From the
Evidence suggests that ATP hydrolysis catalyzed by the
clathrin-coated vesicle proton-translocating ATPase requires at least
four polypeptides of molecular masses of 70, 58, 40, and 33 kDa (Xie,
X.-S., and Stone, D. K.(1988) J. Biol. Chem. 263,
9859-9867). To further investigate the subunit requirements for
ATP hydrolysis, histidine-tagged, 58-kDa polypeptide was expressed in
insect Sf9 (Spodoptera frugiperda) cells. After purification
by Ni
Over the past decade, the importance of the vacuolar
H
V-type ATPases structurally resemble
F
At
the present time, it is not known whether this
Ca
Full definition of the
molecular structure of the enzyme and the function of individual
subunits by a purely biochemical resolution and reconstitution approach
has proven difficult because of limited starting materials for such
experiments. As an alternative, we are currently reconstituting partial
reactions of the V-type ATPase with recombinant polypeptides. By this
strategy, we have recently reconstituted recombinant 40- (19) and 33-kDa (20) subunits to biochemically prepared
fractions that were deficient in these components and have demonstrated
that both are subunits essential for ATP hydrolysis. We have also
cloned and expressed the 70-kDa polypeptide in Sf9 cells. Although this
purified component could not support ATP hydrolysis, it was labeled by
[
With respect
to the 58-kDa component, the subject of this study, cDNAs encoding this
subunit have been cloned from many different sources (22-30). In
addition, there is evidence that isoforms of the 58-kDa subunit are
present in eukaryotic
tissues(22, 23, 26, 28) . Amino acid
sequence analysis reveals that the primary structure of the 58-kDa
subunit is highly conserved and contains an ATP binding motif. In this
respect, the 58-kDa subunit resembles the
We have previously cloned cDNAs coding for the 58-kDa subunit from
human kidney (22) and bovine brain.
The coding region for the 58-kDa subunit of the V-type
H
The bacterial expression vector p
Spodoptera frugiperda (Sf9) cells were grown in monolayer
or in suspension culture at 27 °C in either Grace's or IPL-41
medium with 10% heat-inactivated fetal bovine serum plus 0.1% pluronic
polyol F68. Cells were split 1:5 for propagation every 3-4 days.
Recombinant baculovirus was generated by cotransfection of Sf9 cells by
the lipofection method with purified vector p
For expression and production of the 58-kDa protein,
Sf9 cells (2
500 ml of Sf9 cells were infected with pure recombinant
baculovirus, incubated in suspension culture for 48 h at 27 °C, and
harvested by centrifugation at 4,000
Polyclonal antibody against SDS-denatured, recombinant 58-kDa
subunit, expressed in Escherichia coli BL21(DE3) with
p
The clathrin-coated vesicle H
ATPase activity was measured by the liberation of
Photoaffinity labeling of the recombinant 58-kDa subunit was
performed as described (21, 42) with modification. A
reaction mixture (20 µl) containing 0.2 µg of purified 58-kDa
protein, 10 mM Tris/MES (pH 7.0), 10% glycerol, radioactive
ATP or ADP, and other components as indicated in the legends to figures
was incubated at room temperature for 5 min and then irradiated for 8
min on ice at a distance of 35 mm from the UV source (model R-51,
Spectronics Corp., Westbury, NY). The reactions were terminated by the
addition of 2.2 µl of 10
Protein determination (43) and SDS-PAGE (44) were performed as reported. The sources of all enzymes,
vectors, and chemical reagents used in this study have been previously
described(9, 19, 20, 21) .
As an alternative, we constructed and
purified a recombinant baculovirus containing the cDNA sequence
encoding the 58-kDa protein that was used to infect Sf9 cells. The
recombinant baculovirus directed the expression of a fusion protein
containing a histidine tag at the amino terminus. As shown in Fig. 1, the 58-kDa protein was expressed in Sf9 cells infected
with recombinant virus. The time course of expression of the 58-kDa
protein in Sf9 cells after infection with the virus was studied to
optimize conditions for protein production. At designated time points
over a 96-h course of infection, aliquots of cells were removed and
analyzed by SDS-PAGE and Western blot analysis. Recombinant 58-kDa
protein was detected as early as 36 h postinfection by polyclonal
antiserum against recombinant 58-kDa polypeptide (Fig. 1B, lane3) and was readily
visualized by Coomassie Blue-stained, SDS-PAGE 48-h postinfection (Fig. 1A, lane4). The production of
recombinant 58-kDa protein reached a maximal level by 60-72 h (Fig. 1A, lanes5 and 6),
constituting approximately 3-5% of the total cellular proteins.
