(Received for publication, July 26, 1996, and in revised form, December 13, 1996)
From the Degradation of a protein via the
ubiquitin system involves two discrete steps, signaling by covalent
conjugation of multiple moieties of ubiquitin and degradation of the
tagged substrate. Conjugation is catalyzed via a three-step mechanism
that involves three distinct enzymes that act successively: E1, E2, and
E3. The first two enzymes catalyze activation of ubiquitin and transfer of the activated moiety to E3, respectively. E3, to which the substrate
is specifically bound, catalyzes formation of a polyubiquitin chain
that is anchored to the targeted protein. The polyubiquitin-tagged protein is degraded by the 26 S proteasome, and free and reutilizable ubiquitin is released. In addition to the three conjugating enzymes, targeting of certain proteins requires association with ancillary proteins and/or post-translational modification(s). Using a specific antibody to deplete cell extract from the molecular chaperone Hsc70, we
demonstrate that this protein is required for the degradation of actin,
Degradation of short-lived and key regulatory proteins via the
ubiquitin-proteasome pathway plays important roles in basic cellular
processes. Protein targets of the ubiquitin system include, among
others, cyclins, cyclin-dependent kinases and their
inhibitors, tumor suppressors, oncoproteins, and transcriptional
activators and their inhibitors. Selection of proteins for degradation
can be mediated via primary (constitutive) or secondary signals such as
post-translational modifications or via association with ancillary proteins. These signals are recognized by specific ubiquitin-protein ligases (E3),1 to which the substrate
proteins bind prior to ubiquitination. Thus, the ligases play a key
role in the ubiquitin proteolytic cascade, recognition and selection of
proteins for conjugation and subsequent degradation. Following
formation of the polyubiquitin adduct, the protein moiety is degraded
by the 26 S proteasome complex, and free and reutilizable ubiquitin is
released (reviewed in Refs. 1-4).
Molecular chaperones comprise a set of universally conserved proteins
that bind and stabilize conformers of other proteins. A regulated,
ATP-dependent association-dissociation cycle of the complex
ensures the correct fate of the protein in vivo, which may
be proper folding after synthesis, assembly into a multimeric complex,
or translocation across a variety of intracellular membranes (5-8).
Some of the chaperones are induced by stress, such as heat (heat shock
proteins), and are probably involved in refolding of stress-induced
denatured proteins. However, most chaperones are synthesized
constitutively (heat shock cognate proteins) and play important roles
in normal cellular processes.
Members of the 70-kDa family of molecular chaperones recognize
hydrophobic domains that are exposed in non-native conformations; they
do not bind to native proteins (9-11). To allow the multistep refolding process, the chaperone-substrate complex must undergo several
ATP-dependent association-dissociation cycles. The
substrate binds with high affinity to the heat shock cognate
protein-ADP complex. Exchange of ADP with ATP lowers the affinity and
results in the release of the substrate. The ATPase activity in the
chaperone hydrolyzes the Hsc70-bound ATP to ADP, which results in
rebinding of the released substrate (12). The dissociation of the
substrate is cation-dependent (12). K+, for
example, causes the conformational change in Hsc70 that is necessary
for substrate dissociation; in the absence of the cation, MgATP favors
the stable complex conformation. Activation by cations correlates with
the ionic radii of the ions: those that are within ~0.1 Å of that of
K+, such as NH4+
and Rb+, are active. In contrast, those that are at
least 0.3 Å smaller (Na+ and Li+) or larger
(Cs+) are inactive (13).
It has been suggested recently that molecular chaperones may also be
involved in intracellular protein degradation (reviewed in Ref. 14). In
Escherichia coli, the non-secreted mutant form of alkaline
phosphatase, phoA61, becomes associated with DnaK and is rapidly
degraded by protease La (15). Proteolysis also requires DnaJ and GrpE,
which have been shown to be involved in transport of the wild-type
enzyme across the cell membrane. It was suggested that if the
chaperones fail to successfully promote transport of the protein, they
facilitate its rapid degradation. In another study, it has been shown
that rapid degradation of abnormal proteins in E. coli
involves GroEL and GroES (16). It is interesting to note that in
prokaryotes, the general rate of protein degradation increases
significantly following exposure to a heat stress. This increased
proteolysis cannot be attributed only to stress-induced denaturation of
intracellular proteins and provision of substrates to the proteolytic
system(s). Rather, many of the bacterial proteases, such as the
lon gene product, La (17), and the components of the Clp
protease complex, ClpP, ClpA, ClpB, and ClpX (18, 19), are stress
proteins that are induced concomitantly with generation of their
respective substrates. In eukaryotes, molecular chaperones are involved
in the degradation of proteins that is mediated by different pathways.
