Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M004795200 on July 27, 2000

J. Biol. Chem., Vol. 275, Issue 41, 32331-32337, October 13, 2000
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
275/41/32331    most recent
M004795200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Holliday, L. S.
Right arrow Articles by Gluck, S. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Holliday, L. S.
Right arrow Articles by Gluck, S. L.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

The Amino-terminal Domain of the B Subunit of Vacuolar H+-ATPase Contains a Filamentous Actin Binding Site*

L. Shannon HollidayDagger §, Ming LuDagger , Beth S. Lee||, Raoul D. Nelson**, Suzanne SolivanDagger , Li ZhangDagger , and Stephen L. GluckDagger §

From the Departments of Dagger  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

Received for publication, June 2, 2000, and in revised form, July 19, 2000


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha  and beta  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.

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-/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.

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-/oc- mice, and also with a report that microinjection of myosin II-inhibiting antibodies into osteoclasts greatly decreased their bone resorptive capacity (20).

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.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha -minimum Eagle's medium + 10% fetal bovine serum in the presence of 1,25-dihydroxyvitamin D3 for 6 to 8 days.

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-beta -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).

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-beta -D-galactopyranoside, lysed by sonication, and incubated with glutathione beads (Sigma). The GST fusion proteins were purified and verified as described (27).

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-beta -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).

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.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


View larger version (57K):
[in this window]
[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.

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.


View larger version (89K):
[in this window]
[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).


View larger version (38K):
[in this window]
[in a new window]
  		Fig. 3.   A GST fusion protein containing the amino-terminal half of B1 binds F-actin. A, GST fusion proteins were constructed that containing either the amino-terminal or carboxyl-terminal half of B1, or the full-length E subunit. B, 1 µM F-actin was incubated with GST-agarose beads coated with NT-B1-GST, CT-B1-GST, or E-GST, and the mixtures were centrifuged. The initial unbound material (I) and the pelleted material (P) were analyzed by Coomassie stain. Two polypeptide bands were observed with both B1 fusion proteins (lanes 2 and 4). The upper band is the correct size for the full-length fusion protein, while the lower probably represents a product of proteolytic degradation. A large fraction of the added F-actin associated with the pelleted NT-B1-GST (lane 2), but did not associate either with CT-B1-GST or E-GST, and consequently remained in the supernatant (lanes 3 and 5).

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.


View larger version (26K):
[in this window]
[in a new window]
  		Fig. 4.   Maltose-binding protein fusion proteins containing the amino-terminal 106 amino acids of B1 and 112 amino acids of B2 bind F-actin. A, maltose-binding fusion proteins were constructed that contain the amino-terminal domain of both isoforms of B-subunit. The position of the factor Xa cleavage site is indicated. B, lanes 1-4, MBP (0.6 µM) was incubated for 30 min at room temperature in the absence or presence of 1.0 µM F-actin, and subjected to ultracentrifugation at 200,000 × g for 45 min; lanes 5-8, NT-B2-MBP (0.4 µM) was incubated in the absence or presence of 3.0 µM F-actin, and subjected to ultracentrifugation as above; lanes 9-12, NT-B1-MBP (0.8 µM) incubated in the absence or presence of 4.5 µM F-actin and subjected to ultracentrifugation as above. For all samples, the supernatants (S) and pellets (P) were analyzed by SDS-PAGE. Both NT-B2-MBP and NT-B1-MBP pelleted efficiently in the presence of F-actin (lanes 8 and 12), but neither the fusion proteins alone (lanes 5 and 6 and lanes 9 and 10) nor maltose-binding protein mixed with F-actin (lanes 3 and 4) pelleted under the same conditions. C, combinations of NT-B1-MBP and F-actin were incubated for 30 min at room temperature and subjected to high speed ultracentrifugation, and the supernatants (S) and pellets (P) were analyzed. In lanes 5-8, the 106-amino acid, amino-terminal peptide from B1 was cleaved from maltose-binding protein using the factor Xa cleavage site that precedes the first B subunit-derived amino acid. B subunit-derived peptides pelleted when they were centrifuged with 4.5 µM F-actin (lane 6), but did not pellet in the absence of F-actin (lane 8).

