From the Basic Research Program, SAIC-Frederick,
§ Basic Research Laboratory, National Cancer Institute,
Frederick, Maryland 21702
Received for publication, August 17, 2002, and in revised form, November 19, 2002
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ABSTRACT |
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The 97-kDa valosin-containing protein
(p97-VCP) plays a role in a wide variety of cellular activities, many
of which are regulated by the ubiquitin-proteasome (Ub-Pr)-mediated
degradation pathway. We previously demonstrated that VCP binds to
multi-ubiquitin chains and may act as a molecular chaperone that
targets the ubiquitinated substrates to the proteasome for degradation.
In this report, we show that although the ubiquitin chain-binding
activity, carried out by the N-terminal 200 residues (N domain), is
necessary for the degradation of proteasome substrates, it is not
sufficient. Using in vitro degradation assays, we
demonstrated that the entire VCP molecule, consisting of the N domain
and two ATPase domains D1 and D2, is required for mediating the Ub-Pr
degradation. The ATPase activity of VCP requires Mg2+, and
is stimulated by high temperature. Under optimal conditions, VCP
hydrolyzes ATP with a Km of ~0.33 mM
and a Vmax of ~0.52 nmol
Pi min VCP1 is a member of the
AAA (ATPases associated with a variety of
cellular activities) family, which comprises a highly
conserved group of molecular chaperones that regulate many cellular
functions (reviewed in Refs. 1-4). The family members are
characterized by the presence of either one (type I) or two (type II)
conserved ATPase domains, also referred to as AAA modules. The
200-250-amino acid AAA module encompasses the consensus Walker A
(GxxxxGKT, x = any amino acid) and
Walker B (hhhhDExx, h = hydrophobic residue) motifs (5) that are critical for ATP binding and
hydrolysis, respectively. It is generally thought that the AAA proteins
act as molecular chaperones to assemble or disassemble protein
complexes, unfold or unwind macromolecules, and transport cellular
cargoes. These chaperone or chaperone-like activities are carried out
by the conserved ATPase domains, which provide the needed energy through ATP hydrolysis.
VCP is a type II AAA-ATPase. It is highly abundant, accounting for
about 1% of the total cellular proteins. It is present in all types of
cells in a hexameric ring structure and localizes in the nucleus,
cytoplasm, and perimembrane subcompartments (6). VCP is one of the most
evolutionarily conserved genes, sharing high sequence homology from man
to yeast and archaebacteria (6-9). The high abundance and conservation
suggest that VCP plays an important and fundamental role in cellular
activities. As other AAA proteins, VCP and its orthologs have been
shown to play a role in many seemingly unrelated cellular activities.
Remarkably, almost all these activities have been shown to be regulated
by the Ub-Pr degradation pathway, suggesting that VCP may play a fundamental role in the pathway, which in turn regulates the diverse activities. We previously showed that VCP is involved in the
degradation of a number of Ub-Pr substrates (10-12), physically
associates with the 26 S proteasome (12, 13), and binds the
ubiquitinated substrates directly through the multiubiquitin chains
(10). Using in vitro degradation assays, we showed that
depletion of VCP from cell extracts resulted in an inhibition of the
proteolysis of model substrates, while adding back the recombinant VCP
restored the degradation. Thus, VCP is a critical component of the
Ub-Pr degradation pathway (10, 12, 14). Based on these results, we
propose a model whereby VCP acts as a molecular chaperone that targets
the ubiquitinated substrates to the proteasome for degradation, hence
regulates diverse cell functions through the Ub-Pr pathway. In
agreement with our model, recently a set of papers was published from
several laboratories (15-21), all showing a critical role for VCP in
the degradation of proteins associated with the endoplasmic reticulum
(ER). In both Ub-Pr-dependent and ER-associated
degradation, VCP is believed to dissociate the ubiquitinated substrates
from protein complexes or ER membrane, and the dissociation is
presumably powered by the ATPase activity of VCP. As Ub-Pr and
ER-associated degradation is the underlying cause of many diseases, it
is of high importance to study the detailed mechanism of VCP functions, especially its ATPase activity.
The VCP molecule can be divided into three parts: the N-terminal 200 residues, named N domain, and two conserved ATPase domains, D1 and D2.