In mitochondria, the chaperones Mdj1p and mt-Hsp70 cooperate with Pim1,
the mammalian homolog of La and Clp, in the degradation of abnormal
proteins (20). Selective and stress-induced degradation of proteins in lysosomes involves recognition by Hsc73 of a peptide motif related to
KFERQ. Researchers suggested that a complex between the chaperone and
the recognition motif binds specifically to the lysosomal transmembrane
glycoprotein LGP96, which serves, most probably, as a specific
translocation channel (14, 21, 22). An intralysosomal chaperone, most
likely also Hsc73, is required for the import of the substrate proteins
into the lysosomal lumen (23). Stress proteins may be also involved in
the removal of unassembled subunits of multimeric membrane complexes or
extracellular proteins that are retained, due to a mutation, for
example, in the secretory pathway. Researchers have shown that BiP, the
Hsp70 homolog in the endoplasmic reticulum, is involved in the removal
of a non-secreted variant of the immunoglobulin light chain (24).
Stress proteins have also been implicated in the degradation of
proteins via the ubiquitin-proteasome pathway. A Saccharomyces cerevisiae cell that harbors a temperature-sensitive mutant of Ydj1 demonstrated a large defect in the overall breakdown of
short-lived and abnormal proteins that is mediated by the ubiquitin
system. Degradation of long-lived proteins that is mediated by the
vacuole and proceeds in a ubiquitin-independent manner was unaffected (25). Interestingly, the effect was specific. Whereas the degradation of certain proteins such as several model "N-end rule" substrates and the mitotic cyclin Clb5 not was affected, the degradation of other
substrates (the transcriptional activator Gcn4, for example) required
Ydj1. Concomitantly, the ubiquitination of the
Ydj1-dependent substrates was significantly reduced.
Researchers also found that the chaperone generates a complex with its
target substrate, but due to the limitations of the in vivo
studies, they could not demonstrate that the complex serves as an
essential intermediate in the proteolytic process (25). Degradation of
the yeast cyclin Cln3 is also stimulated by Ydj1. However, in this
case, the chaperone appears to stimulate phosphorylation of the cyclin
by p34cdc28, a post-translational modification that signals the
protein for conjugation and subsequent degradation by the ubiquitin
system (26). Expression of several of the genes encoding components of
the ubiquitin system is induced by heat. These include polyubiquitin genes in chick embryo fibroblasts (27) and yeast (28) and two of the
yeast E2-encoding genes, UBC4 and UBC5 (29).
Mutations in the UBC genes lead to defects that are
characteristic of mutations in heat shock proteins, including
temperature sensitivity and constitutive thermotolerance. Craig
et al. (7) found that overexpression of UBP3, a ubiquitin
C-terminal hydrolase, suppresses the ssa1ssa2 phenotype. It
is possible that lack of the chaperones leads to accumulation of
misfolded proteins in the cytosol, and overexpression of a
rate-limiting enzyme in the ubiquitin pathway ameliorates the situation
by facilitating removal of these proteins. Ohba (30) demonstrated that
a 70-kDa heat shock protein suppresses several of the defects caused by
a mutation in the S. cerevisiae proteasome.
In this study, we show that Hsc70 is required for ubiquitin conjugation
and subsequent degradation of certain proteolytic substrates in
vitro. The chaperone is required in the conjugation step, and a
complex between Hsc70 and the protein substrate serves, most probably,
as an essential intermediate in the proteolytic process.
Materials
FPLC columns and Sepharose-immobilized protein G were from
Pharmacia Biotech Inc. Materials for SDS-PAGE and Coomassie Blue and
silver staining as well as molecular mass markers were from Bio-Rad.
Histone H2A, hexokinase, and ATP Methods
Hsc70 was purified to almost
homogeneity (>95%) from frozen bovine brain using Q-Sepharose Fast
Flow FPLC anion-exchange chromatography, ATP-agarose affinity
chromatography, and Mono Q FPLC anion-exchange chromatography
essentially as described (9, 10). Briefly, a frozen brain (~500 g)
was washed and homogenized in a blender with 1.2 volumes of buffer
containing 10 mM HEPES-KOH, pH 6.5, 150 mM
NaCl, 0.5 mM MgCl2, 1 mM EDTA, 0.5 mM DTT, and 1 mM phenylmethylsulfonyl fluoride.
Following low and high speed centrifugations to remove particulate
material, the supernatant was dialyzed against Buffer A (25 mM Tris-HCl, pH 7.2, 0.1 mM EDTA, and 0.5 mM DTT) and loaded onto a Q-Sepharose Fast Flow column
equilibrated in Buffer A. The adsorbed proteins were eluted in an FPLC
apparatus (Pharmacia) using a linear gradient of 0-0.6 M
KCl in Buffer A. Fractions containing Hsc70 as determined by SDS-PAGE
and silver staining were pooled and dialyzed against Buffer B (20 mM HEPES-KOH, pH 7.0, 25 mM KCl, 10 mM (NH4)2SO4, 0.1 mM EDTA, 0.5 mM DTT, and 2 mM
Mg(CH3COO)2). The sample was applied to an
ATP-agarose column equilibrated in Buffer B, and Hsc70 was eluted by
washing the column with Buffer B containing 5 mM MgATP.