The 58-kDa protein was not detected by SDS-PAGE and Western blot
analysis in the cells that were not transfected with recombinant virus (Fig. 1, A and B, lanes1).
Fig. 3illustrates the influence of various
concentrations of recombinant 58-kDa polypeptide on the reconstitution
activity of ATP hydrolysis. The stimulation of
Ca
A major issue in the field of V-type proton pumps centers
upon the subunit composition of these enzymes and the role of the
individual components in pump function. Reconstitution of function with
biochemically prepared pump fractions suggested that at least four
polypeptides of 70, 58, 40, and 33 kDa are necessary for ATP hydrolysis
(11). As an alternative, we have taken another route for further
resolution of subunit composition and function, in which we clone,
express, and purify the candidate subunits and then attempt to
reconstitute ATPase activity. In this way, we have shown that 40- (19) and 33-kDa (20) polypeptides are essential for ATP
hydrolysis and that they are genuine subunits of the V-type ATPase. We
also demonstrated that recombinant 70-kDa polypeptide alone can be
labeled by [
In the current investigations, we have
turned our attention to the 58-kDa subunit. Previously, we have
successfully purified and renatured the 40- and 33-kDa subunits, which
were expressed in E. coli. Initially, we pursued a similar
approach with the 58-kDa subunit but were unable to renature the
purified protein to yield a reconstitutively active form because of the
aggregation of overexpressed 58-kDa protein in E. coli.
Consequently, we adopted an expression and purification protocol using
baculovirus and Sf9 cells, which we previously utilized for the 33- (20) and 70-kDa subunits(21) .
The 58-kDa component of
V-type ATPase has been considered to be a potential nucleotide binding
protein because of its similarity in amino acid sequence to the
The labeling of the recombinant 58-kDa subunit
with nucleotides is striking in consideration of the observations that
labeling of holoenzyme occurs only on the 70-kDa component. Several
possible explanations for this phenomenon exist. First, the fact that
this polypeptide is recombinant is potentially worrisome in that
improper folding or disordered tertiary structure might arise during
expression itself or during the isolation procedure. By developing a
protocol for harvesting a soluble 58-kDa polypeptide, this possibility
has been minimized. More convincing is the fact that the recombinant
58-kDa polypeptide used for the labeling experiments is
reconstitutively active, as shown in .
A second
potential problem of this nature might arise from the presence of 10
histidine residues at the amino terminus. Attempts at removing these
residues with factor Xa was not successful because of additional
cleavages of the polypeptide. Again, the fact that the histidine-tagged
58-kDa polypeptide is reconstitutively active indicates this is not
likely to cause wholesale distortions in structure. Analysis of the
structure of the
A third possibility, and the mostly
likely, is that under most circumstances, the ATP binding site of the
58-kDa subunit is inaccessible to nucleotide exchange in the native
enzyme and thus is functionally and structurally analogous to the
The most important achievement of this
investigation lies in the reconstitution of purified and recombinant
58-kDa polypeptide. As demonstrated in , recombinant 58-kDa
polypeptide alone or other fractions used for reconstitution in this
experiment have trivial Ca
In addition to
demonstrating, for the first time, the essential role of the 58-kDa
polypeptide in V-type ATPase, this work further underscores our point
that maximal ATP hydrolysis requires multiple components and their
interactions. An accepted view is that ATP hydrolysis actually occurs
on the 70-kDa or 70- and 58-kDa components and that the 40- and 33-kDa
subunits render the catalytic site active. As shown in ,
recombinant 70- and 58-kDa components together did not support
significant ATP hydrolysis, and any three out of four components (70,
58, 40, and 33 kDa) demonstrated only minimal ATPase activity.
Therefore, at least these four subunits are essential for full function
of catalytic sector. With this observation, we are now prepared to
undertake the reassembly of the catalytic sector from wholly
recombinant components.
