From the Department of Biological Sciences and
¶ Departments of Environmental and Occupational Health and of
Pharmaceutical Sciences, University of Pittsburgh,
Pittsburgh, Pennsylvania 15260
Received for publication, September 18, 2000
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ABSTRACT |
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Members of the hsc70 family of molecular
chaperones are critical players in the folding and quality control of
cellular proteins. Because several human diseases arise from defects in
protein folding, the activity of hsc70 chaperones is a potential
therapeutic target for these disorders. By using a known hsc70
modulator, 15-deoxyspergualin, as a seed, we identified a novel
inhibitor of hsc70 activity. This compound, R/1, inhibits the
endogenous and DnaJ-stimulated ATPase activity of hsc70 by 48 and 51%,
respectively, and blocks the hsc70-mediated translocation of a
preprotein into yeast endoplasmic reticulum-derived microsomal
vesicles. Biochemical studies demonstrate that R/1 most likely exerts
these effects by altering the oligomeric state of hsc70.
The ubiquitous hsc70 molecular chaperones bind polypeptides in an
ATP-dependent cycle and facilitate diverse cellular
functions including protein folding, sorting, and translocation
(transport) across organellar membranes (reviewed in Ref. 1).
Specifically, by binding exposed hydrophobic patches in newly
translated proteins, hsc70s maintain secreted proteins in a
translocation-competent state and facilitate proper protein folding by
preventing aberrant interactions. hsc70s also participate in the
quality control process that identifies mis-folded proteins and targets
them for degradation (reviewed in Refs. 2 and 3). In addition, hsc70s
interact with specific native proteins to modulate their oligomeric
state or their interaction with various cofactors (reviewed in Refs. 1,
4, and 5).
Binding and release of polypeptide substrates by hsc70s is governed by
the coordinated actions of their nucleotide- and substrate-binding domains. Polypeptides interact transiently with the substrate-binding domain of ATP-bound hsc70. This interaction stimulates ATP hydrolysis, which results in a conformational change in the substrate-binding domain that increases its affinity for peptide (6, 7). Concomitant with
the exchange of ADP for ATP on hsc70, bound peptides are released and
the cycle repeats. Members of the DnaJ family of molecular
co-chaperones stimulate ATP hydrolysis and promote peptide binding, in
some cases by delivering specific substrates to hsc70 (8, 9). Recent
structural and genetic studies indicate that DnaJ chaperones interact
with the ATPase domain at a site near or coincident with the
peptide-binding domain of hsc70 (10-12).
Because they are involved in protein folding and because several human
diseases arise from protein folding defects (2, 13), the modulation of
hsc70 and DnaJ chaperone activity may be used to combat these
disorders. In addition, cellular hsc70 is co-opted by viral oncogenes
to induce cellular transformation (14-16) and is implicated in the
development of certain tumors (17). Thus, identifying novel
hsc70-regulating compounds could prove beneficial.
One hsc70-interacting compound is 15-deoxyspergualin
(DSG1; see Fig. 1). DSG binds
hsc70 (KD = 4 µM) and stimulates its
ATPase activity (18). We recently found that DSG stimulates the
steady-state ATPase activity of bovine and yeast cytosolic hsc70s by 42 and 22%, respectively, but has no effect on BiP/Kar2p, an hsc70 that
resides in the lumen of the yeast endoplasmic reticulum (ER) (19). In
addition, DSG prevents neither the DnaJ-mediated stimulation of hsc70
ATPase activity nor the ability of hsc70 to bind (and release) peptide
substrates in vitro, even though DSG is hydrophobic and
resembles a peptide (19-21).
DSG is currently being examined as an immunosuppressive agent. The
ability of DSG to reduce tissue rejection in transplant patients may
occur through the inhibition of macrophage function and induction of
cytolytic T and B cells (22). Because the immunosuppressive effects of
some DSG analogs correlate with their binding affinity for hsc70 (21,
23), these activities are thought to be mediated at least in part by
the ability of DSG to modulate the activity of hsc70 (18), In addition,
a recent study suggests that DSG could be the prototype of a new class
of drugs for the treatment of cystic fibrosis. Addition of DSG to cells
expressing a mutant form of the cystic fibrosis transmembrane
conductance regulator ( To identify new and potentially more reactive chemical entities with
hsc70-perturbing effects, we searched the Developmental Therapeutics
Program data base at the National Cancer Institute for DSG-related
compounds. In this study we report the analysis of a compound with some
structural similarities to DSG. This compound, NSC 630668-R/1
(designated R/1), inhibits hsc70 ATPase activity and
hsc70-mediated protein translocation in vitro.
