Yeast V-ATPase Complexes Containing Different Isoforms of the 100-kDa a-subunit Differ in Coupling Efficiency and in Vivo Dissociation*

Shoko Kawasaki-NishiDagger, Tsuyoshi NishiDagger, and Michael Forgac§

From the Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111

Received for publication, November 29, 2000, and in revised form, February 28, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The 100 kDa a-subunit of the yeast vacuolar (H+)-ATPase (V-ATPase) is encoded by two genes, VPH1 and STV1. These genes encode unique isoforms of the a-subunit that have previously been shown to reside in different intracellular compartments in yeast. Vph1p localizes to the central vacuole, whereas Stv1p is present in some other compartment, possibly the Golgi or endosomes. To compare the properties of V-ATPases containing Vph1p or Stv1p, Stv1p was expressed at higher than normal levels in a strain disrupted in both genes, under which conditions V-ATPase complexes containing Stv1p appear in the vacuole. Complexes containing Stv1p showed lower assembly with the peripheral V1 domain than did complexes containing Vph1p. When corrected for this lower degree of assembly, however, V-ATPase complexes containing Vph1p and Stv1p had similar kinetic properties. Both exhibited a Km for ATP of about 250 µM, and both showed resistance to sodium azide and vanadate and sensitivity to nanomolar concentrations of concanamycin A. Stv1p-containing complexes, however, showed a 4-5-fold lower ratio of proton transport to ATP hydrolysis than Vph1p-containing complexes. We also compared the ability of V-ATPase complexes containing Vph1p or Stv1p to undergo in vivo dissociation in response to glucose depletion. Vph1p-containing complexes present in the vacuole showed dissociation in response to glucose depletion, whereas Stv1p-containing complexes present in their normal intracellular location (Golgi/endosomes) did not. Upon overexpression of Stv1p, Stv1p-containing complexes present in the vacuole showed glucose-dependent dissociation. Blocking delivery of Vph1p-containing complexes to the vacuole in vps21Delta and vps27Delta strains caused partial inhibition of glucose-dependent dissociation. These results suggest that dissociation of the V-ATPase complex in vivo is controlled both by the cellular environment and by the 100-kDa a-subunit isoform present in the complex.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The vacuolar (H+)-ATPases (or V-ATPases)1 are a family of ATP-dependent proton pumps found in a variety of intracellular compartments that function in both endocytic and secretory pathways (1-8). Acidification of these compartments is essential for many cellular processes, including receptor-mediated endocytosis, intracellular targeting, protein processing and degradation, and coupled transport. V-ATPases are also present in the plasma membrane of certain specialized cells, including osteoclasts (9), renal intercalated cells (10), and neutrophils (11), where they function in such processes as bone resorption, renal acidification, and pH homeostasis, respectively.

The V-ATPases from fungi, plants, and animals are structurally very similar and are composed of two domains (1-8). The V1 domain is a peripheral complex of molecular mass 570 kDa composed of eight different subunits of molecular mass 70-14 kDa (subunits A-H) that is responsible for ATP hydrolysis. The V0 domain is a 260-kDa integral complex composed of five subunits of molecular mass 100-17 kDa (subunits a, d, c, c', and c'') that is responsible for proton translocation. This structure is similar to that of the ATP synthases (or F- ATPases) that function in ATP synthesis in mitochondria, chloroplasts, and bacteria (12-17). Sequence homology between these classes of ATPase has been identified for both the nucleotide binding subunits (18, 19) and the proteolipid subunits (subunits c, c', and c'') (20, 21). Subunit G has also been shown to have some homology to subunit b of the F-ATPases (22).