We previously showed that the N domain is responsible for
multiubiquitin chain binding, and this activity is necessary in
mediating the in vitro degradation (10). Whether this
substrate binding activity is sufficient for mediating the Ub-Pr
degradation is not known. In other words, whether the D1 and D2 ATPase
domains of VCP are also required in Ub-Pr-mediated degradation has not been determined. Most type II AAA members have one ATPase domain with
high sequence conservation, and the other with divergent sequences. It
has been shown in NSF (N-ethylmaleimide-sensitive factor), a
prototype AAA protein, that the highly conserved D1 domain is
responsible for the ATPase activity, while the less conserved D2 domain
is responsible for oligomerization (22). Interestingly, both D1 and D2
domains of VCP are highly similar to each other as well as to the D1 of
NSF (23), implicating that both have the potential of being an active
ATPase. It is, therefore, of high interests to characterize the ATPase
activity of VCP and to identify the specific functions of the two
ATPase domains.
In this study, we performed in vitro degradation assays to
show that, in addition to the substrate-binding N domain, the ATPase domains of VCP are also required in mediating the Ub-Pr degradation. We
systematically characterize the ATPase activity of VCP and provide the
first evidence that the two domains are not catalytically equal: D2 is
responsible for the major enzyme activity at physiological temperature,
whereas D1 is involved in the regulation of heat-induced ATPase activity.
Generation of VCP Mutants--
All VCP mutants were derived from
the wild type (WT) VCP-His6 fusion construct. ND1 (kindly
provided by A. Shaw) (24) and E (10) deletion mutants, containing only
the D1 and D2 ATPase domain, respectively, have been described
previously. All the site-specific mutants were generated using the
QuikChange site-directed mutagenesis kit (Stratagene) and confirmed by
DNA sequencing. Basically, two types of mutants were made. A-type
mutant harbors a mutation at the ATP-binding site in the Walker A motif
where an invariant lysine is replaced with a threonine. B-type mutant harbors a mutation at the ATP hydrolysis site in the Walker B motif
where a glutamic acid is changed to a glutamine. Mutations were
introduced into either D1 or D2 ATPase domain, or both. The following
oligonucleotides (underlined sequence indicates the triplet
codon for the mutated amino acid) and their corresponding complimentary
strands (not shown) were used in the PCR cloning for individual
mutants: A1-His,
5'-TATGGGCCTCCTGGGCCAGGGACGACCCTGATTGCTCGAGCTGTGGCA-3'; A2-His,
5'-TATGGACCTCCTGGCTGTGGGACAACCTTACTGGCTAAAGCCATTGCT-3'; B1-His,
5'-CCTGCTATCATCTTCATCGATCAGCTTGATGCCATTGCACCCAAAAGA-3'; B2-His,
5'-CCCTGTGTACTCTTCTTTGATCAGTTAGATTCAATTGCCAAGGCCCGT-3'.
Purification of His-tagged Fusion Proteins--
The plasmids
carrying VCP variants were transformed into Escherichia coli
strain M15[pREP4](QIAexpress system, Qiagen), and protein
expression was induced according to the manufacturer. The induced cells
were lysed with lysis buffer containing 50 mM sodium
phosphate, pH 7.4, 1 M NaCl, 20 mM imidazole,
10% glycerol, 10 mM In Vitro Degradation Assay--
In vitro
Ub-Pr-mediated degradation assays were performed as previously
described (10, 12). Human cyclin E was synthesized in wheat germ
lysate-based in vitro transcription/translation system
(Promega) in the presence of [35S]methionine/cysteine and
used as the test substrate. Untreated S100 (50 µg of protein)
isolated from actively growing CA46 cells (25) and differentially
treated S100 preparations were used as the enzyme sources. VCP
depletion was carried out by subjecting S100 to immunoprecipitation
with equal volume of VCP-4 antiserum (12). In a typical VCP
reconstitution experiment, 1-4 µg of recombinant VCP-His fusion
protein was used. The degradation reactions were stopped by boiling in
SDS gel sample buffer and analyzed by SDS-PAGE, Western transfer,
and autoradiography.
ATPase Assay--
Except for specified variations, standard
ATPase assays were carried out in the assay buffer containing 50 mM Tris-HCl (pH 8.0), 20 mM MgCl2,
1 mM EDTA, 1 mM dithiothreitol, 3 mM ATP, and 0.5 µM VCP for 15 min at
37 °C. The inorganic phosphate released by ATP hydrolysis was
measured as previously described (26, 27) with modifications. Briefly,
VCP was added to 50 µl of assay buffer, the reaction was carried out
at 37 °C for 15 min, then 400 µl of dye buffer containing 6 mM ammonium heptamolybdate, 120 µM malachite
green, 0.06% polyvinyl alcohol, and 4.25% sodium citrate was added.