Identification of DSG Analogs--
The structure of DSG
(7-guanidinoheptanoic acid {[4-(3-aminopropylamino)
butylcarbamoyl]hydroxymethyl} amide) and its
GI50 activities in the 60 cell line antitumor screen
performed by the Developmental Therapeutics Program at the
National Cancer Institute were used as seeds to probe for compounds
with similar attributes using the web-based version of the COMPARE
algorithm. In activity-activity comparisons, six compounds with
pairwise correlation coefficients of
R/1 was dissolved in dimethyl sulfoxide (Me2SO) at a
final concentration of 12.2 mM and was stable at 4 °C
for at least 1 month. A 3 mM dilution of the 12.2 mM stock in Me2SO was prepared before each
experiment to avoid precipitation of R/1 upon addition to aqueous solvents.
ATPase Measurements--
Ssa1p, BiP, and Ydj1p were purified as
described previously, and ATPase measurements were performed using
published methods (26-29) except that steady-state reactions
containing R/1 were preincubated 15 min on ice before the addition of
radiolabeled ATP. For single turnover ATPase assays, R/1 was added to
buffer before the addition of ATP-Ssa1p complex, and the reaction was incubated at 30 °C for the indicated times.
In Vitro Translocation Assays--
The synthesis and import
(translocation) into ER-derived vesicles of an unglycosylated form of
yeast prepro-
The integrity of microsomal membranes in the presence of R/1 was
assessed by trypsin accessibility to BiP and translocated yeast
Analysis of R/1-mediated Protein Precipitation--
The
indicated proteins were incubated for 15 min on ice in Buffer A (50 mM HEPES (pH 7.4); 50 mM NaCl; 100 mM dithiothreitol; 2 mM MgCl2; 50 µM ATP) with equal volumes of Me2SO or R/1
(at a final concentration 300 µM) in a total volume of 30 µl. The reactions were then centrifuged at 100,000 × g (40,000 rpm) for 1 h at 4 °C in a TLS-55 Beckman
rotor. Supernatant and pellet fractions were separated and analyzed by
SDS-PAGE followed by silver nitrate staining. BiP and Sse1p were
purified in the Brodsky laboratory (28, 32). Malate synthase, lysozyme,
bovine serum albumin, and citrate synthase were purchased from Sigma.
R/1 Inhibits the Endogenous and J-chaperone-stimulated ATPase
Activity of Ssa1p and BiP--
The therapeutic and biochemical
properties of DSG prompted us to search for additional DSG-like
compounds that could modulate hsc70 activity. One compound, R/1 (Fig.
1), gave a pairwise correlation coefficient of 0.801 using the COMPARE algorithm (see "Materials and
Methods"). A sample of R/1 was consequently obtained from the
Developmental Therapeutics Program at the National Cancer Institute.
To determine whether R/1, like DSG, could modulate hsc70 function, the
steady-state ATPase activity of purified Ssa1p, a yeast cytosolic hsc70
chaperone, was measured in the presence and absence of R/1. At 300 µM, the concentration at which DSG maximally stimulated Ssa1p (19), R/1 caused a 48% decrease in the specific activity of
Ssa1p, from 1.25 nmol·min
We next titrated the effect of R/1 on Ssa1p ATPase activity. In this
assay, half-maximal levels of inhibition were achieved at ~100
µM R/1 (Fig. 3). R/1 also
inhibited the ATPase activity of BiP in a dose-dependent
manner, similar to its effect on Ssa1p (Fig. 3).
