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J. Biol. Chem., Vol. 275, Issue 41, 32331-32337, October 13, 2000
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From the Departments of
Received for publication, June 2, 2000, and in revised form, July 19, 2000
Vacuolar H+-ATPase (V-ATPase)
binds actin filaments with high affinity (Kd = 55 nM; Lee, B. S., Gluck, S. L., and Holliday,
L. S. (1999) J. Biol. Chem. 274, 29164-29171).
We have proposed that this interaction is an important mechanism
controlling transport of V-ATPase from the cytoplasm to the plasma
membrane of osteoclasts. Here we show that both the B1 (kidney) and B2 (brain) isoforms of the B subunit of V-ATPase contain a microfilament binding site in their amino-terminal domain. In pelleting assays containing actin filaments and partially disrupted V-ATPase, B subunits
were found in greater abundance in actin pellets than were other
V-ATPase subunits, suggesting that the B subunit contained an F-actin
binding site. In overlay assays, biotinylated actin filaments also
bound to the B subunit. A fusion protein containing the amino-terminal
half of B1 subunit bound actin filaments tightly, but fusion proteins
containing the carboxyl-terminal half of B1 subunit, or the full-length
E subunit, did not bind F-actin. Fusion proteins containing the
amino-terminal 106 amino acids of the B1 isoform or the amino-terminal
112 amino acids of the B2 isoform bound filamentous actin with
Kd values of 130 and 190 nM,
respectively, and approached saturation at 1 mol of fusion protein/mol
of filamentous actin. The B1 and B2 amino-terminal fusion proteins
competed with V-ATPase for binding to filamentous actin. In summary,
binding sites for F-actin are present in the amino-terminal domains of
both isoforms of the B subunit, and likely are responsible for the
interaction between V-ATPase and actin filaments in
vivo.
V-ATPase1 is an
evolutionarily ancient enzyme that performs such vital functions as
acidification of lysosomes, CURL (compartments for uncoupling of
receptor and ligand) compartments, and a multitude of other
compartments in the vacuolar system of eukaryotic cells (2-5). In
addition, certain cells express copious quantities of V-ATPase on the
plasma membrane. In osteoclasts, V-ATPase is transported to a
specialized domain of the apical plasma membrane, known as the ruffled
membrane, that forms at the site of osteoclast attachment upon
activation to resorb bone (6, 7). V-ATPases in the ruffled membrane
transport protons into the resorption compartment to create the acidic
pH required for bone resorption. V-ATPases are also abundant on the
plasma membrane of the kidney intercalated cell, which is responsible
for hydrogen ion and bicarbonate transport in the cortical and outer
medullary collecting duct (8). In the H+-secreting
intercalated cells, V-ATPase is stored in a specialized intracellular
tubulovesicular compartment, but is recruited rapidly to the apical
plasma membrane in response to systemic administration of nonvolatile
acid (8, 9).
The V-ATPase is composed of at least 13 subunits divided into two major
domains, V1 and V0 (2, 3). V1,
which is composed of proteins peripheral to the membrane, contains the
A, B, C, D, E, F, G, and H subunits. The A and B subunits, which share homology with the Recent studies indicate the cytoskeleton has an important role in
V-ATPase regulation in the osteoclast (18, 19). Nakamura et
al. (19) showed that V-ATPase was associated with the
detergent-insoluble cytoskeleton in bone marrow-derived osteoclasts
from normal mice. In contrast, in the oc We have identified a potential mechanism for the interaction between
the osteoclast V-ATPase and the cytoskeleton, a direct binding
interaction between the V-ATPase and F-actin (1). V-ATPase and F-actin
were shown to co-localize in detergent-solubilized osteoclast remnants,
and actin and myosin II were immunoprecipitated from detergent extracts
of osteoclasts using a monoclonal antibody against the E subunit of
V-ATPase. This association correlated with transport of V-ATPase to
ruffled membranes. We were able to reconstitute binding using purified
V-ATPase and F-actin and reported that pure osteoclast and bovine
kidney V-ATPase bound filamentous actin with high affinity
(Kd = 55 nM). Examination of bovine
kidney V-ATPase bound to F-actin under the electron microscope
suggested binding occurred through a region of the V1
domain that is farthest removed from the membrane insertion site.