After 20 min of incubation at room temperature, 200 µl from each
reaction was transferred to a 96-well plate, and the absorbance at 630 nm was measured using an ELx800 Universal Microplate Reader (Bio-Tek
Instrument). Reactions performed without VCP were routinely included,
and the activity, usually negligible, was subtracted from the
respective experimental data. The inorganic phosphate released was
calculated based on the absorbance standard curve established by
KH2PO4 standards. VCP variants and His-NSF (28)
were affinity-purified to near homogeneity and used in the assays. All
assays were repeated at least three times, and the average activities
with standard errors of measurement (± S.E.) were presented. Kinetic
parameters, Km, Vmax, and Hill coefficient, were derived using the software Prism 3.0 (GraphPad Software, Inc.).
Purification of Wild Type and Mutant VCP-His Proteins--
We
generated a panel of VCP mutants in the form of His fusion proteins
(Fig. 1A, also see Fig. 5 for
gene structure). These include full-length, site-specific mutants and
deletion mutants containing only the D1 (ND1 construct) or D2 (E
construct) ATPase domain. Among the site-specific mutants, mutations
were introduced into the Walker A or Walker B motif of each or both of
the ATPase domains. In naming these mutants, alphabetical A or B
designates the mutation in Walker A or B motif, while numerical 1 or 2 indicates the location of the respective mutation in the first or
second ATPase domain. All mutations were confirmed by DNA sequencing, and all VCP-His fusion variants were expressed in E. coli
and affinity-purified to near homogeneity (Fig. 1A).
Requirement for ATPase Activity of VCP in Ub-Pr-mediated
Degradation--
We previously showed that the N domain of VCP
directly binds to the multiubiquitin chain of proteasome substrates and
is necessary for mediating the Ub-Pr degradation (10). In this study,
we asked whether VCP serves functions other than substrate binding, i.e. whether other domains of VCP are also necessary for the
degradation. We carried out the in vitro degradation assays,
which used the S100 fraction isolated from cell extracts as the Ub-Pr
enzyme source and in vitro synthesized cyclin E as the test
substrate (10). As previously demonstrated, when VCP was depleted from S100, cyclin E degradation was inhibited, whereas adding back the
recombinant WT VCP restored the degradation (Fig. 1B). In contrast, when the substrate-binding N domain (N200) and the
site-specific mutants (A1A2, B1B2, and A2) were used in the
reconstitution, no degradation was restored (Fig. 1B). These
results indicate that although the substrate binding activity is
necessary for an effective Ub-Pr degradation, it is not sufficient.
Functions contributed by the two ATPase domains, D1 and D2, are also
required. Interestingly, although adding back mutant A1 did not fully
restore the degradation, it reproducibly resulted in significant
restoration (Fig. 1B). These data suggest that A1 mutant may
still possess partial activity in mediating the degradation. Based on
the higher restoration obtained in A1 than in A2, we speculated that D2
domain may contribute more to the ATPase activity of VCP.
Characterization of the ATPase Activity of VCP--
To directly
study the ATPase activity, a colorimetric assay was used to measure the
inorganic phosphate (Pi) released from ATP hydrolysis.
Because recombinant VCP exhibited comparable activity as purified
cellular VCP (data not shown), wild type VCP-His fusion protein (WT in
Fig. 1A) was used to characterize the enzyme activity. As
shown in Fig. 2A, wild type
VCP hydrolyzes ATP in a time-dependent manner, with free
phosphate increasing linearly in the initial 30 min. The reaction is
dependent on the concentration of ATP, and the enzyme activity is
saturated when ATP is greater than 3 mM (Fig.
2B). The kinetics were modeled using Enzyme Kinetics template from software Prism 3.0 (GraphPad Software, Inc.). In the
presence of 20 mM magnesium, VCP exhibited a
Km of ~0.33 mM for ATP, a
Vmax of ~0.52 nmol Pi
min Heat-induced ATPase Activity--
Recent studies have suggested an
involvement of VCP/orthologs in stress tolerance pathways (15, 16, 18,
21, 29). We analyzed the in vitro ATPase profile of VCP
under various conditions reflecting environmental stresses (see
"Discussion"). Interestingly, the activity increased significantly
at elevated temperatures and peaked between 50-55 °C (Fig.