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.


View larger version (25K):
[in this window]
[in a new window]
  		Fig. 5.   Binding of NT-B1-MBP and NT-B2-MBP to F-actin approaches saturation at 1 mol of fusion protein/mol of F-actin and the bind with high affinity. A and B, actin was polymerized and diluted into solutions containing phalloidin (10 µM) and either NT-B1-MBP (A) or NT-B2-MBP (B) as described under "Experimental Procedures." The final actin concentrations, indicated by the dashed lines, were 800 nM in A and 200 nM in B. Following incubation and centrifugation as described, the supernatants and pellets were collected and the amount of fusion protein present was determined by densitometry of Coomassie-stained gels. C and D, varying amounts of NT-B1-MBP (C) and NT-B2-MBP (D) were added to F-actin and subjected to high speed centrifugation as described under "Experimental Procedures." Pellets and supernatants were analyzed by SDS-PAGE and densitometry of Coomassie-stained gels. The Kd was calculated from the assumption that the fusion protein binding saturated at 1 mol of fusion protein/mol of actin monomer (established in A and B).

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.


View larger version (43K):
[in this window]
[in a new window]
  		Fig. 6.   NT-B1-MBP and NT-B2-MBP compete with V-ATPase for binding to F-actin. A, actin (1 µM), biotinylated V-ATPase (50 nM), and 1 µM each of NT-B1-MBP and NT-B2-MBP were subjected to high speed ultracentrifugation as described under "Experimental Procedures," either alone or in combination. Samples were analyzed by Coomassie-stained SDS-PAGE gels (left panel), or were electroblotted to nitrocellulose and probed with alkaline phosphatase-conjugated streptavidin (right panel). Lanes 1 and 2, F-actin and biotinylated V-ATPase, respectively, prior to ultracentrifugation. Lanes 3 and 4, F-actin and biotinylated V-ATPase, respectively, were subjected to ultracentrifugation, and the pelleted material was analyzed. Lane 5, a mixture of F-actin and biotinylated V-ATPase was subjected to ultracentrifugation, and the pelleted material was analyzed. Lane 6, F-actin and biotinylated V-ATPase were centrifuged and analyzed as in lane 5, except NT-B1-MBP and NT-B2-MBP were included in the mixture to compete with intact V-ATPase for F-actin binding. A comparison of lanes 5 and 6 in the right panel shows that inclusion of the B subunit fragments reduced the amount of V-ATPase that pelleted. The total amount of biotinylated protein pelleted was determined by densitometry of the blots. B, actin (1 µM), biotinylated V-ATPase (50 nM), 2 µM MBP, and 1 µM each of NT-B1-MBP and NT-B2-MBP were subjected to high speed ultracentrifugation as described above. Lane 1, a mixture of F-actin and biotinylated V-ATPase was subjected to ultracentrifugation, and the pelleted material was analyzed. Lane 2, F-actin and biotinylated V-ATPase were centrifuged and analyzed as in lane 1, except 2 µM MBP was included. This had no effect on V-ATPase binding to F-actin. Lane 3, F-actin and biotinylated V-ATPase were centrifuged and analyzed as in lane 1, except NT-B1-MBP and NT-B2-MBP were included in the mixture to compete with intact V-ATPase for F-actin binding.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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.

