for peptide substrate in the
presence of ATP. However, in the presence of ADP, the mutant Ssc1-2
protein has a lower affinity than wild-type protein. The suppressor
mutation in ssc1-201 substantially corrects this decreased
affinity found in the ssc1-2 protein.
Unfolded DHFR Has a Prolonged Association with Both Wild-type and
Mutant Ssc1 Proteins--
Since the analysis of purified proteins
provided no indication that the mutant proteins had a higher affinity
for peptide substrates, we tested the idea that the failure of DHFR to
fold in the mitochondrial matrix would result in a prolonged
association of wild-type Ssc1. We took advantage of a previously
characterized mutant of mouse DHFR (DHFR*), which encodes three
structurally destabilizing amino acid alterations (29). These amino
acid changes result in an increased susceptibility to protease and diminish the ability of methotrexate, which stabilizes the structure of
wild-type DHFR to block import into mitochondria (17). Upon import into
mitochondria the mutant DHFR was extremely susceptible to digestion
with protease (Fig. 1B). In addition, 30-40% of the mutant
DHFR could be immunoprecipitated using Ssc1-specific antibodies, with
only a slight decrease over time (Fig. 3B). Therefore a
protein known to be defective in folding has a prolonged association
with both mutant and wild-type Ssc1. We conclude that the prolonged interaction observed with Ssc1-201 (and Ssc1-2) mitochondria is a
direct result of the failure of DHFR to fold, thus remaining a
substrate for extended periods of time.
The ATPase Activity of Both Ssc1-2 and Ssc1-201 Are Stimulated
Normally by Peptide--
Hsp70s have two basic activities: binding
hydrophobic peptide segments and ATPase activity. Since the peptide
binding ability of the mutant proteins does not appear to explain the
protein folding defect of Ssc1-2 and Ssc1-201, we analyzed ATPase
activities. For this purpose we used a single turnover assay,
monitoring the hydrolysis of prebound radiolabeled ATP. The ATPase
activity of the three proteins differed less than 2-fold. Wild-type
Ssc1 had a rate constant of 0.063 (±0.0039)
min
1, whereas the observed rate constants for
Ssc1-2 and Ssc1-201 were 0.10 (±0.019) and 0.12 (±0.014)
min
1. This rate is similar to that found for
other Hsp70s (30).
Binding of peptide stimulates the rate of ATP hydrolysis of Hsp70s (31,
32). Therefore, we compared stimulation of wild-type Ssc1 and the
mutant proteins by P5 over a range of concentrations. As can be seen in
Fig. 5A, the ability of
peptide to stimulate the hydrolysis of ATP by the wild-type and mutant
proteins is similar. This result is consistent with the anisotropy
results described above, indicating that Ssc1-2 and wild-type protein have a similar affinity for peptide in the ATP-bound form.

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Fig. 5.
Stimulation of ATPase activity of Ssc1
proteins by P5, Mdj1, and DnaJ. Ssc1-ATP complex (~1
µM) was incubated at 25 °C in the presence of various
concentrations of P5 (A), Mdj1 (B), or DnaJ
(C) as indicated. Samples were removed at 0, 1.5, 3, 6, 11, and 22 min from each reaction, and the fraction of ATP converted to ADP
was determined. The rate of ATP hydrolysis was calculated using Prism
2.0. Fold stimulation was calculated by setting the intrinsic ATP
hydrolysis rate as one.
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Hsp40 Stimulation of the ATPase Activity of Both Ssc1-2 and
Ssc1-201 Are Defective--
Ssc1 functions with co-chaperones Mge1
and Mdj1 (33-35). To assess the nucleotide release activity of Mge1,
we used a single turnover ATPase assay, monitoring the hydrolysis of
radiolabeled ATP prebound to Ssc1. This assay is based on the idea that
release of nucleotide from Ssc1 facilitated by Mge1 will cause a
decrease in hydrolysis of the radiolabeled ATP. Since an excess of
unlabeled ATP is included in the reaction, released radiolabeled
nucleotide will only rarely rebind Ssc1. As previously published (5,
16), the addition of nonradioactive ATP to the reaction had little effect on the hydrolysis of the prebound ATP, indicating that the
nucleotide remains bound throughout the time course of the reaction
(data not shown). However, as previously reported, the addition of
wild-type Mge1 reduced the hydrolysis of radiolabeled ATP when
unlabeled nucleotide was added to prevent rebinding of released
radiolabeled nucleotide (Fig. 6). In the
presence of Mge1 and unlabeled ATP, ADP formation reached a plateau at
only 10% hydrolysis. Very similar results were obtained with Ssc1-2 and Ssc1-201, indicating that these mutant proteins were not defective in their interaction with Mge1.