2H), a temperature at which most enzymes would be
inactivated. The observation is not unique to the low salt
concentration (open circles) because experiments performed
under physiological ionic strength condition also yielded similar
results (filled squares). Comparison between the enzyme kinetics at 37 and 50 °C (Fig. 2I) revealed an ~60%
increase in Vmax, a small decrease in
Km for ATP, and an ~35% increase in the Hill coefficient.
D2 as the Major ATPase Domain--
The high sequence similarity
between D1 and D2 and the sigmoid kinetic curve in VCP suggest that
both D1 and D2 contribute to the ATPase activity. It has been shown in
several type II AAA members that, although not in specific order, one
domain is essential for forming the oligomers and the other responsible
for the major enzyme activity. Since such structure-function
relationship has not been demonstrated for VCP, we used the mutants to
determine which domain is responsible for the major ATPase activity at
physiological temperatures (Fig.
3A and summarized in Fig. 5).
As negative controls, double mutants harboring mutations in ATP-binding
sites (A1A2) or ATP hydrolysis sites (B1B2) in both domains exhibit
near background ATPase activity. A1, B1, and A1B1 mutants, all
containing a mutated D1 and an intact D2, exhibit an activity slightly
lower than the wild type. By contrast, A2, B2, A2B2, and ND1 mutants,
all containing an intact D1 but a mutation in D2, have a low but
detectable activity. Moreover, deletion mutant E, which contains only
the D2 and no D1 domain, displays a reduced but significant ATPase
activity. Together these results suggest that the second ATPase domain, D2, is responsible for the major ATPase activity, a conclusion consistent with the in vitro degradation experiments (Fig.
1B).
NEM Inhibition of the ATPase Activity of D2--
Another line of
evidence supporting D2 as the major ATPase is provided by its
sensitivity to N-ethylmaleimide (NEM), a cysteine-sensitive alkylating agent. It has been shown that VCP orthologs as well as
homologous proteins, such as NSF, SEC18, etc., possess NEM-sensitive ATPase activity (6, 30, 31). As shown in Fig. 3B, NEM
markedly reduced the activity of wild type, A1, B1, A1B1, and E
variants, all of which contain an intact D2 domain. For those variants
containing only the wild type D1 domain (A2, B2, A2B2, and ND1), NEM
did not further lower their activities. It is known that NEM sometimes causes proteins to be denatured and therefore inhibits ATPase activity.
This is not the case for VCP, because gel filtration analysis showed
that wild type VCP still preserves a hexameric structure in the
presence of 5 mM NEM (data not shown).
D1 as the Mediator for the Heat-induced ATPase Activity--
To
investigate the roles of D1 and D2 in heat-induced ATPase activity, we
measured the activity of various mutants at 37 and 50 °C (Fig.
4 and summarized in Fig.
5). Remarkably, A1 displays a temperature
dependence that peaks at ~40 °C, whereas A2 is most active at
~60 °C (Fig. 4A), suggesting that D1 may play a role in
mediating the heat-induced activity. To further substantiate this
notion, various site-specific mutants were assayed. As evidenced in
Fig. 4B, all constructs possessing an intact D1 domain
(e.g. WT, A2, B2, and A2B2) exhibit a heat-induced activity;
but constructs carrying D1 mutations (e.g. A1, B1, A1A2, and
B1B2) do not. To further demonstrate the contrast, A2, B2, and A2B2
were assayed up to 60 °C, at which all variants exhibit a much
higher activity (Fig. 4C). Moreover, different amounts of
the deletion mutants ND1 (containing only D1) and E (containing only
D2) were analyzed at 37 and 50 °C (Fig. 4D). ND1 exhibits
heat-induced activity at every dosage level, whereas E showed even
slightly decreased activity at 50 °C. Combined with the
aforementioned data, our results (summarized in Fig. 5) suggest that at
the physiological temperature, D2 accounts for the major ATPase
activity; but at elevated temperature or heat shock conditions D1
probably mediates the heat-induced activity.
Our study indicates that Ub-binding activity of VCP is necessary
but not sufficient for mediating the Ub-Pr degradation and that the
chaperone activity, executed by the ATPase domains, is also required.