    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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Lee, B. S., Gluck, S. L., and Holliday, L. S. (1999) J. Biol. Chem. 274, 29164-29171
2. Forgac, M. (2000) J. Exp. Biol. 203, 71-80
3. Forgac, M. (1999) J. Biol. Chem. 274, 12951-12954
4. Gluck, S. L., Underhill, D. M., Iyori, M., Holliday, L. S., Kostrominova, T. Y., and Lee, B. S. (1996) Annu. Rev. Physiol 58, 427-446
5. Gluck, S. L. (1993) Int. Rev. Cytol. 137C, 105-137
6. Blair, H. C., Teitelbaum, S. L., Ghiselli, R., and Gluck, S. L. (1989) Science 249, 855-857
7. Väänänen, H. K., Karhukorpi, E.-K., Sundquist, K., Wallmark, B., Roininen, I., Hentunen, T., Tuukkanen, J., and Lakkakorpi, P. (1990) J. Cell Biol. 111, 1305-1311
8. Bastani, B., Purcell, H., Hemkin, P., Trigg, D., and Gluck, S. L. (1991) J. Clin. Invest. 88, 126-136
9. Tisher, C. C., Madsen, K. M., and Verlander, J. W. (1991) Contrib. Nephrol. 95, 168-177
10. Lee, B. S., Holliday, L. S., Ojikutu, B., Krits, I., and Gluck, S. L. (1996) Am. J. Physiol. 39, C382-C388
11. Nelson, R. D., Guo, X.-L., Masood, K., Brown, D., Kalkbrenner, M., and Gluck, S. L. (1992) Proc. Nat. Acad. Sci. U. S. A. 89, 3541-3545
12. Puopolo, K., Kumamoto, C., Adachi, I., Magner, R., and Forgac, M. (1992) J. Biol. Chem. 267, 3696-3706
13. Nelson, N., Perzov, N., Cohen, A., Hagai, K., Padler, V., and Nelson, H. (1999) J. Exp. Biol. 203, 89-95
14. Wax, M. B., Saito, I., Tenkova, T., Krupin, T., Becker, B., Nelson, N., Brown, D., and Gluck, S. L. (1997) Proc. Nat. Acad. Sci. U. S. A. 94, 6752-6757
15. Karet, F. E., Finberg, K. E., Nelson, R. D., Nayir, A., Mocun, H., Sanjad, S. A., Rodriguez-Soriano, J., Santos, F., Cremers, C. W., DiPietro, A., Hoffbrand, B. I., Winiarski, J., Bakkaloglu, A., Ozen, S., Dusunsel, R., Goodyer, P., Hulton, S. A., Wu, D. K., Sskvorak, A. B., Morton, C. C., Cunningham, M. J., Jha, V., and Lifton, R. P. (1999) Nat. Genet. 21, 84-90
16. Breton, S., Smith, P. J., Lui, B., and Brown, D. (1996) Nat. Med. 2, 470-472
17. Skinner, M. A., MacLaren, L. A., and Wildeman, A. G. (1999) J. Histochem. Cytochem. 47, 1247-1254
18. Abu-amer, Y., Ross, F. P., Schlesinger, P., Tondravi, M. M., and Teitelbaum, S. L. (1997) J. Cell Biol. 137, 247-258
19. Nakamura, I., Takahashi, N., Udagawa, N., Moriyama, Y., Kurokawa, T., Jimi, E., Sasaki, T., and Suda, T. (1997) FEBS Lett. 401, 207-212
20. Sato, M., and Grasser, W. (1990) Cell Motil. Cytoskel. 17, 250-263
21. Holliday, L. S., Dean, A. D., Greenwald, J. E., and Gluck, S. L. (1995) J. Biol. Chem. 270, 18983-18989
22. Hemkin, P., Guo, X.-L., Wang, Z.-Q., Zhang, K., and Gluck, S. L. (1992) J. Biol. Chem. 267, 9948-9957
23. Sambrook, J., Fritsch, E. F., and Manatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed. , p. 18, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
24. Gluck, S., and Caldwell, J. (1987) J. Biol. Chem. 262, 15780-15789
25. Spudich, J. A., and Watt, S. (1971) J. Biol. Chem. 246, 4866-4871
26. Luna, E. J. (1998) Methods Enzymol. 298, 32-42
27. Zhang, N., Shen, W., Hawley, R. G., and Lu, M. (1999) Oncogene 18, 2273-2279
28. Lee, B. S., Holliday, L. S., Krits, T., and Gluck, S. L. (1999) J. Bone Miner. Res. 14, 2127-2136
29. Marks, S. C., Jr., Seifert, M. F., and Lane, P. W. (1985) J. Hered. 76, 171-176
30. Toyomura, T., Oka, T., Yamaguchi, C., Wada, Y., and Futai, M. (2000) J. Biol. Chem. 275, 8760-8765
31. Scimeca, J.-C., Franchi, A., Trojani, C., Parrinello, H., Grosgeorge, J., Robert, C., Jaillon, O., Poirier, C., Gaudray, P., and Carle, G. F. (2000) Bone 26, 207-213
32. Li, Y. P., Chen, W., Liang, Y., Li, E., and Stashenko, P. (1999) Nat. Genet. 23, 447-451
33. Frattini, A., Orchard, P. J., Sobacchi, C., Giliani, S., Abinun, M., Mattsson, J. P., Keeling, D. J., Andersson, A. K., Wallbrandt, P., Zecca, L., Notarangelo, L. D., Vezzoni, P., and Villa, A. (2000) Nat. Genet. 25, 343-346
34. Wilkins, S., Vasilyeva, E., and Forgac, M. (1999) J. Biol. Chem. 274, 31804-31810
35. Boekema, E. J., van Breeman, J. F., Brisson, A., Ubbink-Kok, T., Konings, W. N., and Lolkema, J. S. (1999) Nature 401, 37-38
36. Nishi, T., and Forgac, M. (2000) J. Biol. Chem. 275, 6824-6830
37. Abrahams, J. P., Leslie, A. G., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628
38. Brown, D., Gluck, S., and Hartwig, J. (1987) J. Cell Biol. 105, 1636-1648
39. Breton, S., Wiederhold, T., Marshansky, V., Nsuma, N. N., Ramesh, V., and Brown, D. (2000) J. Biol. Chem. 275, 18219-18224
40. Murthy, A., Gonzalez-Agosti, C., Cordero, E., Pinney, D., Candia, C., Solomon, F., Gusella, J., and Ramesh, V. (1998) J. Biol. Chem. 273, 1273-1276