The 100-kDa a-subunit of the V-ATPase is an integral membrane protein possessing an amino-terminal hydrophilic domain and a carboxyl-terminal hydrophobic domain containing multiple putative membrane-spanning segments (23-25). In yeast, the 100-kDa subunit is encoded by two genes, VPH1 and STV1. Vph1p and Stv1p are homologous proteins displaying 54% identity and 71% similarity (23, 24). These proteins show distinct intracellular localization, with Vph1p localized to the vacuole and Stv1p normally localized to some other intracellular compartment, possibly Golgi or endosomes (24). These results suggest that the 100-kDa a-subunit contains information necessary to target the V-ATPase to the appropriate intracellular site. To compare the properties of V-ATPase complexes containing Vph1p and Stv1p, we have taken advantage of the observation that overexpression of Stv1p in a yeast strain deleted in both VPH1 and STV1 results in mislocalization of Stv1p to the vacuole (24). This allowed us to compare the properties of V-ATPase complexes present in the same intracellular compartment (the vacuole) that differed only in the a-subunit isoform present.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials and Strains-- Zymolyase 100T was obtained from Seikagaku America, Inc. Concanamycin A was purchased from Fluka Chemical Corp. Protease inhibitors were from Roche Molecular Biochemicals. The monoclonal antibody 3F10 (directed against the HA antigen), which is conjugated with horseradish peroxidase, was also from Roche Molecular Biochemicals. The monoclonal antibody 8B1-F3 against the yeast V-ATPase A-subunit (26) and the monoclonal antibody 10D7 against the 100-kDa a-subunit (27) were from Molecular Probes. Escherichia coli and yeast culture media were purchased from Difco Laboratories. Restriction endonucleases, T4 DNA ligase, and other molecular biology reagents were from Life Technologies, Inc., Promega, and New England Biolabs. ATP, phenylmethylsulfonyl fluoride, and most other chemicals were purchased from Sigma.

Yeast strain MM112 (MATalpha Delta vph1::LEU2 Delta stv1::LYS2 his3-(Delta 200 leu2 lys2 ura3-52) and plasmid MM322 (VPH1 in pRS316) (24) were used to generate and study the HA-tagged Vph1p. Yeast cells were grown in yeast extract-peptone-dextrose (YPD) medium or synthetic dropout (SD) medium (28).

Cloning of the STV1 Gene-- The STV1 gene was amplified from genomic DNA isolated from yeast strain YPH500 using primers containing the restriction enzyme sites XbaI and BamHI and then cloned into pRS316 using the XbaI and BamHI sites (pRS316-STV1). The sequence of the cloned STV1 gene was confirmed by DNA sequencing using an automated sequencer from Applied Biosystems. The oligonucleotides used for amplification of the STV1 gene were as follows, with the restriction enzyme sites underlined: STV1 forward (XbaI), 5'-GGCCTCTAGATTAGAGGAAATGCAATTTTCTATCATC-3'; STV1 reverse (BamHI), 5'-GGCCGGATCCTATTGGTAGACCACTTGACCGAC-3'.

Introducing HA Tags into Vph1p and Stv1p-- An HpaI site was introduced into VPH1 using polymerase chain reaction, and then the two tandem repeats of the nine-amino acid HA epitope (YPYDVPDYA) were ligated into the HpaI site of pRS316-VPH1 (pRS316-VPH1::HA) and the BglII site of pRS316-STV1 (pRS316-STV1::HA). This resulted in insertion of the tandem HA tags after amino acid residue Asn-185 of Vph1p and the corresponding residue (Leu-227) of Stv1p. A XbaI-BamHI fragment of pRS316-STV1::HA was ligated into XbaI-BamHI sites of the 2-µm plasmid, YEp352 (YEp352-STV1::HA).

Transformation and Selection-- Yeast cells (MM112) lacking functional endogenous Vph1p and Stv1p were transformed using the lithium acetate method (29) with the wild-type pRS316-VPH1 as a positive control, pRS316 vector alone as a negative control, pRS316-VPH1::HA, pRS316-STV1::HA, and YEp352-STV1::HA. The transformants were selected on uracil minus (Ura-) plates as described previously (30). Growth phenotypes of the mutants were assessed on YPD plates buffered with 50 mM KH2PO4 or 50 mM succinic acid to either pH 7.5 or pH 5.5.