Because we isolated myosin II in complex with F-actin and V-ATPase, but
not other myosins, we suggested that myosin II-powered contraction of a
microfilament network transported bound V-ATPase to the site of nascent
ruffled membranes (1). This model is consistent with the findings in
oc Although our previous studies showed that V-ATPase interacts directly
with actin filaments, we were unable to distinguish which of the 13 subunits of V-ATPase bound F-actin. In this study we identify an
F-actin binding site in the amino-terminal domain of the B subunit as a
strong candidate for mediating the binding of V-ATPase to F-actin.
Reagents--
Materials were obtained from Sigma unless
otherwise noted.
Mouse Marrow Cultures--
Osteoclasts were generated from
primary mouse marrow cultures (21). Swiss Webster mice (8-20 g each)
were killed by cervical dislocation, and femora and tibia were
isolated. Marrow was flushed from the bone cavity and grown at a
density of 1 × 106 cells/cm2 on tissue
culture plates in Immunoprecipitation of Mouse Marrow Osteoclast V-ATPase, Partial
V-ATPase Disassembly, and F-actin Binding--
V-ATPase was obtained
from mouse marrow cultures as described previously (1).
Osteoclast-containing mouse marrow cultures were labeled overnight in
90% methionine- and cysteine-free culture medium containing 10%
dialyzed fetal bovine serum and 50 mCi/ml Tran35S-label
(ICN, Costa Mesa, CA). Cells were then washed in phosphate-buffered saline and solubilized in Triton X-100 buffer (1% Triton X-100, 20 mM Tris-Cl, pH 7.4, 1 mM EDTA, 1 mM
dithiothreitol, 10% glycerol, 5 mM sodium azide, and
protease inhibitors). Following centrifugation at low speed (10 min at
20,000 × g, 4 °C) to remove insoluble material, the
extracts were used for immunoprecipitation, which was performed by
first incubating the extracts with protein A-Sepharose (Amersham
Pharmacia Biotech) to clear nonspecifically bound proteins. Extracts
were then incubated with 10 µl of the anti-V-ATPase E-subunit monoclonal antibody E11 ascites (22) for 2 h at 4 °C, and with 20 µl of protein A-Sepharose for an additional 30 min. The immune complexes were pelleted in a microcentrifuge, washed three times in
NET-GEL buffer (23), and eluted with 5 mg/ml E11 target peptide in
F-buffer (20 mM Tris-Cl, pH 7.4, 100 mM NaCl, 5 mM MgCl2, 0.5 mM ATP, 0.2 mM CaCl2, and 0.2 mM
dithiothreitol). The eluate was dialyzed overnight against F-buffer.
Under these conditions the V-ATPase partially disassociated. The
capacity of partially disassociated osteoclast V-ATPase to bind actin
was tested by adding 4.5 µM rabbit muscle F-actin to
these V-ATPase preparations. After incubation for 30 min at room
temperature, the F-actin was pelleted at 200,000 × g
for 45 min, pellets and supernatants were subjected to SDS-PAGE, and
they were analyzed by autoradiography and/or phosphorimaging as
described previously (1).
Other Protein Purification--
Bovine kidney V-ATPase and
bovine microsomes were prepared as described previously (24). All
procedures were performed at 4 °C. 5 mg/ml microsomal protein was
solubilized in 10 mM Tris-Cl, pH 7.0, 1 mM
EDTA, 1 mM dithiothreitol, 0.6 mM CHAPS, 1.5%
n-octyl-
Actin was prepared from rabbit muscle acetone powder by standard
methods (25), and was further purified by two rounds of polymerization-depolymerization and gel filtration on a 2.5 × 100-cm Sephacryl S-300 column (Amersham Pharmacia Biotech).
F-actin Blot Overlays--
F-actin blot overlays were performed
by a modification of the method of Luna (26). Rabbit muscle actin was
polymerized in a buffer containing 50 mM HEPES, pH 7.4, 100 mM NaCl, 1 mM MgCl2, 0.2 mM CaCl2, 0.5 mM ATP and incubated
with sulfo-NHS-LC-biotin (Pierce) at a molar ratio of 1:20 for 30 min
at room temperature. The biotinylated F-actin was depolymerized by
dialysis into buffer G (20 mM Tris, pH 7.4, 0.5 mM ATP, 0.5 mM CaCl2, 0.2 mM DTT). The depolymerized, biotinylated F-actin was spun
at 100,000 × g for 45 min and mixed at a 1:10 ratio
with unlabeled, unpolymerized actin. The mix was polymerized by
addition of 100 mM NaCl and 5 mM
MgCl2 (buffer F) in the presence of 1 mol of gelsolin/25 mol of actin. The polymerized F-actin was stabilized with 10 µM phalloidin. Immunopurified bovine kidney V-ATPase (0.2 µg/lane) was subjected to SDS-PAGE, electroblotted to nitrocellulose,
and probed with 100 µg/ml biotinylated F-actin in buffer F plus 0.5% Tween 20, 10 µM phalloidin, and 5 mg/ml BSA for 2 h
at room temperature. The blots were washed in the same buffer and
probed in the same buffer plus 1 µg/ml alkaline
phosphatase-conjugated streptavidin for 30 min, washed three times for
10 min in the same buffer, and alkaline phosphatase was detected using
a standard substrate containing 5-bromo-4-chloro-3-indolyl
phosphate/nitro blue tetrazolium in 2-amino-2-methyl-1,3-propanediol.