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Fig. 6.
ATP release activity of Mge1 on Ssc1 proteins
in single-turnover ATPase assays. Ssc1-ATP complex (~1
µM) was incubated at 25 °C in the presence or absence
of Mge1 (6 µM) and in the presence of 250 µM ATP. Samples were withdrawn at the indicated times,
and the fraction of ATP converted to ADP was determined. Open
circles, no Mge1 added; closed circles, Mge1 added.
A, WT Ssc1. B, Ssc1-2. C,
Ssc1-201.
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To assess the functional interaction of Hsp40s with Ssc1, we again
utilized the single turnover ATPase assay, as it is well established
that Hsp40s stimulate the hydrolysis of ATP by Hsp70s (32, 36). We
observed a 6-fold stimulation of wild-type Ssc1 upon the addition of
Mdj1 at a concentration of 4 µM; DnaJ stimulated Ssc1
more than 12-fold at the same concentration (Fig. 5, B and C). However, at the same concentration, Mdj1 did not
measurably stimulate the ATPase activity of either Ssc1-2 or
Ssc1-201, whereas DnaJ stimulated less than 2-fold. Thus, both Ssc1-2
and the suppressor protein Ssc1-201 are defective in their interaction
with Hsp40s. In addition, a second suppressor, Ssc1-202, was purified
and analyzed. In this, and other assays described above, Ssc1-202
behaved very similarly to Ssc1-201 (data not shown).
We tested the effect of increasing concentrations of Mdj1 in in
vitro assay to determine whether increasing the concentration of
Mdj1 would overcome the folding defect of Ssc1-2 or Ssc1-201. However, at higher concentrations, Mdj1 was inhibitory, even in the
case of wild-type Ssc1 (Fig. 2B). Perhaps at higher
concentrations more Mdj1 binds to the denatured luciferase, perhaps
inhibiting aggregation but inhibiting a productive folding reaction as well.
 |
DISCUSSION |
Our goal in this study was to better understand the mechanism of
action of molecular chaperones in protein folding in the mitochondrial
matrix. Both mutant Ssc1 proteins studied here, Ssc1-2 and Ssc1-201,
are defective in protein folding in isolated mitochondria as well as in
in vitro folding assays. In addition, both are impaired in
their functional interaction with Hsp40, suggesting not only a
requirement for Hsp40 in the mitochondrial matrix for folding of some
proteins but also a requirement for an interaction between the two
chaperones. This idea is in agreement with previous analyses, as
mitochondria lacking Mdj1 are defective in the folding of imported DHFR
(37). However, even though an interaction between Mdj1 and Ssc1 has
been detected in isolated mitochondria (35), little is understood about
its importance.
Several studies of the E. coli system indicate the
importance of a physical interaction between DnaK and DnaJ. But the
nature of the interaction between Hsp40 and Hsp70 is complex and not fully understood. Evidence suggests an interaction between DnaJ and
both the ATPase and peptide binding domains of Hsp70. The interaction
between the J domain and the Hsp70 ATPase domain is well established.
NMR studies revealed such an interaction (7), and amino acid
alterations in a groove of the DnaK ATPase domain resulted in a
decrease in DnaJ interaction and a decrease ability to refold
luciferase in vitro (38). In addition, an amino acid alteration on the same face of the ATPase domain of DnaK as this groove
was found to suppress not only the temperature-sensitive in
vivo defect caused by an alteration in the J domain of DnaJ but
also the defective physical interaction between the mutant DnaJ and
DnaK (8).
The amino acid alterations in Ssc1-2 and Ssc1-201 occur in the
peptide binding domain. An interaction between Hsp40 and the peptide
binding domain of Hsp70 occurs in vitro. However, the question of the biological relevance of this interaction remains open.