R/1 Inhibits Post-translational Translocation in Vitro--
The
post-translational translocation of preproteins into the ER is an
ATP-dependent, chaperone-mediated process that can be recapitulated in vitro using yeast ER-derived microsomes. In
yeast, two hsc70s are involved in this process; Ssa1p binds unfolded polypeptides in the cytosol to retain them in a soluble,
translocation-competent state (34, 35); and BiP, a lumenal ER protein,
"ratchets" or drives translocating peptides through the ER
translocation pore (36). Ssa1p and BiP interact with specific DnaJ
homologs, Ydj1p and Sec63p, respectively, both of which are also
required for post-translocational translocation (26, 31, 33, 37).
To assess the effect of R/1 on this chaperone-mediated process, we
examined the efficiency with which an in vitro translated yeast pre-pheromone, prepro-
Because R/1 is relatively hydrophobic, it might have prevented
translocation by solubilizing microsomal membranes. To exclude this
possibility, we examined the trypsin accessibility of two lumenal
proteins, BiP and translocated p R/1 Oligomerizes hsc70--
Steady-state ATPase assays measure the
ability of a chaperone to bind, hydrolyze, and rebind ATP. Thus, to
determine whether R/1 specifically affects ATP hydrolysis, we performed
single turnover experiments on pre-formed ATP-Ssa1p complexes. This
allows measurement of initial rates of hydrolysis and is therefore more
sensitive than steady-state experiments. As shown previously (29),
hydrolysis of Ssa1p-bound ATP is rapid (Fig.
6). Surprisingly, when R/1 was included
in the reaction, the initial rate of hydrolysis dramatically increased.
At 300 µM R/1, the initial rate of ATP turnover was ~5-fold higher than that for the reaction without R/1, an increase comparable to that seen by addition of peptide substrates to other hsc70s (38). Although previous experiments performed in our laboratory
demonstrated that 95% of the Ssa1p-ATP complex can be hydrolyzed (29),
the rate of ATP hydrolysis leveled off after ~4 min in the presence
of R/1, suggesting that a fraction of Ssa1p became inactivated by R/1.
At lower R/1 concentrations where translocation was compromised (Fig.
4), the rate of ATP hydrolysis was again enhanced (2.5- and 4.5-fold at
10 µM and 30 µM R/1, respectively), but
after 2 min, the rate of hydrolysis decreased (Fig. 6). These data
indicate that R/1 compromises Ssa1p ATPase activity at concentrations as low as 10 µM, thus providing an explanation for the
inhibition of in vitro translocation at this concentration
(Fig. 4).
King et al. (39, 40) previously found that a rapid burst of
ATP hydrolysis, mediated by the Ydj1p co-chaperone, induces the
formation of metastable Ssa1p polymers. Because R/1 similarly affects
Ssa1p activity (Fig. 4), one scenario for the R/1-mediated inhibition
of Ssa1p ATP hydrolysis is that R/1 oligomerizes Ssa1p. To test this
hypothesis, the amount of hsc70 that precipitates after centrifugation
at 100,000 × g in the presence and absence of R/1 was
examined. In control reactions, ~30% of Ssa1p and BiP precipitated,
whereas in the presence of 300 µM R/1, 60-70% of Ssa1p
and BiP were found in the pellet (Fig.
7). Interestingly, similar results were
observed with yeast Sse1p, a member of the hsp110 family of molecular
chaperones that is 27% identical to Ssa1p (41).
To determine whether the ability of R/1 to precipitate proteins was a
general phenomenon, we examined several other proteins in this assay.
In the presence of R/1 the amount of precipitable malate synthase was
only enhanced ~15% (Fig. 7). Similarly, R/1 had a lesser effect on
the amounts of lysozyme, bovine serum albumin, and citrate synthase
than on the amounts of the Ssa1p, BiP, and Sse1p chaperones found in
the pellet after centrifugation (Fig. 7).
To confirm the chaperone specificity of R/1, the ability of malate
synthase to catalyze the formation of (2S)-malate from acetyl-CoA and glyoxylate in the presence and absence of R/1 was assayed as described (42). We found that the specific activity of
malate synthase was 0.57 and 0.62 µmol·min We report here on the identification of the first specific
inhibitor of posttranslational protein translocation. Single turnover experiments show that hsc70 ATP hydrolysis is initially stimulated but
inhibited at later time points, presumably by hsc70 precipitation and
inactivation. This property is specific for the chaperones examined as
R/1 failed to precipitate several other proteins to the same degree.