ATPase
activities were measured by the liberation of
I thank Drs. Dennis K. Stone, Xiao-Song Xie, and Bill
P. Crider for helpful discussions and suggestions and Ying Zhang for
construction of the bacterial expression vector p
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-nitrolotriacetic acid chromatography, the
58-kDa protein was found to lack significant ATPase activity. However,
the subunit was photoaffinity labeled with
[
-
P]ATP, [
C]ADP, or
S-labeled ADP and UV irradiation in a divalent
cation-dependent manner. The labeling was saturable with an apparent K
of 4 µM for both ATP and
ADP. ATP and ADP competition labeling experiments indicate that the two
nucleotides share the same binding site. When reconstituted with
recombinant 70-kDa subunit and a biochemically prepared catalytic
sector (V
) depleted of the 70- and 58-kDa subunits, the
58-kDa component restores Ca
-activated ATP hydrolysis
to a specific activity of 0.19 µmol P
mg
protein
min
, thus
demonstrating that ATP hydrolysis in vacuolar type proton pumps is
dependent upon the 58-kDa subunit as well as multi-subunit
interactions.
ATPases (V-type ATPase) has become increasingly
apparent. These ATP-driven proton pumps acidify a variety of
intracellular compartments in eukaryotic
cells(1, 2, 3) , and the acidification of
lysosomes, secretory vesicles, and storage vacuoles plays a critical
role in many basic cellular processes. Vacuolar acidification is
necessary for protein degradation in lysosomes, ligand-receptor
processing in endosomes, peptide maturation in secretory vesicles,
neurotransmitter uptake in synaptic vesicles, and the concentration of
nutrients in storage vacuoles. From a biomedical standpoint, V-type
ATPases are of interest because of their role in the pathogenesis of
osteoporosis (4, 5, 6) and the systemic acidosis
of uremia (7). More recent evidence suggests that V-type ATPase may be
involved in resistance to chemotherapeutic agents in cultured tumor
cells(8) .
-F
ATP synthetases and are complex
hetero-oligomers with molecular masses of greater than 500 kDa. At
least 10 polypeptides are considered candidate subunits; these have
apparent molecular masses of 116, 70, 57, 58, 50, 40, 38, 34, 33, and
17 kDa(9, 10) . Attempts to determine subunit
composition by means of the resolution and reconstitution approach has
led to the definition of several functional domains of the
clathrin-coated vesicle H
-ATPase. These are a
peripheral ATP hydrolytic sector (V
)(11) , a
bafilomycin sensitive proton channel
(V
)(12, 13) , and a soluble regulatory
element(14) . Dissociation of the peripheral components of
purified pump leads to the generation of an ATP hydrolytic sector,
which functions in the presence of Ca
-ATP but not
Mg
ATP(11) . Although the components of this
active catalytic sector, V
, are under active investigation,
the relevance of Ca
-activated ATPase activity to
native enzyme function was established with the demonstration that
dissociated pump components could be reassembled and transformed from a
Ca
-activated to Mg
-activated mode
by a novel, 50-57-kDa polypeptide heterodimer(10) .
-activated ATPase activity is physiologically
important. Myosin and kinesin switch from a
Mg
-activated ATP-hydrolytic state to a
Ca
-activated mode with removal of actin and
microtubules, respectively(15, 16) . This transition
serves to prevent futile ATP hydrolysis, and such a phenomenon may
occur during the biogenesis of V-type H
-ATPases.
Alternatively the Ca
-ATPase activity of V
may resemble that of chloroplast F
(CF
),
wherein Ca
-ATPase activity is manifested through
several nonphysiological manipulations, such as protease and detergent
treatment, or heating(17, 18) . Irrespective of this
issue, however, analysis of the polypeptide requirements for
Ca
-activated ATP hydrolysis has provided a useful
assay for subunit identification in V-type proton pumps. Moreover, the
subunit-dependent reversibility of the transition (10) further
supports its potential cellular importance.
-
P]ATP and UV irradiation. When
reconstituted to a 70-kDa-depleted fraction, it restored
Ca
-activated ATP hydrolytic activity. This evidence
is consistent with the proposal that the 70-kDa subunit is the actual
ATP hydrolytic center of the V-type ATPase(21) .
subunit of
F
-ATPase. Although several laboratories have conducted
labeling experiments with ATP, nucleotide analogues, and/or inhibitors
(31-35), the 58-kDa component was labeled in only one study,
where it was found that the photoactivable ATP analogue
-
P-labeled 3-O-(4-benzoyl)benzoyl-ATP bound
to this subunit(31) . However, in this instance, the 70-kDa
polypeptide, which was consistently labeled by nucleotide in all other
studies(32, 33, 34) , was not labeled.