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
H. Feng, T. Cheng, N. J. Pavlos, K. H. M. Yip, A. Carrello, R. Seeber, K. Eidne, M. H. Zheng, and J. Xu
Cytoplasmic Terminus of Vacuolar Type Proton Pump Accessory Subunit Ac45 Is Required for Proper Interaction with V0 Domain Subunits and Efficient Osteoclastic Bone Resorption
J. Biol. Chem., May 9, 2008; 283(19): 13194 - 13204.
[Abstract] [Full Text] [PDF]

Home page
 J. Exp. Biol.Home page
J. Zuo, S. Vergara, S. Kohno, and L. S. Holliday
Biochemical and functional characterization of the actin-binding activity of the B subunit of yeast vacuolar H+-ATPase
J. Exp. Biol., April 1, 2008; 211(7): 1102 - 1108.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
R. Supino, G. Petrangolini, G. Pratesi, M. Tortoreto, E. Favini, L. D. Bo, P. Casalini, E. Radaelli, A. C. Croce, G. Bottiroli, et al.
Antimetastatic Effect of a Small-Molecule Vacuolar H+-ATPase Inhibitor in in Vitro and in Vivo Preclinical Studies
J. Pharmacol. Exp. Ther., January 1, 2008; 324(1): 15 - 22.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. Voss, O. Vitavska, B. Walz, H. Wieczorek, and O. Baumann
Stimulus-induced Phosphorylation of Vacuolar H+-ATPase by Protein Kinase A
J. Biol. Chem., November 16, 2007; 282(46): 33735 - 33742.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Renal Physiol.Home page
S. Breton and D. Brown
New insights into the regulation of V-ATPase-dependent proton secretion
Am J Physiol Renal Physiol, January 1, 2007; 292(1): F1 - F10.
[Abstract] [Full Text] [PDF]