The ability of R/1 to inhibit translocation does not arise from the
increase in trypsin-resistant pp The simplest explanation for the effect of R/1 on hsc70 is that the
length and hydrophobic nature of R/1 (Fig. 1) enable it to mimic a
peptide substrate. This would explain the initial burst of ATP
hydrolysis observed in the single turnover experiments (Fig. 6) and the
inhibition of posttranslational translocation (Fig. 4) if R/1 competes
effectively with pp Despite their structural similarity (Fig. 1), there are several
differences in the activities of DSG and R/1. First, DSG stimulates the
steady-state ATPase activity of Ssa1p (19), whereas R/1 inhibited Ssa1p
steady-state activity. Second, DSG inhibits neither the ATPase activity
of BiP (19) nor the posttranslational
translocation,2 in contrast
to R/1. Third, although R/1 may be a peptide mimic (see above), DSG
does not appear to interact with the substrate-binding domain of
hsc70 as it does not prevent Ssa1p or mammalian hsc70 from binding
polypeptide substrates (19, 20). Finally, there is no evidence that DSG
inactivates or alters the oligomeric state of hsc70. Thus, DSG and R/1
probably modulate hsc70 activity by distinct mechanisms.
Our discovery of a small molecule that is capable of inhibiting a
hsc70-mediated, DnaJ-stimulated process is profound. Because molecular
chaperones influence the biogenesis and degradation of many medically
relevant protein substrates (2, 13), R/1 and R/1 analogs could prove to
be therapeutically valuable. Future studies will be directed toward
identifying and characterizing these compounds.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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F508-CFTR) enhances the plasma membrane
cAMP-stimulated chloride current (24). It is possible that DSG
dissolves a fraction of the hsc70-
F508-CFTR complexes that are
trapped in the ER. Similarly, Rubenstein and Zeitlin (25) reported that
phenylbutyrate decreases the cellular concentration of
hsc70-
F508-CFTR complexes, resulting in a rescue of the
F508-CFTR defect.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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0.8 were found. NSC 630668-R/1
({6-[6-(5-ethoxycarbonylaminocarbonyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-hexyloxycarbonylamino]hexyl}carbamic acid
6-(5-ethoxycarbonylaminocarbonyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)hexyl ester) bore some structural resemblance to DSG (see Fig. 1) and was
thus selected for biochemical analyses.
-factor (
Gpp
F) was performed essentially as
described (30). In brief, translocation reactions were assembled with
Gpp
F, yeast ER-derived microsomes, and R/1 or Me2SO
and preincubated on ice for 30 min before the addition of an
ATP-regenerating system. Import reactions were performed at 20 °C
for 40 min and split, and half was treated with trypsin at a final
concentration of 0.2 mg/ml for 30 min on ice to determine the extent of
membrane-enclosed, signal sequence-cleaved substrate. All samples were
treated with trichloroacetic acid, and the precipitates were analyzed
by SDS-polyacrylamide gel electrophoresis (PAGE) followed by autoradiography.
Gpp
F. For BiP protection experiments, mock translocation reactions were assembled with equal volumes of R/1 (to a final concentration of 300 µM), Me2SO, Triton X-100
(to a final concentration of 1%) or with Buffer 88 (20 mM
HEPES (pH 6.8); 150 mM KOAc; 5 mM MgOAc; 250 mM sorbitol). Following a 45-min incubation at 20 °C,
translocation reactions were split, and one-half was treated with
trypsin at a final concentration of 0.35 mg/ml for 60 min on ice. All
samples were trichloroacetic acid-precipitated and loaded onto a 10%
SDS-polyacrylamide gel. After electrophoresis, proteins were
transferred to a nitrocellulose membrane (Schleicher & Schuell) and
probed with anti-BiP antibody (31) followed by horseradish
peroxidase-conjugated anti-rabbit antiserum (Amersham Pharmacia
Biotech) and the SuperSignal West Chemiluminescence system from Pierce.