Isolation of Vacuolar Membrane Vesicles-- Vacuolar membrane vesicles were isolated using a modification of the protocol described by Uchida et al. (31). Yeast were grown overnight at 30 °C to 1 × 107 cells/ml in 1 liter of selective medium. Cells were pelleted, washed once with water, and resuspended in 100 ml of 10 mM dithiothreitol and 100 mM Tris-HCl, pH 9.4. After incubation at 30 °C for 15 min, cells were pelleted again, washed once with 100 ml of YPD medium containing 0.7 M sorbitol, 2 mM dithiothreitol, and 100 mM MES-Tris, pH 7.5, resuspended in 100 ml of YPD medium containing 0.7 M sorbitol, 2 mM dithiothreitol, 100 mM MES-Tris, pH 7.5, and 2 mg of zymolase 100T and incubated at 30 °C with gentle shaking for 60 min. The resulting spheroplasts were osmotically lysed, and the vacuoles were isolated by flotation on two consecutive Ficoll gradients. Protein concentrations were measured by the BCA protein assay (Pierce).

Analysis of the 100-kDa Subunit Expression and V-ATPase Assembly-- Yeast were grown to log phase at 30 °C in selective medium, whole cell lysates were prepared as described previously (26), and the proteins were separated by SDS-PAGE on 8% acrylamide gels. The expression of the 100-kDa subunit was detected by Western blotting using the horseradish peroxidase-conjugated monoclonal antibody 3F10 against HA, whereas subunit A was detected by monoclonal antibody 8B1-F3 directed against subunit A followed by a horseradish peroxidase-conjugated secondary antibody (Bio-Rad). Assembly of the V-ATPase was assessed by measurement of the amount of subunit A present on isolated vacuolar membranes (27, 32). Vacuolar membrane proteins were separated by SDS-PAGE on 8% acrylamide gels, and the 100-kDa subunit, subunit A, and subunit d were detected by Western blotting as described above. Blots were developed using a chemiluminescent detection method obtained from Kirkegaard and Perry Laboratories (Gaithersburg, MD).

Measurement of ATPase Activity and Examination of Effects of Inhibitors on ATPase Activity-- ATPase activity was measured using a coupled spectrophotomeric assay as described previously (33) with some modification. To determine the KmATP and the Vmax for V-ATPase complexes containing Vph1p or Stv1p, ATPase activity was measured over a range of ATP concentrations from 0.1-2 mM ATP, whereas MgSO4 was maintained at 4 mM. Vacuoles isolated from the vph1Delta stv1Delta strain expressing Vph1p (3 µg of protein) or Stv1p (10 µg of protein) were incubated in ATPase assay buffer (50 mM NaCl, 30 mM KCl, 20 mM HEPES-NaOH, pH 7.0, 0.2 mM EGTA, 10% glycerol, 1 mM MgCl2, 1.5 mM phosphoenolpyruvate, 0.35 mM NADH, 20 units/ml pyruvate kinase, and 10 units/ml lactate dehydrogenase) with 0.1% Me2SO or 1 µM concanamycin A at room temperature for 10 min. The assay was then started by the addition ATP at the indicated concentrations, and the absorbance at 341 nm was measured continuously using a Kontron UV-visible spectrophotometer. V-ATPase activity was defined as that fraction of the ATPase activity inhibited by 1 µM concanamycin A (typically 90% for Vph1p-containing vacuoles and 50% for Stv1p-containing vacuoles). KmATP and Vmax were calculated from double-reciprocal plots of ATP concentration versus V-ATPase activity expressed in µmol ATP/min/mg of protein. To examine the effect of inhibitors on ATPase activity, isolated vacuolar membranes were incubated in ATPase buffer containing 0.1% Me2SO, 1 µM concanamycin A, 0.1 mM sodium vanadate, or 0.5 mM sodium azide at room temperature for 10 min followed by initiation of the assay by the addition of 0.5 mM ATP. ATP-dependent proton transport activity was measured by quenching of ACMA fluorescence using a PerkinElmer LS50B spectrofluorometer as previously described (34).