To characterize the ATPase activity, we generated a variety of VCP
mutants in the form of His-tagged fusion proteins. We noticed that the
purity of each variant is of extreme importance in determining the
ATPase activity, because even trace amount of the co-purified bacterial
ATPases would introduce significant background activities (data not
shown). Both conventional column chromatography and affinity
chromatography with step elution were employed to purify the fusion
proteins. The repeated affinity chromatography proved sufficient to
yield high purity products because the quality of such purified
proteins is similar to that obtained from serial combinations of
conventional chromatography (data not shown).
AAA family members all possess weak ATPase activities. Consistent with
this notion, VCP indeed exhibits an ATPase activity ~10-fold weaker
than Na+/K+-ATPase (data not shown). The
estimated Km of ~0.33 mM for ATP and
Vmax of ~0.52 nmol Pi
min The chaperone activity of AAA family proteins is often modulated by
cellular environment. We tested the ATPase activity of VCP under
conditions reflecting environmental stresses, including heat, pH,
osmotic, alcohol, and oxidative stresses. We found that the activity is
stimulated by high pH values, but inhibited by the presence of salt
(Fig. 2G), ethanol, ADP, and hydrogen peroxide (data not
shown). Most interestingly, the activity is significantly stimulated by
high temperature (Fig. 2, H and I), peaking
around 50-55 °C. Since most enzymes are inactivated at such
temperature, the stabilization of VCP may be attributed to its own
chaperone activity, namely the prevention of heat-induced denaturation
of substrates (itself in this case)
(34).2
Because of the essential nature and high conservation of VCP, it is
important to characterize the specific functions of each domain. In
this study, we report the first structure-function analyses in VCP. Our
conclusion of D2 as the major ATPase domain is supported by several
lines of in vitro evidence: 1) deletion and site-specific
mutants containing only the intact D2 exhibit significant activity; 2)
mutants lacking an intact D2 have severely reduced activity; 3)
sequence comparison among VCP, NSF, and FtsH (a type I AAA-ATPase)
reveals that the "Second Region of Homology" (35) in D2, but not in
D1, of VCP shares high sequence similarity with those in the identified
ATPase domain of NSF and FtsH; 4) depletion and add-back experiment
shows that A1, but not A2, mutant restores the Ub-Pr-mediated
degradation significantly; and 5) our recent studies further indicate
that D1 of VCP plays a critical role in mediating hexamerization
of VCP.3 The importance of
D2 has also been demonstrated in in vivo studies. A yeast
mutant harboring a point mutation in D2 exhibited typical apoptotic
phenotypes (36). Expression of VCP with D2 mutation (equivalent to our
A2 mutant) created massive cytoplasmic vacuoles and cell death in a
neurodegenerative disease model (37). Moreover, a recent study on
trypanosome showed that D2, but not D1, is essential for TbVCP activity
and cell viability (38).
The identification of D2 as the major ATPase domain raised the question
of how other domains of VCP influence the enzyme activity. Previous
reports show that tyrosine phosphorylation at the C terminus of VCP
correlates with nuclear translocation in a cell cycle-specific manner
(39) and that phosphorylated VCP promotes its own release from
associated membranes (40). These findings seem to suggest that the
C-terminal domain of VCP may also regulate its biological/enzymatic functions. Nevertheless, mutation of these phosphorylation sites did
not affect the ATPase activity (Ref. 41 and data not shown). Physical
binding of specific antibody to the C terminus of VCP also did not
impair the ATPase activity (data not shown). Therefore, tyrosine
phosphorylation and the conformation of the C terminus of VCP likely do
not play a significant role in regulating its ATPase activity. However,
our recent trypsin digestion study suggested that the C-terminal ~100
residues of VCP form a loosely structured tail.3 Thus, it
is formally possible that association of this relatively flexible C
terminus with other cellular proteins could modify the overall enzyme
activity of VCP.
Does the D1 oligomerization domain affect the ATPase activity at
physiological temperature? It has been established in several AAA
proteins that an oligomeric structure is a prerequisite to the ATPase
activity and that these proteins require the presence of nucleotide to
form oligomers (22, 33, 42, 43). Consistently, monomeric VCP does not
have any enzyme activity (44).3 However, in contrast to
these AAA proteins, VCP does not require the presence of nucleotide to
form hexamers.3 The hexameric VCP is highly stable, and
many attempts have failed to dissociate it into stable monomers. This
unusually high preponderance to form functional hexamers in VCP further
emphasizes its essential nature in the cell. This can also be observed
in our mutants, A1, B1, and A1B1, all containing mutations in D1 yet
still appearing to be hexamers.3 Nevertheless, these
mutations do introduce conformational alterations to the ring
structure. A lower ATPase activity in these mutants (relative to the
wild type) (Fig. 3A) likely resulted from the lacking of a
perfect hexameric structure and a proper intersubunit communication.