Therefore, to date, the view that the 58-kDa subunit of the V-type
ATPase is consistently a nucleotide binding polypeptide is equivocal.
(
)In
the current study, we have expressed the 58-kDa protein in insect Sf9
cells with a recombinant baculovirus. When purified, the recombinant
58-kDa subunit can be photoaffinity labeled with
[
-
P]ATP, [
C]ADP, and
S-labeled ADP and UV irradiation in a divalent-cation
dependent manner, indicating that the 58-kDa subunit contains a
nucleotide binding domain. When reconstituted with biochemically
prepared 40- and 33-kDa subunits and a recombinant 70-kDa polypeptide,
the 58-kDa subunit restores Ca
-activated ATP
hydrolysis to a specific activity of 0.19 µmol of P
mg of protein
min
, thus demonstrating that ATP hydrolysis in
vacuolar-type proton pumps is dependent upon the 58-kDa subunit as well
as multi-subunit interactions.
Plasmid Construction
-ATPase was amplified by polymerase chain reaction
using cloned cDNA (22) as a template and two primers
(5`-CTGCCATATGGCCATGGAGATAGACAGCAGG-3` and
5`-GCGTGATCATCTAGAGCGCAGTGTCAGGCGCGAGG-3`), which were designed to
contain NdeI and BclI restriction sites and initiator
and stop codons at their 5`-ends, respectively, to enable cloning into
the expression vector p
(36) . The amplification
reaction was performed in a Gene Machine thermal cycler using the
following conditions: 1 min at 94 °C and 5 min at 68 °C, for a
total of 30 cycles. The polymerase chain reaction product was size
fractionated by agarose gel electrophoresis, from which a single
1.6-kilobase fragment was identified and purified. The fragment was
identical to that previously reported(22) , as determined by
restriction mapping and direct DNA sequencing by the dideoxy
termination method(37) . Expression vector p
was constructed by replacing the NdeI and BamHI
fragment of p
with the polymerase chain
reaction-amplified fragment, which had been digested with NdeI
and BclI. Another expression vector, p
,
which expresses a fusion protein containing 10 histidine residues and a
factor Xa recognition sequence at the amino terminus of the 58-kDa
protein, was constructed by replacing the NdeI and HindIII fragment of p
with NdeI-HindIII fragment of p
.
Expression of the 58-kDa Subunit in Insect Sf9 Cells
Using a Baculovirus Vector
was
digested with XbaI, and the resultant 1.6-kilobase fragment,
which contained the entire coding region for the 58-kDa subunit, was
cloned into XbaI sites of the baculovirus expression vector
p
. The resultant plasmid, p
,
was characterized by restriction mapping and partial DNA sequencing.
Plasmid DNA was prepared with alkaline lysis and purified by two rounds
of cesium chloride gradient centrifugation(38) .
DNA and linearized AcRP23-LacZ viral DNA(21, 39) .
Positive recombinant viral clones were isolated by plaque assay and
identified by their ability to direct the expression of the 58-kDa
protein, as determined by SDS-PAGE
(
)and Western
blot analysis.
10
cells/ml medium) were grown in
suspension culture at 27 °C, infected with pure recombinant virus,
and maintained in culture for designated time periods to optimize
expression.
Purification of the Recombinant 58-kDa Subunit from
Sf9 Cells
g for 15 min. The
cells were then resuspended in 30 ml of sonication buffer consisting of
50 mM NaH
PO
(pH 8.0), 500 mM NaCl, 0.1 mM phenymethysulfonyl fluoride, and sonicated
for 1 min. The preparation was examined under the microscope to achieve
efficient cell lysis with minimal sonication. The lysate was then
centrifuged at 40,000 rpm for 1 h at 4 °C with a Beckman 70 Ti
rotor. The resultant supernatant was used to purify the recombinant
58-kDa protein by the following steps.
Step 1: Ni
A 3-ml Ni-NTA Resin
Chromatography
-NTA resin column
was prepared and equilibrated with sonication buffer as
described(40) . Supernatant (30 ml) was loaded onto the column,
which was then washed with 30 ml of sonication buffer, 30 ml of wash
buffer (20 mM Tris/HCl (pH 8.0), 0.01%
C
E
, and 20% glycerol), and 200 ml of wash
buffer containing 20 mM imidazole (pH 8.0). The protein was
eluted with 20 ml of wash buffer plus 200 mM imidazole, and
1-ml fractions were collected and analyzed by SDS-PAGE.