In Vivo Dissociation of the V-ATPase in Response to Glucose Deprivation-- Dissociation of the V-ATPase in response to glucose depletion was measured as described previously (35) with some modifications. The vph1Delta stv1Delta strain MM112 expressing Vph1p or Stv1p from the single copy plasmid pRS316 or Stv1p using the high copy plasmid YEp352 was grown in selective medium overnight to an absorbance at 600 nm of <1.0. The yeast strains SF838-1D (MATalpha leu2-3, 112 ura3-52 his4-519 ade6 gal2 pep4-3), SGY79 (MATalpha leu2-3, 112 ura3-52 his4-519 ade6 gal2 pep4-3 vps21::Kanr), and SGY73 (MATalpha leu2-3, 112 ura3-52 his4-519 ade6 gal2 pep4-3 vps27::LEU2) (36) were grown under the same conditions except using YPD media. The cells were converted to spheroplasts by treatment with zymolase 100T and incubated in YEP media with or without 2% glucose for 40 min at 30 °C. Spheroplasts were pelleted and lysed in phosphate-buffered saline containing 1% C12E9, protease inhibitors, and 1 mM dithiobis(succinimidyl propionate). An aliquot (corresponding to ~6 × 105 cells) was removed to allow analysis of proteins present in the whole cell lysate. The V-ATPase complexes were immunoprecipitated from the remainder of the lysate (corresponding to ~3 × 106 cells) using 8B1-F3 against the A-subunit and protein G-agarose followed by separation on 8% acrylamide gels and transfer to nitrocellulose. Western blotting was then performed separately using the horseradish peroxidase (HRP)-conjugated monoclonal antibodies 3F10 against HA or 10D7-A7 against Vph1p to detect the V0 domain or antibody 8B1-F3 against the A-subunit to detect the V1 domain followed by an HRP-conjugated secondary antibody. Dissociation of the V-ATPase complex is reflected as a reduction in the amount of the 100-kDa subunit immunoprecipitated using the antibody directed against subunit A (located in the V1 domain). Blots were developed using the chemiluminescent detection system from Kirkegaard and Perry Laboratories.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We wished to compare the properties of V-ATPases containing Vph1p and Stv1p, the two isoforms of the yeast 100-kDa a-subunit. Because these isoforms normally reside in different intracellular compartments, it was necessary to devise a strategy that would allow complexes containing these two isoforms to be targeted to the same intracellular membrane. It has previously been shown that overexpression of Stv1p in a strain disrupted in both VPH1 and STV1 causes a significant amount of Stv1p to appear in the vacuolar membrane (24). Because Vph1p normally localizes to the vacuole, this finding allowed us to directly compare the properties of V-ATPase complexes present in the same intracellular compartment that differed only in the a-subunit isoform present. To detect the expression level of each a-subunit isoform, tandem HA epitope tags were inserted in the amino-terminal region of both Vph1p and Stv1p. Expression of the HA-tagged form of either Vph1p or Stv1p using the low copy plasmid pRS316 in the vph1Delta stv1Delta double-deletion strain MM112 led to a wild-type growth phenotype. That is, cells showed normal growth at both pH 7.5 and 5.5. This was also true using expression of Stv1p from the high copy plasmid YEp352. Western blotting of whole cell lysates (Fig. 1) indicated that Stv1p is expressed at a lower level from the single copy plasmid pRS316 than is Vph1p. Expression of Stv1p using the high copy plasmid YEp352 led to a significant increase in expression levels of Stv1p.