Fractions containing Hsc70 (see above) were pooled and equilibrated by
dialysis in Buffer A. The pooled fractions were applied to a Mono Q
anion-exchange chromatography column equilibrated in Buffer A. Hsc70
was eluted by a linear gradient of 0.1-0.6 M KCl in Buffer
A, and fractions containing the chaperone were pooled, dialyzed against
Buffer B (lacking Mg(CH3COO)2), and stored in
small aliquots at Rat monoclonal anti-Hsc70
antibodies were prepared from 1B5 rat hybridoma cells (31). Cells were
grown as a monolayer at 37 °C in Dulbecco's modified Eagle's
medium supplemented with 20% fetal calf serum. Cultures were
harvested, and the cells were washed twice with phosphate-buffered
saline. Washed cells were injected (5 × 106 cells in
2 ml) into male Harlan Sprague Dawley rats that had been injected
intraperitoneally with pristane 8 days earlier. Ascites fluid,
developed within 1 week, was collected and subjected to precipitation
by 45% (NH4)2SO4 followed by
dialysis against a buffer containing 25 mM Tris-HCl, pH
7.2, and 75 mM NaCl.
300-µl aliquots of reticulocyte
lysate (see below; contains ~30 µg of Hsc70 as determined by
quantitative Western blot analysis) were incubated at 4 °C for 90 min in the presence of 125 µg of 1B5 antibody (prepared as described
above) or 125 µg of preimmune rat IgG. Following incubation, the
mixtures were added to 25 µl of packed protein G-Sepharose beads
(washed with 20 mM Tris-HCl, pH 7.2, and 75 mM
NaCl) and swirled at 4 °C for 45 min. After a brief centrifugation
to remove the beads, the supernatants were tested for the presence of
Hsc70 by Western blot analysis. Detection was performed with the ECL
system (Amersham Corp.) using 1B5 as the primary antibody and rabbit
anti-rat peroxidase-conjugated IgG as the secondary antibody.
Similarly, Hsc70 was depleted also from 100-µl aliquots of Fraction
II or IIA (see below; both also contain ~30 µg of Hsc70).
RCM- Reticulocyte-rich blood was induced in rabbits by
successive injections of phenylhydrazine, and reticulocyte lysate was
prepared as described (33). Lysates were resolved by anion-exchange
chromatography on DEAE-cellulose into unadsorbed material (Fraction I)
and high salt eluate (Fraction II) as described (33). E1 was purified to homogeneity from Fraction II following affinity chromatography on
immobilized ubiquitin as described (33). E2-F1 was purified to
homogeneity from Fraction I, and Fraction IIA was prepared from
Fraction II by (NH4)2SO4
precipitation (0-38%) as described (34). UbcH5 cDNA (35) was
expressed in E. coli BL21 cells, and the enzyme was purified
from the bacterial extracts following induction as described (36).
Degradation and
conjugation assays of the 125I-labeled protein substrates
were performed essentially as described (32, 34). Briefly, reaction
mixtures contained (in a final volume of 25 µl) the following: 10 µl of reticulocyte lysate (~1 mg of protein) (crude; dialyzed
(against a buffer containing 20 mM Tris-HCl, pH 7.2, and 1 mM DTT); Hsc70-depleted; dialyzed and Hsc70-depleted; or
preimmune rat IgG-treated, as indicated), 40 mM Tris-HCl,
pH 7.6, 5 mM MgCl2, 2 mM DTT, 5 µg of ubiquitin, and 0.05-0.1 µg of 125I-labeled
protein substrate (~100,000 cpm). For inhibition of ubiquitin
C-terminal hydrolases, conjugation reactions contained 0.5 µg of
ubiquitin aldehyde (37). To deplete ATP, hexokinase (0.4 µg) and
2-deoxyglucose (10 mM) were added. To provide energy, conjugation assays contained 5 mM ATP Crude or
Hsc70-depleted reticulocyte lysates were dialyzed at 4 °C against a
buffer containing 20 mM Tris-HCl, pH 7.2, and 1 mM DTT.
For
monitoring complex formation between the chaperone and the substrates,
Hsc70 and the various 125I-labeled protein substrates (0.1 µg, ~104 cpm; following centrifugation at 13,000 × g for 15 min) were incubated (at a molar ratio of ~4:1,
respectively) for 45 min at 37 °C in a buffer containing 20 mM Tris-HCl, pH 7.2, and 1 mM DTT (incubation
buffer). Reaction mixtures (25 µl) were resolved electrophoretically
via nondenaturing 5-15% polyacrylamide gradient gel as described (34)
or via gel filtration chromatography on a 1 × 60-cm Sepharose
6B-CL column equilibrated in incubation buffer containing 1 mg/ml
ovalbumin and 75 mM NaCl. Here, we monitored shift in
radioactivity to a high molecular mass region of the column in the
presence of the chaperone. For monitoring the role of the
Hsc70·RCM- Protein concentration was
determined by the Bradford method (38) using BSA as a standard.
We noted that the degradation in
reticulocyte lysate of 125I-labeled actin,
Effect of K+ on the degradation of protein substrates of the
ubiquitin proteolytic system
Degradation of the various 125I-labeled substrates was
monitored by the release of radioactivity into trichloraacetic
acid-soluble fractions as described under "Experimental
Procedures." Complete lysate + ATP represents a system that was
incubated in the presence of ATP and an ATP-regenerating system.