Step 2: CM-Sepharose (CL-6B)
Chromatography
Selected fractions (5 ml) from
Ni-NTA chromatography were combined and diluted with
3 volumes of buffer A (10 mM Tris/MES, pH 7.0, 1 mM EDTA, 0.01% C
E
, 1 mM dithiothreitol, 20% glycerol) and loaded onto a 2-ml CM-Sepharose
column, which had been pre-equilibrated with buffer A. The pass-through
fraction was saved for the next step.
Step 3: DEAE-Sepharose (CL-6B)
Chromatography
The fraction from step 2 (20 ml) was loaded
onto a pre-equilibrated 2-ml DEAE-Sepharose column, which was
sequentially washed with 10 ml of buffer A and 20 ml of buffer A plus
100 mM NaCl. The 58-kDa subunit was eluted with 10 ml of
buffer A containing 400 mM NaCl, and 1 ml fractions were
collected and analyzed by SDS-PAGE. The peak fractions were dialyzed
against 300 volumes of buffer A without dithiothreitol and used for
reconstitution of ATP hydrolysis and nucleotide labeling experiments.
Antibody Preparation and Western Blot Analysis
, was raised in a New Zealand White female rabbit
with the same method to prepare polyclonal antibodies against 40- and
33-kDa proteins(19, 20) . For Western blot analysis,
cell lysates and proteins were separated on a 11% SDS-polyacrylamide
gel and transferred electrophoretically to nitrocellulose paper.
Immunodetection was performed using Amersham ECL Western blotting
system.
Purification of Clathrin-coated Vesicle
H
-ATPase and Generation of a Fraction Depleted of
the 70- and 58-kDa Subunits
-ATPase complex
was solubilized with C
E
and purified from
clathrin-coated vesicles of bovine brain as described(9) .
Generation of the 70- and 58-kDa-depleted fraction was performed
generally as follows. Briefly, proton pump complex (750 µg of
purified protein) from the final glycerol gradient was treated with 3 M urea on ice for 1 h. The mixture was adjusted to pH 7.1 with
MES and loaded on a 15-ml CM-Sepharose column, which was
pre-equilibrated with 3 M urea in a buffer containing 30
mM Tricine-Na, pH 7.5, 0.02% C
E
, 0.5
mM EDTA, 3 mM dithiothreitol, and 10% glycerol. The
proteins were eluted with 0-500 mM NaCl in the same
buffer without urea. Fractions depleted of 70- and 58-kDa polypeptides
were combined, saturated (NH
)
SO
was
added to a final concentration of 65%, and the mixture was centrifuged
at 80,000
g for 25 min. The pellet was dissolved in
the above buffer and was layered over a 10-30% linear glycerol
gradient prepared in the same buffer (as above, without urea, and
without glycerol). After centrifugation at 180,000
g for 22 h at 4 °C, fractions were harvested from the bottom of
the tube, subjected to SDS-PAGE, analyzed for
Ca
-activated ATPase activity, and used for
reconstitution experiments described under ``Results.''
Purified proton-translocating ATPase and the 70- and 58-kDa
subunit-depleted fraction utilized for reconstitution experiments are
illustrated in Fig. 2. The protein bands illustrated in Fig. 2were quantitatively evaluated on an LKB 2202 Ultrascan
Laser Densitometer to determine the degree of subunit depletion.
Figure 2:
SDS-PAGE of purified recombinant 58- and
70-kDa proteins and biochemically prepared proton pump and
polypeptides. SDS-PAGE was performed as described under
``Experimental Procedures.'' Lane1,
purified clathrin-coated vesicle proton pump from bovine brain; lane2, purified ATP catalytic sector
(V); lane3, biochemically prepared
V
depleted of 70- and 58-kDa polypeptides; lane4, purified recombinant 58-kDa protein; lane5, purified recombinant 70-kDa
subunit.
Measurement of ATPase Activity
P
from [
-
P]ATP.
The assay solution consisted of 30 mM KCl, 50 mM Tris/MES (pH 7.0), 2.5 mM CaCl
, and 2 mM [
-
P]ATP (200-400 cpm/nmol).
ATPase samples were preincubated with 2.5 µg of phosphatidylserine
for 10 min at room temperature. Reactions were initiated by the
addition of 100 µl of assay solution. After incubation for 30 min
at 37 °C, the reactions were terminated by the addition of 1 ml of
1.25 N perchloric acid, and liberated
P was
extracted and counted as described(41) .