For analysis of translocated pro-
-factor, translocation reactions
were performed in the absence of R/1. Subsequently, the reactions were
spun at 13,000 rpm in a Sorvall Biofuge Pico microcentrifuge for
2 min at 4 °C, washed with 1 volume of Buffer 88, and spun again as
above before being resuspended in the same volume of Buffer 88. The
reactions were divided into thirds, and equal volumes of R/1 (to a
final concentration 300 µM), Me2SO, or Triton
X-100 (to a final concentration of 1%) were added. Typsin-treated and
untreated reactions were trichloroacetic acid-precipitated and loaded
onto an 18% urea/SDS-polyacrylamide gel (30). After electrophoresis,
the gels were fixed, dried, and analyzed by PhosphorImager analysis
using the MacBas software program (version 2.4) from Fuji Photo Film Inc.
RESULTS
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ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Chemical structures of NSC 630668-R/1 and
DSG. Structures were drawn in a way to roughly depict potential
structural superimposition. Conformational analysis and superimposition
were performed using Cerius version 4.2MS software (MSI/Biosym).
Consensus alignment was achieved employing flexible root mean square
atoms fitting after finding a global conformational minimum energy
structure of R/1 with the Adopted Basis Newton-Raphson coordinate
minimization method and a systematic search of the 35 torsions in the
molecule.
1·mg
1
in the absence of R/1 to 0.65 nmol·min
1·mg
1
in its presence (Fig. 2). Purified Ydj1p,
the cytosolic DnaJ partner of Ssa1p (27, 33), increased the specific
activity of Ssa1p 6.2-fold in this experiment, a result consistent with previously published data (14, 27, 28). Addition of R/1 decreased the
Ydj1p-mediated stimulation of Ssa1p from 9 to 4.4 nmol·min
1·mg
1,
a 51% reduction (Fig. 2). Thus, R/1 compromises the endogenous and
DnaJ-stimulated ATPase activity of Ssa1p.
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Fig. 2.
R/1 inhibits the endogenous and
co-chaperone-stimulated ATPase activity of Ssa1p. Steady-state
measurements of Ssa1p ATPase activity were performed in reactions
containing equal volumes of R/1 (at a final concentration of 300 µM) or Me2SO and in the presence or absence
of Ydj1p. The molar ratio of Ssa1p to Ydj1p used in this experiment was
1:2. The mean specific activities from three independent experiments
(±S.D.) are expressed in nanomoles of ATP hydrolyzed per min per mg of
Ssa1p.
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Fig. 3.
Dose dependence of R/1-mediated inhibition of
Ssa1p and BiP ATPase activities. Steady-state ATPase measurements
of Ssa1p (open triangles) and BiP (open circles)
were performed as in Fig. 2 with the indicated concentrations of R/1.
Each data point represents the mean (±S.D.) of four independent
measurements. The mean specific activities of Ssa1p and BiP in these
experiments were 1.5 and 1.8 nmol·min 1·mg
1,
respectively.
-factor (pp
F), was translocated into
yeast ER-derived microsomes in the presence of increasing concentrations of R/1. Translocation of pp
F into microsomes was assessed by cleavage of the signal sequence of pp
F to form
pro-
-factor (p
F) and by the subsequent resistance of p
F to
trypsin digestion due to its membrane enclosure. As shown in Fig.
4, A and B, we found that translocation was almost completely blocked at 300 µM, and the half-maximal level of inhibition occurred at
~6 µM.
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Fig. 4.
R/1 inhibits posttranslational
translocation. Translocation reactions with radiolabeled, in
vitro translated pp F were performed as described under
"Materials and Methods" with the indicated concentrations of R/1.
A, after translocation, one-half of the reaction was
trichloroacetic acid-precipitated (lane 1), and the other
half was treated with trypsin (lane 2) before precipitation.
Trypsin-resistant pp
F accumulated in reactions containing higher
concentrations of R/1 (see "Discussion") (B). The
percentage of mature p
F in translocation reactions containing the
indicated concentrations of R/1 was assessed by quantification of
trypsin-protected p
F in relation to the total input pp
F. Shown
are the means from three independent experiments performed at each R/1
concentration, ±S.D.