Complete lysate
Effect of K+, NH4+, Rb+, Cs+,
Na+, and Li+ on the degradation of actin,
Degradation of the various substrates was monitored as described in the
legend to Table I. When indicated, the chloride salts of the
appropriate cations were added to the reaction mixture to a final
concentration of 10 mM. Results represent net
ATP-dependent degradation. ATP-independent degradation did
not exceed 20% of total degradation, and the results were corrected
accordingly. Department of Biochemistry and the Rappaport
Institute for Research in the Medical Sciences, Bruce Rappaport
Faculty of Medicine, Technion-Israel Institute of Technology, Haifa
31096, Israel, the § Section on Cancer Biology,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENT
REFERENCES
-crystallin, glyceraldehyde-3-phosphate dehydrogenase,
-lactalbumin, and histone H2A. In contrast, the degradation of bovine serum albumin, lysozyme, and oxidized RNase A is
Hsc70-independent. Mechanistic analysis revealed that the chaperone is
required for the conjugation reaction; however, it does not substitute
for E3. Involvement of the chaperone in the proteolytic process
requires complex formation with the substrate. Formation of this
complex appears to be essential in the proteolytic process. In
addition, the proper function of the chaperone in the proteolytic
process requires the presence of K+, which allows
rapid cycles of dissociation and association of the complex. The
chaperone may act by binding to the substrate and unfolding it to
expose a ubiquitin ligase-binding site. In addition, it can also act
directly on the ubiquitination machinery.
S were from Boehringer Mannheim.
Lysozyme, BSA, GAPDH,
-crystallin,
-lactalbumin, ubiquitin, ovalbumin, phenylmethylsulfonyl fluoride, DTT, ATP, ATP-agarose, phosphocreatine, creatine kinase, 2-deoxyglucose,
2,6,10,14-tetramethylpentadecane (pristane), preimmune rat IgG, Tris
buffer, and HEPES were purchased from Sigma. Actin was from
Worthington. DEAE-cellulose (DE52) was from Whatman. Ammonium sulfate
was obtained from Life Technologies, Inc. Na125I was from
DuPont NEN. Dulbecco's modified Eagle's medium and fetal calf serum
were from Biological Industries (Kibbutz Beth Ha'emek, Israel). All
other chemicals were of high analytical grade.
70 °C. All purification procedures were carried
out at 2-4 °C.
-LA was prepared as
described (32). 125I-Labeled proteins were prepared by the
chloramine-T method as described (32).
S or 5 mM ATP, whereas degradation assays contained ATP (0.5 mM) and an ATP-regenerating system (10 mM
creatine phosphate and 2 µg of creatine phosphokinase). When indicated, Hsc70 (2.5 µg or as indicated) and KCl, NH4Cl,
RbCl, CsCl, LiCl, or NaCl (10 mM) were added. All assays
were incubated at 37 °C for either 30 (conjugation) or 120 (degradation) min. For monitoring conjugation, reaction mixtures were
resolved via SDS-PAGE (12.5%), and the gels were dried and exposed to
BioMax X-ray film (Eastman Kodak Co.) or to a bioimaging analyzer
(PhosphorImager, Fuji). Degradation of iodinated protein substrates was
determined by measuring the radioactivity released into trichloroacetic
acid-soluble fractions as described (32, 34). Degradation values are
expressed as percentage of the radioactivity released. 100% is the
radioactivity in the trichloroacetic acid-insoluble fraction of a
substrate that was not incubated. Conjugation assays containing
purified components were performed in the presence of 2 µg of E1, 0.5 µg of E2-F1 (or UbcH5), and the indicated amount of Fraction IIA protein.
-LA complex as an intermediate in the proteolytic process, ~10 µg of iodinated RCM-
-LA (~10 × 106 cpm) were centrifuged at 13,000 × g
for 15 min and incubated for 45 min at 37 °C in a volume of 100 µl
in incubation buffer in the presence or absence of 150 µg
of purified Hsc70 (~4-fold molar excess of the chaperone over the
labeled substrate). Following incubation, the reaction mixtures were
resolved on a 16 × 60-cm Superdex 200 HR FPLC column equilibrated
in incubation buffer containing 1 mg/ml ovalbumin and 75 mM
NaCl. The radioactive substrate was detected by a
-counter. In the
presence of the chaperone, radioactivity shifted almost completely to a
molecular mass region of ~100 kDa. 20% of the radioactivity migrated
in two smaller molecular mass peaks, one (~10%) that corresponds to
the mass of the free unincorporated substrate and one (~10%) that
corresponds to free unincorporated iodine. In the absence of the
chaperone, ~90% migrated as free substrate and 10% as free iodine.
~2% migrated as high molecular mass aggregates in the void volume
region of the column. The vast majority of the substrate isolated from
the void peak region (~90%) migrated in gel electrophoresis in
nondenaturing gel as a high molecular mass complex (see below) and not
as a free protein. Degradation of the Hsc70-bound substrate and the free substrate was monitored as described above.