Nucleotide Labeling of the Recombinant 58-kDa Subunit
SDS-PAGE sample buffer. After
SDS-PAGE, the gel was dried and autoradiographed on Kodak X-Omat film
at -80 °C for 24-48 h. Incorporation of
[
-
P]ATP was determined by scanning
autoradiograms with an Image Quan densitometer (Molecular Dynamics 300A
computing densitometer). Relative radioactive nucleotide incorporation
was quantified as absorbance area on the basis of whole band analysis.
Exposure time of the film was adjusted to ensure that the signal would
fall well within the linear range of detection.
Miscellaneous
Expression of the 58-kDa Subunit of V-type
ATPase
Two bacterial expression vectors, p and p
, each containing the entire encoding
region for the 58-kDa subunit, were used to express the 58-kDa protein
in E. coli. However, the resultant recombinant 58-kDa protein
in E. coli was aggregated and functionally inactive. Repeated
attempts to solubilize, denature, and refold the protein were
unsuccessful (data not shown).
Figure 1:
SDS-PAGE and Western blot
analysis of the 58-kDa polypeptide expressed in Sf9 cells transfected
with a recombinant baculovirus. Total lysates of infected Sf9 cells
were subjected to SDS-PAGE analysis (panelA). PanelB illustrates the Western blot analysis of the
soluble portion of the cell lysates, defined as the supernatant
obtained after centrifuging the total cellular lysates at 150,000
g for 1 h. Lane1, non-transfected
Sf9 cells, lanes2-7, Sf9 cells infected with
recombinant baculovirus for 24, 36, 48, 60, 72, or 96 h,
respectively.
Purification of the Recombinant 58-kDa Protein from
Sf9 Cells
Although significant expression of the 58-kDa
protein was obtained in Sf9 cells 48-72 h postinfection (Fig. 1), recombinant protein was largely aggregated. Western
blot analysis of soluble fractions of Sf9 cell lysates demonstrated
that infected Sf9 cells produced detectable soluble recombinant protein (Fig. 1B) 36-72 h after infection and that optimal
production of soluble 58-kDa protein (Fig. 1B, lane4) occurred at 48 h. The 58-kDa protein was partially
purified from soluble extract by Ni-NTA column
chromatography(20, 40) . Further purification was
achieved by CM-Sepharose and DEAE-Sepharose column chromatography, as
illustrated in Fig. 2, lane4. As expected, the
recombinant protein is of a slightly greater molecular mass than that
obtained from native bovine tissue because of the additional histidine
tag and factor Xa site residues at the NH
terminus.
Generation of the 58- and 70-kDa Subunit-depleted
Catalytic Core of Vacuolar H
Proton-translocating ATPase, purified from
clathrin-coated vesicles of bovine brain (Fig. 2, lane1), was partially dissociated with 3 M urea.
After (NH-ATPase from Bovine
Brain and Reconstitution of ATP
Hydrolysis
)
SO
precipitation and
glycerol gradient centrifugation, a fraction consisting of at least
four polypeptides (70-, 58-, 40-, and 33-kDa components) was obtained (Fig. 2, lane2). By CM-Sepharose
chromatography and glycerol gradient centrifugation, a fraction
depleted of 58- and 70-kDa subunits, which consisted largely of the 40-
and 33-kDa polypeptides, was generated (Fig. 2, lane3). Densitometric analysis of this fraction, compared
with that of whole enzyme, revealed that the fraction was depleted of
70- and 58-kDa components by approximately 96%. To date, we have been
unable to achieve selective depletion of only the 58-kDa subunit from
V
. This problem, however, was overcome by the experiments
shown in . In this study, recombinant 70-kDa subunit (Fig. 2, lane5)(21) , the 58-kDa
subunit, and a biologically prepared V
depleted of 70- and
58-kDa subunits ( Fig. 2lane3) were tested
singly and in combination for their ability to support
Ca
-activated ATP hydrolysis. As shown in , the recombinant 58-kDa, 70-kDa, or biochemically prepared
fraction alone cannot support significant ATP hydrolysis. The mixture
of the 70- and 58-kDa components or the addition of either the 58- or
70-kDa subunit to the biochemically prepared fraction restored only
trivial ATP-hydrolytic activity. However, when both the 70-
and 58-kDa polypeptides were added to the biochemically prepared
fraction, significant Ca
-activated, N-ethylmaleimide-sensitive ATP-hydrolytic activity (0.45 nmol
P
min
assay
; 0.19 µmol P
mg of
protein
min
) was
observed. There was no significant Mg
-activated
ATPase activity when all these components were reconstituted under
these conditions.