As a control, some V-ATPase blots were incubated with 1 mg/ml
unlabeled, gelsolin-capped F-actin together with 100 µg/ml
biotinylated gelsolin-capped F-actin. Quantitation was by densitometry
using a Fluorchem 8000 in reflectance mode (Alpha Innatech Corp., San
Leandro, CA).
Fusion Protein Preparation--
To localize the domain in the B1
subunit of V-ATPase responsible for interaction with F-actin, we
generated two B1 subunit-GST fusion constructs by ligating in frame DNA
fragments corresponding to B1 subunit amino acids 1-268 or 249-513
with the GST gene in pGEX-2TK (Amersham Pharmacia Biotech). A construct
containing the entire coding region of the E subunit of V-ATPase
ligated in frame with the GST gene was used as a negative control.
Bacteria containing the recombinant plasmids were cultured in LB
medium, induced with
isopropyl-1-thio-
To construct the NH2-terminal B1 subunit maltose-binding
protein fusion protein (NT-B1-MBP), we amplified by polymerase chain reaction (PCR) a 329-base pair DNA fragment encoding the amino-terminal 106 amino acids from human B1 using the sense and antisense primers 5'-ATGGCCATGGAGATAGACAGCAGG-3' and 5'-GCTCTAGACTAGCAAGTGGTCTTCC-3' with
a human B1 subunit cDNA clone as the template. The same strategy was used to construct a fusion protein containing the amino-terminal 112 residues from human B2 (NT-B2-MBP). A 347-base pair DNA fragment was amplified by PCR using the sense and antisense primers
5'-ATGGCGCTGCGGGCGATGCGGGGG-3' and
5'-GCTCTAGACTAACAGGACGTTTTCTT-3' with a human B2 subunit cDNA clone as the template.
200 pmol each of the sense and antisense primers were added to 100 ng
of cDNA template in 50 mM KCl, 10 mM
Tris-HCl, pH 9.0, 1.0% Triton X-100, and 0.2 mM dATP,
dCTP, dGTP, and dTTP. PCR was carried out for 30 cycles at 94 °C for
1 min, 50 °C for 1 min, and 72 °C for 1 min. The PCR product was
ligated into the vector pMALc (New England Biolabs, Beverly, MA).
Colonies of XL-1 Blue Escherichia coli (Stratagene, La
Jolla, CA), transformed with the ligation products, were selected for
inducible expression of the fusion proteins with 0.3 mM
isopropyl-1-thio- Fusion Protein Binding Assays--
GST fusion proteins were
precipitated with glutathione beads from bacterial extracts (26) and
were washed in buffer F. Actin (70 µM) was polymerized in buffer F,
prespun at 15,000 × g for 15 min, and diluted to 1 µM in buffer F. 100 µl of the diluted F-actin was mixed
with 25 µl of GST beads containing bound GST fusion protein. The mix
was incubated with agitation for 25 min at room temperature, and the
beads were pelleted by centrifugation at 200 × g for 1 min. The supernatants were collected and beads were washed in 1 ml of
buffer F, and pelleted at 5,000 × g for 1 min. The
wash procedure was repeated three times, and proteins were solubilized
from the beads using SDS-PAGE sample buffer. The initial unbound
material and the proteins that bound the beads after washing were
subjected to SDS-PAGE and detected by Coomassie stain.
The affinity and stoichiometry of NT-B1-MBP and NT-B2-MBP for actin was
determined by quantitative binding assays. Protein concentrations of
actin and the fusion proteins were determined by BCA assay (Pierce).