Thus, under physiological conditions, the D1 domain does play a minor
role in the steady-state kinetic behavior of the ATPase domain.
In contrast to the physiological condition, D1 probably plays an
important role in mediating an elevated activity in heat-stressed condition (Figs. 4 and 5). As the wild type VCP exhibits heat-induced activity, which peaks around 50-55 °C, mutation in D1 abolishes this heat-inducibility. On the other hand, mutants containing a mutated
D2 and an intact D1 consistently exhibit a low activity at 37 °C but
a significantly higher activity at a temperature greater than 50 °C.
It is noted that when comparing the activity of the wild type with that
of the sum of A1 and A2, the former is always greater than the latter,
especially in the high temperature range (data not shown). This
observation suggests that D1 and D2 rings communicate and cooperate
with each other in hydrolyzing ATP (as discussed above), and this
cooperative effect is more profound at elevated temperature. While the
molecular basis for this heat response is unknown, we speculate that
the structure of the two hexameric rings plays an important role.
Previous studies revealed that it is relatively easy to obtain crystal
structure of the oligomerization domain of type II AAA proteins,
probably because the ring made up from such domains adopts a more
stable and compact structure. In contrast, to this date, there is no crystal structure available for the ATPase domain, likely due to its
more flexible and relaxed conformation. Since VCP undergoes significant
conformational changes during ATPase cycles (45), it is conceivable
that upon heat treatment, the originally compact D1 ring expands and
becomes more flexible and accessible to the solvent, thus facilitating
ATP hydrolysis more readily.
Based on the heat-induced ATPase activity, VCP may be qualified as a
heat-shock protein, and may have heat-stimulated chaperone activity
which directly mediates the heat-shock responses. Interestingly, Hsp104, a two-domain-containing AAA super family chaperone, has been
characterized to have many biochemical properties similar to those of
VCP (46). The ATPase activity of Hsp104 is strongly influenced by
factors that vary with cell stress, e.g. temperature, pH,
and ADP. It is crucial for stress tolerance in Saccharomyces cerevisiae, and both of its ATPase domains are required in
mediating the stress responses. Hsp104 is essential in protecting cells at extreme temperatures, e.g. 50 °C, and this protection
is related to its ability to promote the solubilization and
reactivation of damaged and aggregated proteins (47, 48). So far, no
mammalian Hsp104 proteins have been found. Based on the importance of
Hsp104 and the similar biochemical properties observed in the two
chaperones, it will be of profound interest to examine whether VCP
possesses similar biological functions.
1 µg
1. At a
physiological temperature, mutation in D2 significantly inhibits the
ATPase activity, while that in D1 has little effect. Interestingly,
mutations in D1, but not D2, abolish the heat-stimulated ATPase
activity. Thus, we provide the first demonstration that the ATPase
activity of VCP is required for mediating the Ub-Pr degradation, that
D2 accounts for the major ATPase activity, and that D1 contributes to
the heat-induced activity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol, and protease inhibitor mixture for His-tagged proteins (Sigma, number P8849). The
cleared cell lysates were mixed with nickel-nitrilotriacetic acid-agarose beads for 1 h at 4 °C, and the fusion proteins
were step-eluted with the elution buffer (lysis buffer containing 50, 100, 250, or 500 mM imidazole). Repeated affinity
purification was often performed to obtain the high-purity product. The
eluted fractions were analyzed by SDS-PAGE, and the purified proteins were dialyzed against 20 mM Tris-HCl, pH 8.0 with 10%
glycerol. His-NSF fusion protein was expressed and purified with
similar procedures. The purified proteins were quantified by BCA assay (Pierce) and analyzed by SDS-PAGE and Coomassie Blue staining.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Expression and purification of VCP-His fusion
variants. A, purified wild type and mutant VCP proteins. WT
and mutant (as indicated on the top) VCP-His fusion proteins
were expressed in E. coli and purified as described under
"Experimental Procedures." The purified proteins (2.5 µg in each
lane) were resolved by SDS-PAGE and stained with Coomassie
Blue. The molecular size standards are shown on the right.