View larger version (29K):
[in this window]
[in a new window]
 
Fig. 1.   Western blot analysis of expression levels of HA-tagged 100-kDa a-subunits expressed using low and high copy plasmids. Whole cell lysates prepared from the vph1Delta stv1Delta strain MM112 transformed with the single copy plasmid pRS316 alone, HA-tagged Vph1p in pRS316, HA-tagged Stv1p in pRS316, or HA-tagged Stv1p in the high copy plasmid YEp352 were subjected to SDS-PAGE on an 8% acrylamide gel followed by transfer to nitrocellulose and Western blot analysis using the HRP-conjugated monoclonal antibody 3F10 against the HA epitope or the monoclonal antibody 8B1-F3 against the 69-kDa A-subunit followed by an HRP-conjugated secondary antibody. Also shown are the growth phenotypes of cells transformed with each of the indicated plasmids on YPD plates buffered to pH 7.5.

To confirm previous reports that overexpression of Stv1p led to its presence in the vacuolar membrane, vacuoles were isolated from each of the strains shown in Fig. 1, separated by SDS-PAGE, and analyzed by Western blotting using anti-HA antibodies. As shown in Fig. 2, although Vph1p was detectable in the vacuolar membrane using the single copy plasmid, Stv1p was not. By contrast, when Stv1p was expressed at higher levels using the multicopy plasmid, it was detectable in the vacuolar membrane at levels comparable with Vph1p. Overexposure of the gel shown in Fig. 2 revealed very low levels of Stv1p in the vacuolar membrane when expressed using pRS316. Quantitation using densitometric analysis indicated that although the ratio of Vph1p to Stv1p using pRS316 in whole cell lysates was 3:1 (Fig. 1, second and third lanes from the left), the ratio in the vacuolar membrane was 30:1 (Fig. 2, longer exposure, not shown). These results confirmed that although Stv1p is normally not targeted to the vacuole, overexpression results in the appearance of a significant amount of Stv1p in the vacuole.


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 2.   Western blot analysis of levels of HA-tagged Vph1p and Stv1p and the 69-kDa A-subunit on purified vacuolar membranes. Vacuolar membranes were isolated from the vph1Delta stv1Delta strain MM112 transformed with pRS316 alone, HA-tagged Vph1p in pRS316, HA-tagged Stv1p in pRS316, or HA-tagged Stv1p in Yep352, an aliquot (0.5 µg protein) was subjected to SDS-PAGE on an 8% acrylamide gel, and the levels of the HA-tagged Vph1p, HA-tagged Stv1p, and the 69 kDa A-subunit were determined by Western blot analysis as described in the legend to Fig. 1. The amount of A-subunit present on the vacuolar membrane is one measure of assembly of the V-ATPase complex.

To assess the degree of assembly of V-ATPase complexes containing Vph1p and Stv1p, vacuoles isolated from each strain were also probed using an antibody directed against the 70-kDa A-subunit. It has previously been shown that the A-subunit (and other V1 subunits) only associates with the vacuolar membrane if assembly of the V-ATPase complex is normal (27, 32). As shown in Fig. 2, although the level of Stv1p in the vacuolar membrane using the high copy plasmid is comparable with that of Vph1p using pRS316, the level of A-subunit associated with Stv1p is much lower than that associated with Vph1p. Quantitation by densitometry indicates an 8-fold reduction in the level of A-subunit associated with vacuoles containing Stv1p relative to vacuoles containing Vph1p. This result indicates that, at least when present in the environment of the central vacuole, V-ATPase complexes containing Stv1p are less assembled than V-ATPase complexes containing Vph1p.

To determine whether introduction of the HA epitope tags into Vph1p and Stv1p altered assembly of the V-ATPase complex, vacuolar membranes from strains expressing either tagged or untagged versions of Vph1p and Stv1p were probed by Western blot using antibodies against HA, Vph1p, subunit A, and subunit d (a V0 subunit). No antibodies specific for the untagged version of Stv1p are currently available. As can be seen from Fig. 3, the presence of the HA tag had virtually no effect on the amount of Vph1p, subunit A, or subunit d present in the vacuolar membrane, indicating that introduction of the tag did not disrupt assembly of complexes containing Vph1p. Similarly, for Stv1p-containing complexes, the amounts of subunit A and subunit d present in the vacuoles was unchanged by the introduction of the epitope tag. Thus, the presence of the HA tag does not alter the assembly of V-ATPase complexes. Interestingly, the ratio of subunit A to subunit d staining in the vacuoles is almost the same for Vph1p and Stv1p (Fig. 3) despite the much lower ratio of subunit A to 100-kDa subunit for Stv1p than for Vph1p (Fig. 2). This result suggests that the lower assembly of V1 and V0 observed for Stv1p-containing complexes may be the result of reduced association of Stv1p with subunit d, at least in the environment of the vacuolar membrane.