Purified rabbit muscle actin was polymerized at a concentration of 70 µM in buffer F, and diluted in F-buffer immediately prior
to the experimental procedure. F-actin alone (0.8 µM) or
F-actin plus varying concentrations of fusion protein dialyzed against
F-buffer were incubated for 1 h at room temperature. The samples
were then subjected to ultracentrifugation at 200,000 × g for 45 min, and pellets and supernatants were collected,
separated by SDS-PAGE, and stained with Coomassie Blue, and the amounts of fusion protein in the supernatants and pellets were determined by
absorbance densitometry using a Fluorchem 8000.
V-ATPase Biotinylation and Fusion Protein
Competition--
Bovine kidney V-ATPase in PBS was biotinylated with
NHS-LC-biotin at a molar ratio of 2.5 mol of biotin/mol of V-ATPase for 30 min at room temperature. The biotinylated V-ATPase was then dialyzed
into buffer F. Actin (0.8 µM) was polymerized in the same
buffer and pre-incubated with 1.0 µM NT-B1-MBP and 1.0 µM NT-B2-MBP, or 1 µM MBP as a control, for
45 min. Biotinylated V-ATPase (0.05 µM) was then added,
and the mix, or actin alone as a control, was incubated for 30 min at
room temperature. The samples were then subjected to
ultracentrifugation at 200,000 × g for 45 min,
supernatants and pellets were collected, and the proteins were
separated by SDS-PAGE on 12.5% acrylamide gels. Some samples were
electroblotted to nitrocellulose, and biotinylated protein was detected
using alkaline phosphatase-conjugated streptavidin, which was detected
using a standard substrate containing 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium in 2-amino-2-methyl-1,3-propanediol. Quantitation of total biotinylated protein was performed by reflectance densitometry using a Fluorchem 8000.
V-ATPase was immunoprecipitated from the low speed supernatants of
metabolically labeled, osteoclast-containing mouse marrow cultures
using the monoclonal anti-E subunit antibody, E11 (Fig. 1, lane 1).
V-ATPase isolated by this method is enriched in the A, B, and E
subunits and has associated F-actin and myosin II (1). It also contains
subunits C, D, and c, and is capable of bafilomycin-sensitive ATPase
activity (28). We do not detect the a subunit by this method. The
complexes were eluted by competition with the E11 target peptide and
dialyzed overnight in a buffer containing physiologic salt, magnesium,
and ATP. We have observed previously that this treatment induced
partial disassembly of the
V-ATPase.2 After overnight
dialysis, the complexes were supplemented with rabbit muscle actin
(Fig. 1, lane 2) to ensure that F-actin was present in excess, and subjected to ultracentrifugation. The resulting supernatants (Fig. 1, lane 3) and pellets (Fig.
1, lane 4) were analyzed by SDS-PAGE and
autoradiography. 95.7% of the actin (by phosphorimaging) and 96.2% of
the B subunit pelleted under these conditions, but only 63.4% of A
subunit and 5.1% of E subunit were detected (Fig. 1, lane
4). In this procedure, only trace amounts of other subunits
were detected. The simplest explanation for this result is that the B
subunit bound F-actin, and other subunits were recovered in the pellet
only when complexed with the B subunit.
F-actin overlays were used as a second and independent method for
identifying actin-binding sites on the V-ATPase. Bovine kidney V-ATPase
was separated by SDS-PAGE, and electroblotted to Immobilon P. The blots
were probed with biotinylated, phalloidin-stabilized F-actin, and
detected using alkaline phosphatase-conjugated streptavidin (Fig.
2). The biotinylated F-actin bound
only the two B subunit isoforms of the bovine kidney V-ATPase (Fig. 2,
lanes 4 and 5). Co-incubation of blots
with biotinylated F-actin together with an excess of unlabeled F-actin
reduced binding by biotinylated-F-actin by 82% (Fig. 2,
lane 7). The ability of F-actin to specifically bind B subunit in blot overlay experiments provided a second line of
evidence that the B subunit contains a binding site for F-actin.
The Amino-terminal Domain of the B Subunit of Vacuolar
H+-ATPase Contains a Filamentous Actin Binding Site*
§¶,,
,,, and§
Medicine and
§ Anatomy & Cell Biology, University of Florida College of
Medicine, Gainesville, Florida 32610, the Department of Internal
Medicine, Washington University School of Medicine, St. Louis, Missouri
63110, and the ** Department of Pediatrics, University of Utah,
Salt Lake City, Utah 84132
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and 
subunits of the ATP synthase, are thought to form a hexameric barrel of alternating subunits. The A subunit contains a site for ATP hydrolysis; in conjunction with the B subunit,
it is thought to undergo a conformational change during ATP hydrolysis,
which generates the force to turn an inner rotating "cam" composed
of multiple subunits other than A and B (2, 3). Rotation of the cam is
thought to drive proton movement through the V0 domain.