-activated ATPase activity by the 58-kDa component
was saturable, and maximum activity was reached when 4 µg of 58-kDa
polypeptide was added to a mixture of recombinant 70 kDa (1 µg) and
biochemically prepared V
depleted of 70- and 58-kDa
subunits (0.4 µg).
Figure 3:
Effect of various concentrations of 58-kDa
polypeptide on reconstitution of Ca-activated ATP
hydrolysis. ATPase activities were measured by the liberation of
P
from [
-
P]ATP as
described under ``Experimental Procedures,'' using 0.4 µg
of biochemically prepared V
depleted of 70- and 58-kDa
polypeptides, 1 µg of 70-kDa protein, and different concentrations
of 58-kDa polypeptide as indicated.
Nucleotide Labeling of the Recombinant 58-kDa
Protein
Nucleotide labeling of recombinant 58-kDa protein
was attempted using radioactive ATP or ADP and ultraviolet irradiation.
As shown in Fig. 4A, the 58-kDa protein was
photoaffinity labeled by [-
P]ATP when the
reaction mixture contained divalent cations (Mg
or
Ca
). Although the 58-kDa protein is labeled when no
additional divalent was added to the reaction mixture (Fig. 4A, lane3), this labeling was
eliminated by the addition of 5 mM EDTA (Fig. 4A, lane4), indicating that the
ATP labeling is truly divalent dependent. In contrast, N-ethylmaleimide, an inhibitor of V-type
H
-ATPase, did not inhibit ATP-labeling, even at 10
mM (data not shown). Bafilomycin, a specific inhibitor of
V-type H
-ATPase, also did not inhibit ATP labeling
either even at 10 µM, a concentration of about 1,000-fold
greater than that required to inhibit V-type H
pumps
(data not shown).
Figure 4:
Photoaffinity labeling of the recombinant
58-kDa polypeptide. Reaction mixtures (20 µl) containing 0.2 µg
of the 58-kDa protein, 10 mM Tris/MES, pH 7.0, 10% glycerol,
radioactive nucleotide, and other components as indicated were UV
irradiated for 8 min on ice, followed by SDS-PAGE and autoradiography
as described under ``Experimental Procedures.'' PanelA, [-
P]ATP labeling. All lanes contained 1 µl of
[
-
P]ATP (10 µCi/µl, 3000 Ci/mmol).
Specific reaction conditions were: lane1, 2
mM MgCl
; lane2, 2 mM CaCl
; lane3, no additional divalent
cation; lane4, 5 mM EDTA. PanelB, radioactive ADP labeling. Lane1,
S-labeled ADP (1 µl, 5 µCi/µl, 1095 Ci/mmol),
2 mM CaCl
; lane2,
[
C]ADP (10 µl, 1 µCi/50 µl, 57.1
mCi/mmol), 2 mM CaCl
.
In addition, the 58-kDa polypeptide was also
labeled by [C]ADP and
S-labeled
ADP, as demonstrated in Fig. 4B. Photoaffinity labeling
of the 58-kDa polypeptide by [
-
P]ATP and
S-labeled ADP exhibited saturation kinetics. 20 µM of either ATP or ADP was sufficient to saturate photolabeling as
demonstrated in Fig. 5. The apparent K
for both ATP and ADP, determined from double reciprocal
plots of response curve shown in Fig. 5, was 4 µM,
indicating that the ATP and ADP had similar binding affinity to the
58-kDa polypeptide.
Figure 5:
Kinetics of photoincorporation of
radioactive nucleotides into recombinant 58-kDa polypeptide. The 58-kDa
polypeptide was UV irradiated in the presence of 2 mM MgCl and indicated concentrations of
[
-
P]ATP (0.25 µCi/µm ATP,
-
) or
S-labeled ADP (0.2
µCi/µM ADP,
-
). Incorporation of
radioactive nucleotides into the 58-kDa polypeptide was determined from
the densitometric scans of the autoradiograms as described under
``Experimental Procedures.''
The competition of cold ATP and cold ADP on
photoaffinity labeling of the 58-kDa polypeptide by
[-
P]ATP is shown in Fig. 6.