Effects of Cations on the Degradation of Certain Protein Substrates
of the Ubiquitin System
-crystallin,
histone H2A, GAPDH,
-LA, and RCM-
-LA is inhibited following a
short period of dialysis in low molecular mass cutoff dialysis tubing
(molecular mass cutoff= 1.0 kDa). Addition of KCl reconstituted the
inhibited activity (Table I). The degradation of BSA,
lysozyme, and oxidized RNase A was not affected by the removal and
resupplementation of KCl (Table I; results for oxidized RNase A are not
shown). Titration of the cation shows that the maximal stimulating
effect is attained at ~20 mM (Fig. 1; see
"Discussion"). We have previously shown that all these substrates
are targeted for degradation by the ubiquitin proteolytic system
in vitro (32, 34, 39, 40). In an attempt to characterize
better the cations that can reconstitute proteolysis, we found that
NH4+ can substitute for
K+ efficiently, Rb+ is less effective,
Cs+ is significantly less active, and Na+ and
Li+ are inactive (Fig. 1 and Table II). In a
search for a potential role of the cations in the proteolytic process,
we noted that several functions of the 70-kDa heat shock cognate
protein (Hsc70), such as peptide binding and release and clathrin
uncoating, require K+ (12, 13, 41). The cation is required,
most probably, to allow the change in the conformation of the chaperone
that is necessary for substrate dissociation. Examination of the effect of other monovalent cations on the activity of Hsc70 revealed an
identical profile to that observed for stimulation of
ubiquitin-dependent degradation (Ref. 13 and Table II).
Thus, we set out to examine directly the effect of Hsc70 on the
function of the ubiquitin system.
ATP represents a system that was preincubated
for 5 min at 37 °C in the presence of 2-deoxyglucose and hexokinase
prior to addition of the labeled substrate.
Substrate
Complete lysate
Dialyzed
lysate
+ATP
ATP
+ATP
+ATP/K+
Actin
25.1
4.7
7.2
26.5
-Crystallin
16.4
2.8
4.1
15.6
Histone
H2A
29.6
5.9
8.2
31.1
GAPDH
24.5
3.8
6.9
21.6
-LA
17.3
2.6
11.6
17.1
RCM-
-LA
39.1
7.3
9.1
36.8
BSA
43.0
2.6
40.9
41.6
Lysozyme
31.8
6.7
27.3
27.8
Fig. 1.
Cation-dependent degradation of
actin and BSA. Degradation of 125I-labeled actin (,
,
) and BSA (
) was monitored in a dialyzed reticulocyte lysate
in the presence of increasing concentrations of K+ (
,
), NH4+ (
), or Na+
(
). Degradation was monitored by measuring the release of
radioactivity into trichloroacetic acid-soluble fractions as described
under "Experimental Procedures." 100% reflects net
ATP-dependent degradation values. ATP-independent
degradation did not exceed 20% of total degradation in a complete
system, and the results were corrected accordingly.
[View Larger Version of this Image (17K GIF file)]
-crystallin, histone H2A, GAPDH, and RCM-
-LA
Substrate
Complete lysate
Dialyzed
lysate
Dialyzed lysate
+K+
+NH4+
+Rb+
+Cs+
+Na+
+Li+
Actin
22.2
3.7
23.8
24.5
15.6
5.2
3.1
2.9
-Crystallin
15.7
1.9
16.7
13.2
11.5
4.7
2.2
1.4
Histone
H2A
21.6
3.2
24.7
18.9
13.2
5.6
3.0
2.1
GAPDH
19.5
3.3
18.4
16.7
12.9
6.9
4.1
2.8
RCM-
-LA
34.7
6.4
30.1
28.6
17.6
10.4
7.6
6.9
To test the hypothesis that the
cation requirement for proteolysis reflects a requirement for the
molecular chaperone Hsc70, we used a monoclonal antibody to deplete
Hsc70 from reticulocyte lysate, the proteolytic extract that is used
most frequently for monitoring degradation in a cell-free system. As
can be seen in Fig. 2, treatment of the extract with the
specific antibody results in almost complete removal of Hsc70.
Quantitative assessment of the chaperone in the lysate revealed a
concentration of ~100 µg/ml. Depletion of Hsc70 resulted in
significant inhibition of degradation of the cation-sensitive
substrates (Table III; similar results were obtained for
the degradation of histone H2A (data not shown)). Depletion of Hsc70
did not affect the proteolytic sensitivity of lysozyme (Table III).
Similarly, the degradation of BSA and oxidized RNase A was not affected
(data not shown). It is interesting to note that while the effects of
cation and heat shock cognate protein depletion on the degradation of
RCM--LA are dramatic, the effects on
-LA are significantly lower,
and the degradation of this substrate is much less dependent upon these
two components (Tables I and III). Similar quantitative differences
between the two substrates were obtained also when we monitored the
effect of Hsc70 on ubiquitin conjugate formation and on generation of chaperone-protein complexes (data not shown; see Fig. 8 and
"Discussion"). Resupplementation of Hsc70 reconstituted the
activity almost completely and, in the case of actin and
-crystallin, even beyond the basal value measured in a complete,
untreated lysate (Table III; see below). As expected, addition of the
cation or the chaperone alone is not sufficient to reconstitute
activity, and proteolysis resumes only following simultaneous addition
of the two factors (Table IV). The experiments described
in Tables I, II, III, IV still leave open the possibility that the cation and
chaperone exert their effects on the proteolytic process via two
distinct and independent mechanisms. However, the identity in cation
profiles for Hsc70 activation and proteolysis makes this possibility
highly unlikely. It should be noted that the maximal effect of
K+ in stimulating the ATPase activity and the
association-dissociation cycle of the Hsc70-peptide complex is attained
at ~100 mM (13), whereas the maximal effect on
proteolysis is attained already at ~20 mM (Fig. 1). This
apparent discrepancy can be explained by the difference in the systems
used in the two studies. The study on the ATPase activity of Hsc70 was
carried out using purified Hsc70 and synthetic short peptides as
binding substrates. In contrast, the proteolytic assays are carried out
in a crude extract using intact proteins as substrates. The
K+ concentration in the cell is ~150 mM, and
the activity of the chaperone in the cell appears to be maximal
anyway.