Apart from its putative function in ATPase activity, the function of
the B subunit is unknown. There are two isoforms of the B subunit
encoded by different genes. The B2 "brain" isoform is expressed
ubiquitously (5), and at high levels in osteoclasts (10), macrophages
(10), proximal tubules of the kidney (11, 12), and neurons (13). The
distribution of the B1 "kidney" isoform is more restricted. It is
found at high levels in intercalated cells of the kidney (11, 12), in
the ciliary epithelium of the eye (14), and in the ear (15), epididymis
(16), and placenta (17). The difference in the expression of these
isoforms suggests that the B subunit may function in V-ATPase regulation.
/oc
"osteosclerotic" mouse, which has severe bone deformities due to
abnormal osteoclast bone resorption, osteoclasts appeared to lack
V-ATPase association with the detergent-insoluble cytoskeleton, and
were unable to transport V-ATPase to the ruffled membranes (19). These
results suggest that interactions between the osteoclast V-ATPase and
the detergent-insoluble cytoskeleton are essential for recruitment of
V-ATPase to the ruffled membrane, and for normal bone resorption.
/oc mice, and also with a report that
microinjection of myosin II-inhibiting antibodies into osteoclasts
greatly decreased their bone resorptive capacity (20).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-minimum Eagle's medium + 10% fetal bovine serum
in the presence of 1,25-dihydroxyvitamin D3 for 6 to 8 days.
-D-glucopyranoside, and 10%
glycerol. The mixture was centrifuged at 150,000 × g
for 1 h, and the clear supernatant containing solubilized V-ATPase was collected. Immunoaffinity purification of V-ATPase from bovine kidney was performed exactly as described (24).
-D-galactopyranoside, lysed by sonication, and incubated with glutathione beads (Sigma). The GST
fusion proteins were purified and verified as described (27).
-D-galactopyranoside. Inducible
products with predicted sizes of 54,000 and 55,000 for B1 and B2,
respectively, were demonstrable by SDS-PAGE analysis of whole cell
extracts. Each fusion protein construct was verified by
dideoxynucleotide sequencing. The fusion proteins were purified on
amylose columns, using protocols supplied by the manufacturer (New
England Biolabs). Homogeneity was demonstrated by SDS-PAGE, and the
products were further confirmed by proteolytic cleavage of the fusion
protein using factor Xa, which cleaves a site just amino-terminal to
the first residue of the B subunit-derived portion. The B subunit
peptides were separated from maltose-binding protein by SDS-PAGE on
17% bisacrylamide gels, electroblotted to Immobilon P (Pierce), and
the amino-terminal 20 amino acids were sequenced by Edman degradation
(Protein Chemistry Core Facility, Interdisciplinary Center for
Biotechnology Research, University of Florida).
RESULTS
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DISCUSSION
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View larger version (57K):
[in a new window]
Fig. 1.
F-actin preferentially binds B subunit from
partially disrupted V-ATPase. V-ATPase was isolated from
metabolically labeled mouse marrow cultures by immunoprecipitation
using the monoclonal antibody, E11, eluted using the target peptide for
E11 (lane 1), and dialyzed overnight against
F-buffer. The next day 4.5 µM unlabeled rabbit muscle
actin was added (lane 2), and the sample was
subjected to centrifugation at 200,000 × g for 45 min.
The supernatants (lane 3) and pellets
(lane 4) were collected and subjected to
SDS-PAGE, and proteins were detected by autoradiography. The relative
amount of protein recovered in the pellet was determined by
phosphorimetry.
View larger version (89K):
[in a new window]
Fig. 2.
F-actin binds B subunit in blot
overlays. F-actin was biotinylated and used to probe blots of
bovine kidney V-ATPase as described under "Experimental
Procedures." Panels 1-4 are four identical
blots containing 0.2 µg of bovine kidney V-ATPase probed as follows:
panel 1, rabbit anti-V-ATPase antibody that
detects A, B1, B2, and E subunits; panel 2, no
probe; panel 3, 100 µg/ml unlabeled F-actin;
panel 4, 100 µg/ml biotinylated F-actin.