Half-maximal protection against photo-incorporation of
[
-
P]ATP into 58-kDa polypeptide occurred
with cold ATP and ADP at approximately 280 and 300 µM,
respectively, and complete protection was afforded by 1 mM of
either ATP or ADP. This result implies that ATP and ADP share the same
binding site.
Figure 6:
Competition by cold ATP or ADP for the
[-
P]ATP binding to the recombinant 58-kDa
polypeptide. The 58-kDa protein was UV irradiated in the presence of 2
mM of MgCl
, 1 µl of
[
-
P]ATP (10 µCi/µl, 3000 Ci/mmol),
and indicated concentrations of cold ATP (
) or cold ADP (
).
Photoincorporation of [
-
P] ATP into the
58-kDa polypeptide was determined from the densitometric scans of the
autoradiograms as described under ``Experimental
Procedures.''
-
P]ATP and is necessary for ATP
hydrolysis(21) .
(and
) subunit of F
-ATPase and specifically because of
the presence of a nucleotide binding motif. To date, several
laboratories have attempted to label V-ATPase with radioactive
nucleotides or analogues. In fact, in only one instance has the 58-kDa
subunit been labeled by a nucleotide analogue(31) . The current
study demonstrates that the 58-kDa subunit can be labeled by
radioactive nucleotides and that labeling requires a divalent cation
(Mg
or Ca
) (Fig. 4). The
divalent cation dependence of the labeling is similar to that of the
subunit of F
-ATPase from E.
coli(45) . Earlier investigations indicated that the
subunit binding by nucleotide was divalent cation
independent(46) . Based on results of current and previous
experiments(21) , the 58-kDa polypeptide demonstrates a higher
ATP affinity (K
of 4 µM ATP)
than the 70-kDa subunit (K
of 35
µM ATP).
subunit of F
, which is presumably
analogous to the 58-kDa subunit of V-type pumps, reveals that the amino
terminus of the
subunit protrudes from the top of F
and that there is evidentially a high degree of freedom in
rotation in this terminal domain(47) . Thus, it is possible that
the amino terminus of the 58-kDa subunit of the V-type proton pump is
structurally noncontiguous (and noncommunicative) with the nucleotide
binding domain and that the presence of additional amino terminus
residues is inconsequential.
subunit of F
, which contains a
``nonexchangeable'' ATP (and divalent metal) binding site.
Thus, in isolated V-type pumps, this site may already be occupied by
nucleotide, and only by preparing nascent, recombinant protein can the
site become accessible to labeling. At present, there is no proven role
for nucleotides occupying nonexchangeable sites in F
,
although it has been proposed that these sites become occupied during
the biogeneses of the catalytic sector and may play a stabilizing,
structural assembly role. If so, it will be of interest to determine if
this is the case in V-type pumps by analyzing the nucleotide
requirements, if any, for the assembly or association of the 70- and
58-kDa components.
-activated ATPase activity.
When reconstituted to a biochemically prepared V
depleted
of 70- and 58-kDa subunits and recombinant 70-kDa fraction, the 58-kDa
polypeptide resulted in a significant Ca
-activated
ATP hydrolysis to a specific activity of 0.19 µmol P
min
mg of
protein
. Without the 58-kDa subunit, the
reconstitution with other polypeptides demonstrated only minimal ATPase
activity. These data indicate that the 58-kDa component is necessary
for maximal ATP hydrolysis. Like the na-tive catalytic sector from
bovine brain-coated vesicles, the reconstituted ATPase activity was N-ethylmaleimide sensitive ().
Table: Reconstitution of
Ca-activated ATPase with recombinant 58-kDa
subunit and other polypeptides of the V-type ATPase
P
from [
-
P]ATP as described under
``Experimental Procedures,'' using 1 µg of purified
recombinant 58-kDa protein, 1 µg of recombinant 70-kDa protein
(21), 0.4 µg of biochemically purified Vc depleted of 70- and
58-kDa polypeptides, and 5 mMN-ethylmaleimide as
indicated. Denaturation of the 58-kDa subunit was achieved by boiling
for 10 min. All of the fractions utilized in this experiment are as
shown in Fig. 2.
E
, polyoxyethylene 9-lauryl ether; MES,
2-(N-morpholino)ethanesulfonic acid; NTA, nitrolotriacetic
acid.
.
Superb technical and administrative assistance were provided by
Shung-Ching Tsai and Kay Martin.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.