|
|
Degradation of a
protein via the ubiquitin system proceeds via two distinct steps,
conjugation of ubiquitin to the target protein and degradation of the
tagged protein by the 26 S proteasome complex. To identify the cation-
and Hsc70-dependent step, we monitored conjugation in
dialyzed and K+-resupplemented lysates as well as in
Hsc70-depleted and -resupplemented lysates. As can be seen in Figs.
3A and 4A, removal
of ions inhibits conjugation of GAPDH and -crystallin, respectively.
Addition of K+ reconstitutes adduct formation. Similarly,
addition of NH4+ and, to a lesser
extent, Rb+ also restores activity (data not shown). As demonstrated in
Figs. 3B and 4B, depletion of Hsc70 also inhibits
conjugate formation. Resupplementation of the chaperone restores
activity. Fig. 5 demonstrates that the conjugation of actin requires both K+ and Hsc70; addition of any of these
components alone is not sufficient to promote conjugation. Similar to
the effect of ions and Hsc70 on degradation (Fig. 1 and Tables I and
III), the effects of these factors on conjugation are also specific for
a certain subset of substrates: dialysis and depletion of Hsc70 do not
affect conjugation of lysozyme (Fig. 6) and oxidized
RNase A (data not shown). The conjugating system contained either
ATP
S or ATP (see "Experimental Procedures"). The ATP analog is
utilized in conjugation assays since it promotes conjugation (via
activation of E1), but inhibits degradation (since the 26 S proteasome
can utilize only ATP), and therefore leads to accumulation of ubiquitin
adducts (42). We were surprised to find that the nonhydrolyzable
nucleotide analog is active, although not more than ATP, in promoting
Hsc70- and cation-dependent conjugation that is apparently
dependent on ATP hydrolysis. This may be due to the presence of
endogenous ATP and ADP in the lysate and to the activity of adenylate
kinase that generates ATP from ADP. Slight hydrolysis of ATP
S can
also generate ADP. Also, the vast molar excess of the chaperone over the labeled substrate may allow presentation of the substrate to the
conjugating machinery after only a single cycle of association. Thus,
the reaction may not be heavily dependent upon the presence of ATP. We
noted that addition of Hsc70 to a complete lysate stimulated conjugation to
-crystallin (Fig. 4B, compare lanes
3 and 4) and actin (data not shown). In contrast,
addition of the chaperone had no effect on the conjugation of GAPDH
(Fig. 3B, compare lanes 3 and 4),
histone H2A,
-LA, and RCM-
-LA (data not shown). As noted before,
resupplementation of excess Hsc70 had a stimulatory effect on the
degradation of these substrates (Table III). These results can be
explained by the possibility that the affinity between actin and
crystallin and the chaperone is weaker than that of the other protein
substrates. Most of the endogenous chaperone contained in the lysate is
occupied by native cellular proteins. Therefore, exogenous substrates
with low affinity cannot displace the endogenous substrates from the
chaperone and are partially dependent for their conjugation and
degradation upon the addition of excess exogenous and substrate-free
Hsc70. The high affinity substrates can displace the endogenous
proteins and are not dependent upon the addition of free exogenous
chaperone for their conjugation and subsequent degradation.
Conjugation Requires, in Addition to Hsc70, All Three Conjugating Enzymes, E1, E2, and E3
Conjugation of proteolytic substrates
involves specific complex formation with their cognate E3 enzymes,
which serve, among other functions, as docking proteins for the
substrates during the tagging process. Thus, it was of interest to
study whether the molecular chaperone, which also recognizes specific
motifs in the substrate, can substitute for E3 in the conjugation
reaction. In this case, catalysis of conjugation would require E1, E2,
and the chaperone, but not E3. As demonstrated in Fig.
7 (lanes 1 and 2), addition of E1,
E2-F1 (or UbcH5; data not shown), and the chaperone to a cell-free
reconstituted system is not sufficient to restore conjugation.
Conjugate formation is dependent upon the addition of Fraction IIA
(lanes 3 and 4), which contains all the known E3
enzymes (34, 36, 43). Depletion of Hsc70 from Fraction IIA inhibits
conjugation (lane 5), whereas, as expected, addition of
purified Hsc70 to the system restores activity. Thus, Hsc70 cannot
function as an E3 enzyme and is not involved directly in the
conjugation process.