Panels 5-7 represent a second experiment. These
panels are as follows: panel 5, 0.05 µg of
bovine kidney V-ATPase probed with 100 µg/ml biotinylated F-actin;
panel 6, 0.05 µg of bovine serum albumin probed
with 100 µg/ml biotinylated F-actin; panel 7,
0.05 µg of bovine kidney V-ATPase was incubated with 1 mg/ml
unlabeled F-actin and 100 µg/ml biotinylated F-actin. Binding of the
antibody in panel 1 was detected using a goat
anti-rabbit secondary antibody conjugated to alkaline phosphatase;
binding of the probes in panels 2-7 was detected
with alkaline phosphatase-conjugated streptavidin as described under
"Experimental Procedures."
In order to study interaction between V-ATPase and F-actin in greater
detail, we constructed GST fusion proteins containing the amino- and
carboxyl-terminal halves of the B1 isoform of the B subunit (NT-B1-GST,
CT-B1-GST) and full-length E subunit (E-GST), depicted in Fig.
3A. Mixtures of these fusion
proteins with phalloidin-stabilized F-actin (1.0 µM) were
assayed by precipitation with glutathione beads (Fig. 3B).
No actin was detected associated with beads coated either with
CT-B1-GST or E-GST (Fig. 3B, lanes 4 and 6). In contrast, a large amount of actin bound the
NT-B1-GST coated beads (Fig. 3B, lane
2).
|
On the basis of these data, we identified the amino terminus of the B
subunit as the most likely location for the actin-binding site that
mediates the high affinity interaction between V-ATPase and F-actin. We
made additional fusion proteins incorporating the amino-terminal 106 and 112 amino acids of the B1 and B2 isoforms, respectively (NT-B1-MBP;
NT-B2-MBP) using a maltose-binding protein expression system to guard
against possible GST-related artifacts (depicted in Fig.
4A). Each fusion protein, or
unaltered MBP as a control, was subjected to ultracentrifugation either
alone, or in a mixture with F-actin (Fig. 4B). The fusion
proteins pelleted with F-actin (Fig. 4B, lanes
5-12), whereas purified MBP did not (Fig. 4B,
lanes 1-4). These results indicate that an actin
binding site is located within the first 112 amino acid residues of the amino terminus of the B subunit.
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The fusion proteins prepared using the MBP vector contain a factor Xa cleavage site preceding the first amino acid of the B subunit-derived peptide chain. To determine if the actin binding was the result of interaction between the amino-terminal peptide and F-actin, we examined the effect on actin binding of cleavage of NT-B1 with factor Xa (Fig. 4C). Intact or cleaved NT-B1-MBP was mixed with F-actin, and binding was assayed by ultracentrifugation (Fig. 4C). The NT-B1 peptides bound F-actin, but binding of MBP was lost after cleavage with factor Xa. Similar results were obtained using cleaved NT-B2-MBP (data not shown). These results confirmed that binding of the fusion proteins was mediated through the B subunit peptides, rather than MBP.
The binding of the B1- and B2-MBP fusion proteins to F-actin was
characterized in more detail (Fig. 5).
Saturation was approached by both NT-B1-MBP and NT-B2-MBP at a 1:1
stoichiometry (1 mol of fusion protein:1 mol of F-actin subunit; Fig.
5, A and B). Using this stoichiometry,
dissociation constants were determined from Haines plots (Fig. 5,
C and D). Both fusion proteins bound F-actin with
high affinity; the Kd of NT-B1-MBP binding was 130 nM, and the Kd of NT-B2-MBP binding was
190 nM.
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Finally, to test whether the interaction of intact V-ATPase with
F-actin was mediated through the B subunit, we incubated saturating
levels of NT-B1-MBP and NT-B2-MBP with F-actin for 30 min, and then
added biotinylated-V-ATPase. After an additional 30 min, the mixtures
were centrifuged. The supernatants and pellets were subjected to
SDS-PAGE, blotted to nitrocellulose, and the biotinylated V-ATPase was
detected with alkaline phosphatase-conjugated streptavidin (Fig.
6). Binding of V-ATPase to F-actin was
reduced by 71.2 ± 9% (by densitometric analysis,
n = 4), compared with control values when F-actin was
pretreated with saturating amounts of the fusion proteins (Fig.