B, requirement for ATPase domains for in vitro
degradation of cyclin E by the ubiquitin-proteasome degradation
pathway. In vitro degradation assays were performed using
radioactively labeled cyclin E as the substrate and untreated or
differentially treated (as indicated on top) S100 as the
source of enzymes for the Ub-Pr system. The reactions were terminated
at the indicated periods of time and analyzed by SDS-PAGE, Western
transfer, and autoradiography. The test substrate cyclin E is
indicated.
1 µg
1, and a Hill coefficient of 1.2. Since VCP almost always adopts a stable hexameric ring structure (data
not shown), the derived Hill coefficient probably suggests a
cooperative nature in hydrolyzing ATP among the subunits. Comparison
between VCP and NSF revealed that VCP exhibits a near 5-fold activity
over NSF (Fig. 2C) under our experimental condition. The
activity is VCP dose-dependent up to 1 mM and
displays a sigmoid kinetic profile (Fig. 2D), probably reflecting the activities of the two ATPase domains. The ATPase activity requires the presence of divalent cations, e.g.
Mg2+ (Fig. 2E). Although Mn2+ and
Co2+ at a low concentration also moderately support the
ATPase activity, other biologically relevant divalent cations including
Cu2+, Cd2+, Cr2+, Ca2+,
Fe2+, and Zn2+ do not (Fig. 2E).
Titration experiments further determine the optimum concentration for
Mg2+ to be 20 mM (Fig. 2F). Notably,
at concentrations greater than 20 mM, the ATPase activity
is markedly suppressed. This probably reflects a general inhibitory
effect of the high salt conditions rather than a
Mg2+-specific phenomenon. The presence of NaCl (Fig.
2G) and KCl (data not shown) actually suppresses the ATPase
activity, but does not significantly change the hexameric structure of
VCP (data not shown). Based on the initial characterization of the
ATPase reaction, 0.5 µM VCP, 3 mM ATP, 20 mM Mg2+, and a 15-min reaction time were
routinely used in the rest of the study.
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Fig. 2.
Characterization of the ATPase activity of
VCP. A, time dependence of ATPase activity of VCP. ATP
hydrolysis was carried out at 37 °C for the indicated time
(min). The released inorganic phosphate was quantified by
malachite green assay. Activities were taken from the average of three
independent experiments. B, ATPase activity of VCP at
various ATP concentrations. Standard ATPase assay (as described under
"Experimental Procedures") was carried out using 0.5 µM VCP and the indicated concentration of ATP.
C, comparison of ATPase activity of VCP and NSF. Equal
amount (0.5 µM) of VCP-His or His-NSF fusion protein was
used in the standard in vitro ATPase assay. D,
ATPase activity at various concentrations of VCP. Standard assay was
carried out using the indicated amount of wild type VCP. E,
requirement of divalent cations for the enzyme activity. ATPase
activity was measured in the presence of different divalent cations at
a concentration of either 2 mM (filled bar) or
20 mM (open bar). Control (Ctrl), no
cation included. F, determination of the optimal
Mg2+ concentration for ATPase activity. Standard assay was
performed in the presence of indicated concentration of
MgCl2. Note that the concentrations below and above 50 mM are presented in different scales. G,
inhibitory effect of salt on ATPase activity. The ATPase activity was
measured in the presence of the indicated concentration of NaCl at
37 °C. H, temperature effect on the ATPase activity of
VCP. The enzyme activity was measured at the indicated temperatures
with either 0 (open circle) or 135 mM NaCl
(filled square). I, kinetic study at different
temperatures. Reactions were carried out in the presence of varying
amounts of ATP at either 37 (open circle) or 50 (filled circle) °C. The data represent an average of three
independent experiments.
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Fig. 3.
D2 as the major ATPase domain in VCP.
A, ATPase activity of VCP variants. Purified VCP variants
(as shown in Fig. 1A) were used in the standard ATPase
assays at 37 °C. Activities were taken from the average of three
independent experiments. Activity detected in the wild type is assigned
as 100%, and those detected in other variants are expressed as % of
the wild type. B, inhibition of ATPase activity of VCP by
NEM. Standard ATPase assays were carried out using the indicated VCP
variants in the absence (filled bar) or presence (open
bar) of 5 mM NEM. Relative activity is presented with
the activity detected in the wild type without NEM as 100%.