Possible Mechanism of Involvement of Hsc70 in Ubiquitin-mediated Proteolysis: Formation of a Chaperone-Substrate Intermediate Complex
All known activities of molecular chaperones are mediated
via complex formation with their substrates. Thus, it was important to
determine whether Hsc70 generates a complex with the
chaperone-dependent proteolytic substrates, but not with
those that are degraded in a chaperone-independent manner. As can be
seen in Fig. 8, -LA and RCM-
-LA, two proteins that
require Hsc70 for their conjugation and subsequent degradation (see
Table III; results for conjugation of both proteins are not shown),
generate a complex with Hsc70. Similarly, actin,
-crystallin, GAPDH,
and histone H2A associate with Hsc70, whereas BSA, oxidized RNase A,
and lysozyme do not (data not shown; association of Hsc70 with these
proteins was followed by monitoring the shift of radioactivity of these
proteins to a higher molecular mass region of a gel filtration
chromatography column as described under "Experimental
Procedures"). It is interesting to note that a larger proportion of
RCM-
-LA became associated with the chaperone compared with the
nonmodified protein (Fig. 8, compare lanes 2 and
4). This difference is probably due to the higher affinity
that the completely unfolded protein has for the chaperone. This high
affinity is most probably reflected also in the greater sensitivity of
the modified protein to cation (Table I) and Hsc70 (Table III)
depletion. Hsc70-protein complexes were also obtained when the
substrates were incubated in the presence of a complete proteolytic
extract (devoid of ATP to prevent conjugation and degradation) (data
not shown). Utilization of Hsc70-depleted lysate did not yield a high
molecular mass complex (data not shown). This observation demonstrates
that the only protein in the mixture that associates with the substrate
is the chaperone.
To test for the possible role of the chaperone-substrate complex as an essential intermediate in the proteolytic process, we isolated the complex and subjected it to degradation in a complete lysate as well as in a lysate from which Hsc70 and K+ were removed and subsequently supplemented. As can be seen in Table V, addition of K+ to the doubly depleted lysate stimulated the degradation of the Hsc70-bound substrate 3-fold, whereas the degradation of the free substrate was stimulated by only 35%. It should be noted, however, that the 3-fold stimulation could be increased by a further 2-fold by addition of excess free chaperone. Addition of the free chaperone reconstituted almost completely the degradation of the free substrate as well. The fact that the complex was not sufficient to promote maximal degradation of the bound substrate is due, most probably, to the lack of molar excess of the chaperone. Following one cycle of dissociation, the reassociation of the substrate with the chaperone in the presence of an equimolar concentration of the chaperone is less efficient compared to the same reaction in the presence of a molar excess of the stress protein. Needless to say, all the proteolytic activities were dependent upon the presence of K+.
|
We have shown that the conjugation and degradation of certain
proteolytic substrates by the ubiquitin system in vitro
require K+ and the molecular chaperone Hsc70.
Immunodepletion of the protein from reticulocyte lysate results in a
significant inhibition of conjugation and subsequent proteolysis of
actin, -crystallin, GAPDH, histone H2A,
-LA, and RCM-
-LA. In
contrast, the conjugation and degradation of yet another set of
proteolytic substrates (BSA, lysozyme, and oxidized RNase A) are
chaperone-independent. The chaperone forms a complex with the target
substrate that serves as an essential intermediate in the proteolytic
process. However, this complex cannot substitute for E3, which also
forms a complex with the substrate prior to tagging it with ubiquitin.
We noted that the chaperone is required for formation of high molecular mass ubiquitin conjugates of histone H2A and RCM-
-LA; formation of
low molecular adducts of these proteins is Hsc70-independent (data not
shown). It is interesting to note that formation of low molecular mass
conjugates is also E3-independent and requires only E1 and E2 (reviewed
in Ref. 4). Also, only the high molecular mass adducts serve as
proteolytic substrates for the 26 S proteasome complex. Taken together,
these findings indicate that the chaperone and E3 act in concert to
generate the appropriate multiply ubiquitinated substrates that are
recognized and subsequently proteolyzed by the 26 S proteasome (see
also below). While the cations are most probably required for the
activity of the chaperone, it is still possible that the two components
act via two distinct and independent mechanisms. However, as noted, the
identity between the cation profiles required for Hsc70 activation and
proteolysis makes this possibility unlikely.
The mechanistic basis for the substrate specificity of Hsc70 is not
known. It may be due to specific structural features of the proteins
that allow their binding to the chaperone. We noted that all
three "Hsc70-independent" substrates (lysozyme, BSA, and oxidized
RNase A) are primary "N-end rule" proteins that are recognized
and targeted via direct binding of their free N-terminal and
"destabilizing" amino acid residue (44). It is possible that in
these proteins, the recognition domain that is extremely short and well
defined is exposed and is directly recognized by the ubiquitin-protein
ligase E3. Therefore, chaperone-assisted unfolding of the protein to
expose the E3 recognition domain is not necessary. Structural analysis
of labeled lysozyme has shown that at least for this protein, the
N-terminal domain is indeed freely accessible (45). For the
Hsc70-dependent substrates, it is possible that their E3
recognition site is hidden, and the binding to the chaperone is
necessary to uncover it. Thus, for certain substrates, the
rate-limiting step in the multistep ubiquitin cascade may be their
recognition by the ligase. The rate of this step may vary from
substrate to substrate, and the role of the chaperone is to facilitate
recognition for the "chaperone-dependent" substrates.