Several dnaK mutant proteins having amino acid alterations in the peptide binding domain resulting in a lowered affinity for
peptide substrates have been tested for DnaJ interactions. All tested
mutants have an impaired interaction with DnaJ (8, 9, 21, 39). Several
issues are unresolved. It remains unclear whether in vitro
Hsp40 binds as a substrate in the peptide binding cleft or if there is
a unique (or overlapping) site in the C terminus at which a Hsp40
binds. Also, the interaction of Hsp40s with the peptide binding region
may be an in vitro artifact, not a biologically relevant
interaction. At any rate, for in vitro detection of most Hsp40-Hsp70 interactions, an interaction of the Hsp40 at or near the
peptide binding cleft of Hsp70 appears to be required.
Ssc1-201 is unusual in that it is defective in interaction with Hsp40s
even though interaction with peptide substrates appears normal, as
measured by affinity for peptide and the ability of peptide to
stimulate hydrolysis of Hsp70-bound ATP. Therefore, for the first time,
an amino acid alteration in the peptide binding domain separates
peptide binding from interaction with Hsp40. Two different explanations
could account for this separation. First, this alteration could be
revealing the site of interaction with Hsp40 that is distinct (or
perhaps overlapping) with the site of interaction with peptide. If so,
our results suggest that the interaction of Hsp40 with the peptide
binding domain is biologically meaningful. Alternatively, the
alterations might alter the interaction between the ATPase and peptide
binding domains such that the interaction of the J domain with the N
terminus is affected. There are several arguments against this second
possibility as some aspects of communication between the domains are
normal in both Ssc1-2 and Ssc1-201. Peptide binding stimulates the
ATPase activity of the N-terminal domain normally, and binding of ATP
results in the rapid dissociation of peptide substrates. In addition,
both Ssc1-2 and Ssc1-201 seem able to undergo normal conformational
changes in response to nucleotide binding as measured by tryptophan
fluorescence (data not shown). At the moment, technical limitations
prohibit us from definitively discriminating between these two
alternatives. However, mutants such as ssc1-201 may be
useful in dissecting the importance of the interaction of Hsp40s with
the peptide binding domain of Hsp70 in the future.
An interesting characteristic of the Ssc1-2 protein is its altered
binding to peptide substrates. Our data indicate that peptide binding
is normal in the ATP but not the ADP-bound form. In the ADP-bound form,
Ssc1-2 has a 5-fold lower affinity for a peptide substrate. This
reduced affinity is mainly due to a decreased on-rate. However,
interaction with the ATP form appears normal, as indicated by
stimulation of ATPase by peptides and the association of fluorescent
peptide with the ATP-bound form of the protein. Extensive work by Bukau
and co-workers (27) has recently shown a positive correlation
between the affinity of binding of substrates to ADP-bound DnaK and the
ability of these substrates to stimulate ATPase activity. This
correlation held regardless of whether DnaK lacked the
-helical lid
over the peptide binding domain or had alterations in either the
"arch" over the peptide binding cleft or in the cleft itself (27).
Our preliminary analysis with alterations in the peptide binding domain
of Ssc1 has indicated a similar correlation; a decrease in affinity for
peptide substrates in the ADP form is accompanied by a decreased
ability of peptides to stimulate ATP hydrolysis and function in
vivo (data not shown). Therefore, Ssc1-2 may represent a new
class of peptide binding mutants, and the ability to isolate second
site suppressors may indicate that this class of mutants will be
powerful in dissecting the importance of the ADP-bound form of Hsp70s
in protein folding.
According to the models of the cycle of interaction between substrate
polypeptides and Hsp70s/Hsp40s (2, 3), the on-rate of substrates for
the Hsp70·ATP form and the off-rate in the Hsp70·ADP form
are the important parameters for the cycle. Accordingly, the on-rate in
the Hsp70·ADP form should be inconsequential, as binding occurs only
in the ATP-bound form, which is then converted to the high affinity
Hsp70·ADP form by stimulation of hydrolysis. Our data raise the
question as to whether the binding to substrate in the ADP-bound form
has some physiological role. In fact, the ssc1-201
suppressor mutation does allow some recovery of in vitro folding activity although still far from wild-type levels. The mutants
studied here as well as others isolated in the future having similar
differential effects on the interaction of substrates with the two
nucleotide-bound forms should allow this question to be addressed.
In summary, our results suggest that the defect in protein folding in
the mitochondrial matrix of a Hsp70 mutant is due to a defective
interaction with Hsp40. Such comparisons between defects in the complex
milieu of the mitochondria and analysis of biochemical defects found in
purified systems will be important in understanding the mechanism of
action of molecular chaperones in complex physiological processes such
as protein translocation and folding.