6A). As an additional control, pretreatment of F-actin with
maltose-binding protein had no effect of V-ATPase binding to actin
(Fig. 6B). These data indicate that the B subunit fusion
proteins were able to compete with V-ATPase for binding sites on
F-actin, indicating that the B subunit contains an F-actin binding site
in the intact V-ATPase.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
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The present study shows that the amino terminus of both isoforms of the B subunit of V-ATPase contain high affinity F-actin binding sites. The B subunit was first identified as a potential F-actin-binding protein in centrifugation assays, which revealed preferential pelleting of B subunits when partially disrupted V-ATPase was mixed with F-actin. Independent evidence for an interaction between the B subunit and F-actin was found in F-actin blot overlays, in which F-actin bound bovine kidney B1 and B2 isoforms, but not other V-ATPase subunits. On the basis of these results, we prepared fusion protein constructs to test different domains of the B subunit for F-actin binding. Fusion proteins incorporating the amino-terminal domain of either B subunit isoform bound F-actin with high affinity. This is consistent with our earlier finding that V-ATPase containing either B1 or B2 isoforms bound F-actin indistinguishably (1). Binding of the fusion proteins approached saturation at an equimolar ratio with actin monomers. In contrast, we reported previously that binding of F-actin to the intact V-ATPase approached saturation at a molar ratio of 1 V-ATPase per 8 F-actin subunits. This difference is most likely due to steric hindrance of F-actin binding to the intact V-ATPase complex. Pre-incubation of amino-terminal B subunit fusion proteins with F-actin largely blocked subsequent binding of intact V-ATPase, providing strong evidence that these actin binding sites mediate the interaction between isolated V-ATPase and F-actin that we identified previously (1). Failure of the fusion proteins to block V-ATPase binding completely may be due to the higher affinity of the V-ATPase for F-actin, allowing it to successfully compete with fusion proteins. It is also possible that the V-ATPase associates to some degree with the fusion proteins, or that V-ATPase binds F-actin at additional sites.
We originally became interested in the interaction between V-ATPase and
F-actin based on a report that failure of V-ATPase to associate
normally with the detergent-insoluble cytoskeleton of osteoclasts was a
characteristic of the defective osteoclasts in
oc/oc mice (29). Osteoclasts from
oc/oc mice differentiate in normal numbers
and appear to express the typical compliment of osteoclast marker
proteins, but fail to form ruffled membranes when they contact bone,
and do not resorb bone (19). These osteoclasts do not transport
V-ATPase to the apical plasma membrane, a prerequisite for bone
resorption, despite the enzyme being present at normal levels as
assayed by measuring V-ATPase-specific, ATPase activity (19). These
findings suggested to us that identification of the mechanism by which
V-ATPase associates with the detergent-insoluble cytoskeleton of
osteoclasts would provide insight into how V-ATPase is transported to
ruffled membranes.
Our present studies extend our initial investigations on the nature of
the interaction between V-ATPase and the detergent insoluble
cytoskeleton. In principle, and as proposed in the literature (18, 19),
V-ATPase could associate with either the microfilament- or
microtubule-based cytoskeletons, or both. We found that interaction between V-ATPase and the microfilament cytoskeleton was most likely defective in oc/oc mice. V-ATPase
co-localized with F-actin in both inactive and active wild-type
osteoclasts, and this co-localization persisted in experiments
identical to those performed by Suda and colleagues (19), which showed
a defect in V-ATPase association with the detergent-insoluble
cytoskeleton in oc/oc mice. In addition,
F-actin and myosin II, but not tubulin or tubulin-based motors, were
recovered by immunoprecipitation from osteoclast detergent-extracts
using a monoclonal anti-E subunit antibody (1). Finally, we identified
a tight binding interaction between isolated V-ATPase and F-actin (1).
Therefore, we believe that a plausible explanation for the inability of
V-ATPase to associate with the detergent-insoluble cytoskeleton
observed in oc/oc mice is a failure of
V-ATPase to bind normally to microfilaments. This does not rule out a
role for microtubules in establishing V-ATPase distribution in
osteoclasts (18, 30).
Recently, the genetic defect responsible for the
oc/oc phenotype was identified as a
deletion in the gene encoding the a3 isoform of the V-ATPase (31). This
result was consistent with experiments in which the a3 isoform was
deleted by homologous recombination (32). In addition, a subset of
human autosomal recessive osteopetrosis patients were found to have
mutations in the a3 isoform, suggesting that a3 is also crucial to
osteoclast function in humans (33).