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Fig. 4.
D1 as the mediator for the heat-induced
ATPase activity. A, temperature dependence of the ATPase
activity in A1 and A2. An equal amount (1.5 µM) of A1
(open circle) or A2 (filled circle) was measured
for the ATPase activity at the indicated temperatures. A representative
experiment is shown. Relative activity (in %) is shown with the peak
activity of A1 as 100%. B, correlation between the
heat-induced activity and intact D1. VCP variants at 0.5 µM were tested for the ATPase activity at 37 °C
(filled bar) or 50 °C (open bar). Data
represent the mean ± S.E. (n = 3). Relative
activity is shown with the maximum activity of WT at 50 °C as 100%.
C, heat-induced activity in A2, B2, and A2B2 mutants.
Indicated variants (0.5 µM) were tested for the ATPase
activity at 37 °C (open bar), 50 °C (gray
bar), or 60 °C (filled bar). The activity detected
in B2 at 60 °C is assigned as 100%. D,
heat-induced activity in deletion mutant ND1 but not E. Various amounts
of ND1 (circle) and E (square) were tested for
the ATPase activity at either 37 °C (open) or 50 °C
(filled). The relative activity is presented with the
highest activity in E as 100%. A representative experiment is
shown.
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Fig. 5.
Schematic representation of VCP variants and
summary of the ATPase activities at 37 °C and the heat inducibility
at 50 °C. The A-type (K to T) and B-type (E to Q) site-specific
mutations are depicted. The ATPase activities at 37 °C (from Fig. 3)
are summarized as +++, ++, +, , and
, which indicate high to low
activities. The results from the heat inducibility study (from Fig. 4)
are also summarized in which + or
indicates the presence or
absence of the heat-induced ATPase activity.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 µg
1 are in agreement with a
Km of 0.62 mM and
Vmax of 0.3 nmol Pi
min
1 µg
1 previously reported for the rat
liver p97 (32). The ATPase activity reaches the maximum at an ATP
concentration of 3 mM (Fig. 2B). Thus, the
enzyme activity can be modulated by the level of ATP within a normal
physiological concentration of ATP (0.5-2 mM). At a
physiological temperature, VCP has a significantly higher activity than
NSF (Fig. 2C). Since both proteins were purified in the
absence of ATP, which is required for hexamerization of NSF (22, 33)
but not VCP (data not shown), the low activity in NSF is possibly a
result from the lack of hexamers. However, this is not the case because
the ATPase assay using NSF preincubated with ATP (thus preassembled
hexamer) did not yield significantly higher activity. It is likely that
the presence of ATP rapidly facilitates the hexamerization of NSF in
our assay. While both VCP and NSF play an essential role in mediating
membrane fusion, it is not clear how the difference in ATPase activity
translates into functional significance. Nevertheless, it is tempting
to speculate that the involvement of VCP in the Ub-Pr pathway requires a higher enzyme activity than that in the membrane fusion. This is
supported by the observation that p47, a cofactor required in membrane
fusion events but not in Ub-Pr degradation, suppresses the enzyme
activity of VCP (32).
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ACKNOWLEDGEMENT |
---|
We thank R. Dai for excellent technical assistance, Dr. S. A. Whiteheart for NSF plasmid, Drs. A. Shaw and X. Zhang for ND1 plasmid, and Drs. S. Gottesman, M. Maurizi, S. K. Singh, and S. Wickner for helpful discussions.
![]() |
FOOTNOTES |
---|
* This work has been supported with Federal funds from the NCI, National Institutes of Health under Contract No. NO1-CO-56000. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organization imply endorsement by the United States Government.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.
¶ To whom correspondence should be addressed. Tel.: 301-846-1478; Fax: 301-846-7034; E-mail: licc@ncifcrf.gov.
Published, JBC Papers in Press, November 20, 2002, DOI 10.1074/jbc.M208422200
2 C. Song, Q. Wang, and C.-C. H. Li, unpublished data.
3 Q. Wang, C. Song, and C.-C. H. Li, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: VCP, valosin-containing protein; Ub-Pr, ubiquitin-proteasome; ER, endoplasmic reticulum; NSF, N-ethylmaleimide-sensitive factor; WT, wild type; NEM, N-ethylmaleimide.
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