For other substrates, this may not be the rate-limiting step, and their
recognition proceeds unimpaired even in the absence of the chaperone
and the ion. An alternative explanation for the selective effect of
Hsc70 may involve the degree of unfolding (denaturation) or the
aggregation state of the substrate. It is clear that, at least for the
pair
-LA and RCM-
-LA, there is a strong and direct correlation
between the degree of unfolding of the protein and its sensitivity to
cation and Hsc70 depletion: completely unfolded RCM-
-LA shows a
greater dependence on K+ and Hsc70 for its conjugation
(data not shown) and subsequent degradation compared with its cognate,
untreated protein (Tables I and III). It also associates at a higher
efficiency with the chaperone (Fig. 8, compare lanes 2 and
4). In a different approach to the problem of "substrate
specificity," we noted that the chaperone-dependent (but
not the chaperone-independent) substrates appear to be in an
aggregated form (as determined by the proportion of the substrate that
can be precipitated by a short, high speed centrifugation). Our initial
experiments indicate that Hsc70 may be involved in solubilization of
these aggregates (data not shown). It should be noted that in the cell,
Hsc70 may serve to prevent aggregation rather than to resolubilize
aggregated proteins. It is clear that all the substrates we utilized
are denatured to a smaller or larger degree, but the association of the
chaperones with proteins appears to be mostly with unfolded proteins.
Frydman and Hartl (46) noted recently in their study on the association
of chaperones with nascent truncated actin that although caution should
be exercised when extrapolating from in vitro studies to the
functions of chaperones in the cell, the interactions of chemically
denatured proteins with chaperones may resemble those occurring in the
cell following its exposure to various forms of stress such as elevated
temperature.
Our findings also indicate that the involvement of the chaperone in the proteolytic process requires complex formation with the targeted substrate and that the complex that serves as an essential intermediate in the proteolytic process must undergo cation-dependent cycles of association-dissociation. Like other proteins that bind to Hsc70, it is possible that without the cation, the proteolytic substrate is "locked" into the chaperone and therefore cannot be released for presentation to the conjugating machinery.
An important problem involves the mechanism of action of the chaperone in the proteolytic system. One possibility is that Hsc70 binds the substrate and actively unfolds it in a manner that exposes the ligase-binding domain. The cation is required for release of the substrate from the chaperone and its transfer to the ligase. Here, the chaperone serves to "hold" the substrate in an unfolded, but E3-bindable state. An alternative (and apparently antithetical) explanation involves repeated cycles of binding and release with an attempt to refold the protein to its native form. It is highly likely that many cellular proteins are denatured to a certain degree. One well known and carefully studied function of molecular chaperones involves refolding of denatured proteins to their native form. The process is gradual and involves repeated, cation-dependent cycles of association-dissociation (5, 12, 13). It is possible that Hsc70 is involved in an attempt to refold denatured proteolytic substrates. Failure to renature the proteins leads to their presentation to the ligase. The "decision" to present the denatured protein to the scavenging system may be based on the rate of successful collisions between the Hsc70-substrate complex and E3. A successful collision is one in which the chaperone presents to the ligase a protein with an exposed recognition domain and that results in transfer of the chaperone-bound substrate to E3. Successful refolding of the protein to a form in which this domain is no longer exposed will lead to an infertile collision and eventually to release of a native refolded protein from the chaperone. Failure to refold the protein and continuous exposure of the ligase recognition motif increase the chances for a successful collision. This "refold or degrade" hypothesis is clearly distinct from the first, "holding" hypothesis. Kinetic measurements as well as utilization of different chaperones and proteolytic substrates in the cell-free system can be now used to test these two mechanistic hypotheses. An additional explanation for the involvement of the chaperone in the proteolytic process is that it affects, in addition to the substrate, one or more of the components of the system. It should be noted that the identity of the E3 enzymes that are involved in the conjugation of all the chaperone-dependent proteins is not known. Therefore, experiments with purified components are not possible at this stage, and a detailed analysis of the mechanism of involvement of Hsc70 in ubiquitin-mediated proteolysis will have to await further characterization of the system.
It is not known whether Hsc70 affects only conjugation or whether it is involved also in degradation of the conjugates by the 26 S proteasome. Also, it is not known whether, as in protein synthesis (as determined in a cell-free reconstituted system and not in the intact cell), other chaperones are also involved in the proteolytic process. Folding of nascent polypeptide chains requires high molecular mass assembly with Hsc70, Hsc40, and TRiC (47). The three chaperones are required for cotranslational formation of all the domains of the protein and completion of folding following release of the chain from the ribosome. Depletion of any of the chaperones leads to improper folding of the nascent chain and release of an inactive protein.
We thank Dr. Lawrence E. Hightower (University of Connecticut, Storrs, CT) for advice and for providing reagents in the initial phase of the study.