F, after the addition of R/1 to
microsomes. Both proteins remained resistant to trypsin after treatment
with R/1 or Me2SO (Fig.
5A, lanes 4 and 6;
Fig. 5B, lanes 4 and 5) but were degraded by
trypsin when microsomes were permeabilized with detergent (Fig.
5A, lane 8, and 5B, lane 6). These results
indicate that R/1 does not prevent translocation by destroying
microsome integrity.
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Fig. 5.
Microsomal membrane integrity is unaffected
by R/1. A, microsomes were incubated in buffer,
Me2SO, R/1, or Triton X-100 as indicated, and the
susceptibility of BiP to protease was assessed by immunoblot analysis.
B, P F-loaded microsomes were incubated in
Me2SO, R/1, or Triton X-100 and then treated with
(lanes 1-3) or without trypsin (lanes 4-6) on
ice for 20 min.
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Fig. 6.
R/1 initially enhances but then inhibits
single turnover ATP hydrolysis. Ssa1p-ATP complexes were incubated
at 30 °C for the indicated times, and the percentages of ATP
hydrolyzed over time in the absence (closed circles) or
presence of 10 µM R/1 (open circles), 30 µM R/1 (open squares), and 300 µM R/1 (open triangles) were analyzed.
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Fig. 7.
R/1 precipitates molecular chaperones.
The indicated proteins were incubated with either Me2SO
(DMSO) or R/1 and pellet (P) and supernatant
(S) fractions after centrifugation of the reactions were
analyzed by SDS-PAGE.
1·mg
1
in the presence of Me2SO and 300 µM R/1, respectively.
DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
F observed (Fig. 4A, lane
2) as ~26% of pp
F became resistant at 60 µM
R/1, and translocation was inhibited by ~90% at this concentration. Therefore, we conclude that the abrogation of pp
F translocation is
most likely mediated by the ability of R/1 to inhibit the activities of
two hsc70s, Ssa1p and BiP, that are required to engineer preprotein transport into the yeast ER (31, 33-36, 43).
F for occupation of the substrate-binding site of
Ssa1p and/or BiP. Alteration of the oligomeric state of hsc70 could
result indirectly from precipitation of hsc70-bound R/1 molecules (Fig.
7). Alternatively, R/1 might promote the formation of hsc70 polymers by
binding to a site on hsc70 distinct from that which interacts with
peptides. Support for the latter hypothesis derives from the
observation that DnaJ homologs can polymerize hsc70 in the absence of
substrate (39, 40). This phenomenon may arise from hsc70 homopolymer formation. In the absence of a substrate, hsc70s may bind one another
to form metastable polymers (40). In our experiments, the initial burst
of ATP hydrolysis caused by R/1 could promote the formation of hsc70
polymers, which in turn would inhibit ATP hydrolysis of existing
ATP-Ssa1p complexes.
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ACKNOWLEDGEMENTS |
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We thank the Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, and Division of Cancer Treatment at the National Cancer Institute for providing R/1 and Amie J. McClellan, Michael W. Morrow, and Jennifer L. Goeckeler for providing purified chaperone proteins and technical assistance.
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FOOTNOTES |
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* This work was supported in part by Grants MCB-9904575 from the National Science Foundation and RPG-99-267-01 from the American Cancer Society (to J. L. B.) and by Grant CA78039 (to B. W. D.) from the National Institutes of Health.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.
§ Supported by National Research Service Award 1 F32 CA83270-01 from the NCI, National Institutes of Health.
To whom correspondence should be addressed. Tel.:
412-624-4831; Fax: 412-624-4759; E-mail: jbrodsky@pitt.edu.
Published, JBC Papers in Press, October 17, 2000, DOI 10.1074/jbc.M008535200
2 J. L. Brodsky, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: DSG, 15-deoxyspergualin; ER, endoplasmic reticulum; CFTR, cystic fibrosis transmembrane conductance regulator; PAGE, polyacrylamide gel electrophoresis; BiP, IgG heavy chain binding protein.
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