These results raise the question of how deletion of a3 could affect the interaction between B subunit and F-actin that we propose to be essential for actin binding and V-ATPase transport. We suggest that the a subunit regulates access to the F-actin binding site in the amino terminus of the B subunit. This idea is consistent with what is currently known about V-ATPase structure (2, 3, 34, 35), but higher resolution models of both the physical structure of V-ATPase and V-ATPase bound to F-actin are now required to test this idea. In osteoclasts, we suggest that the a1 isoform, which is also expressed (30), can substitute for a3 in allowing V-ATPase assembly, but does not allow the F-actin binding necessary for proper transport of V-ATPase to ruffled membranes. This fits with recently described differential distribution between a1 and a3 in resting osteoclasts (30). If our hypothesis is correct, other a isoforms may allow F-actin binding under other regulatory circumstances. Alternatively, a3, which is expressed in a variety of tissues (30, 36), may uniquely be able to regulate interaction between B subunit and F-actin. If the presence of other a subunit isoforms in V-ATPase normally blocks binding to F-actin, it would explain why V-ATPase from cell lines such as LLC-PK1 and tissues such as bovine kidney is not normally recovered bound to F-actin (1, 28), despite the presence of high affinity F-actin binding sites in the B subunits. However, during our immunoaffinity purification of V-ATPase, the a subunit is lost, which may allow the enzyme we purify from a variety of sources to bind F-actin in in vitro assays.
The F-actin binding site that we identified in the amino-terminal domain of the B subunit is ideally located to mediate binding of V-ATPase to F-actin. The amino terminus of the B subunit is predicted, by homology to the F-ATPase crystal structure (37), to be in the region of the V1 headpiece that extends away from the plasma membrane insertion. This location is consistent with our previous electron microscopic images of V-ATPase binding F-actin (1), and would allow the F-actin association observed between V-ATPase-packed vesicles from the region of toad urinary bladders, which was detected by freeze-fracture, deep-etch electron microscopy (38).
We found high affinity F-actin binding sites in both isoforms of B subunit, and have proposed that the binding between F-actin and V-ATPase in osteoclasts, which contains only B2, is important for transport of V-ATPase to osteoclast ruffled membranes (1). That B1 also has a high affinity F-actin binding site suggests that its interaction with F-actin may be important to its function. B1 has a second potential route for interaction with F-actin. Breton et al. (39) recently showed that the B1 isoform is a PDZ domain-binding protein, which they linked to the ability of B1 to be transported to the plasma membrane of renal intercalated cells and to co-localize with Na+/H+-exchanger regulatory co-factor (NHE-RF). Because NHE-RF interacts with the actin cytoskeleton through members of the ERM (ezrin, radixin, moesin) family of actin-binding proteins (40), it seems that V-ATPase may have both direct and indirect routes for interaction with the microfilament-based cytoskeleton.
In summary, we have identified F-actin binding sites in the
amino-terminal domain of the B1 and B2 isoforms of the B subunit of the
V-ATPase. The binding sites identified are ideally located to mediate
interaction between membrane-bound V-ATPase and F-actin. Efforts now
must be focused on confirming that these binding sites, which were
identified by an in vitro biochemical approach, are functionally relevant. Circumstantial evidence, based on a reasonable interpretation of our data in the context of data from
oc/oc mice, suggests that binding between
V-ATPase and F-actin may be important for V-ATPase transport in
osteoclasts. Progress will now depend on identifying the F-actin
binding sites in the B subunit isoforms precisely, and using molecular
approaches to dissect their functions in vivo.
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ACKNOWLEDGEMENTS |
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We thank Dr. Stephen Sugrue and the Department of Anatomy and Cell Biology of the University of Florida College of Medicine for graciously providing us with temporary laboratory and office space, and Dr. Frederick Southwick for allowing us to use his TL-100 mini-ultracentrifuge.
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FOOTNOTES |
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* This work was supported by an Arthritis Investigator award from the Arthritis Foundation (to L. S. H.) and by National Institutes of Health Grants R01 DK52131 (to B. S. L.) and R01 DK38848 (to S. L. G.).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: Dept. of Medicine, Div. of Nephrology, 1600 Archer Rd., Campus Box 100224, University of Florida College of Medicine, Gainesville, FL 32610. Tel.: 352-392-2568; Fax: 352-392-3581; E-mail: hollils@medicine.ufl.edu.
Published, JBC Papers in Press, July 27, 2000, DOI 10.1074/jbc.M004795200
2 B. S. Lee, S. L. Gluck, and L. S. Holliday, unpublished observations.
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ABBREVIATIONS |
|---|
The abbreviations used are: V-ATPase, vacuolar H+-ATPase; F-actin, filamentous actin; PAGE, polyacrylamide gel electrophoresis; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; GST, glutathione S-transferase; MBP, maltose-binding protein; PCR, polymerase chain reaction.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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