View larger version (38K):
[in this window]
[in a new window]
 
Fig. 3.   Comparison of assembly competence of HA-tagged and untagged versions of Vph1p and Stv1p by Western blot analysis of vacuolar membranes. Vacuolar membranes were isolated from the vph1Delta stv1Delta strain MM112 transformed with Vph1p in pRS316, HA-tagged Vph1p in pRS316, Stv1p in YEp352, or HA-tagged Stv1p in YEp352, an aliquot (20 µg protein) was subjected to SDS-PAGE on an 8% acrylamide gel, and the levels of the HA-tagged Vph1p, HA-tagged Stv1p, Vph1p, subunit A, and subunit d were determined by Western blot analysis as described in the legend to Fig. 1.

To compare the kinetic properties of V-ATPase complexes containing Vph1p and Stv1p, vacuoles were isolated from the vph1Delta stv1Delta strain expressing either Vph1p using pRS316 or Stv1p using the high copy plasmid. In this case, the untagged versions of Vph1p and Stv1p were expressed. As indicated in Table I, the Km for ATP was ~250 µM for complexes containing both Vph1p and Stv1p. Although the Vmax for ATP hydrolysis for Stv1p-containing complexes was lower than for Vph1p-containing complexes, the 8.6-fold reduction in activity could be entirely accounted for by the lower degree of assembly of V-ATPases containing Stv1p (Fig. 2). Measurement of proton transport using ACMA fluorescence quenching (Table I) indicated an even lower level of proton transport for Stv1p-containing vacuoles relative to Vph1p-containing vacuoles (the ratio of Vph1p:Stv1p was 55:1). This result indicates that the ratio of proton transport to ATP hydrolysis is ~4-5-fold lower for Stv1p-containing complexes compared with Vph1p-containing complexes.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Kinetic analysis of V-ATPases containing Vph1p and Stv1p

The inhibitor sensitivity of V-ATPase complexes containing Vph1p and Stv1p was also compared. As shown in Table II, ATPase activity for Vph1p-containing vacuoles was 90% inhibited by 1 µM concanamycin A but was only 8% inhibited by 0.1 mM vanadate and was not affected by 0.5 mM azide. By contrast, ATPase activity in Stv1p-containing vacuoles was only 44% inhibited by concanamycin A and 25% inhibited by vanadate. This apparent discrepancy between Vph1p and Stv1p may be partially accounted for by the presence of one or more concanamycin-resistant ATPase activities in the vacuolar membrane, one of which is vanadate-sensitive. Under normal conditions (i.e. with vacuoles containing Vph1p), these other activities are quite minor since the bulk of ATPase activity in the vacuolar membrane corresponds to the V-ATPase. By contrast, because of the much lower levels of V-ATPase activity observed with Stv1p (Table I), the percentage of the total ATPase activity in the vacuolar membrane that corresponds to these "other" ATPases is significantly larger.

                              
View this table:
[in this window]
[in a new window]
 
Table II
Effects of inhibitors on ATPase activity of isolated vacuoles containing Vph1p and Stv1p
ATPase assays were carried out on isolated vacuolar membranes at 0.5 mM ATP in the presence or absence of the indicated inhibitors as described under "Experimental Procedures." Activities are expressed relative to the control sample, which received 0.1% Me2SO. Both Vph1p and Stv1p were untagged.

To compare the affinity of V-ATPase complexes containing Vph1p and Stv1p for concanamycin A, ATP-dependent proton pumping was measured in vacuoles using ACMA fluorescence quenching. As shown in Fig. 4, the concentration of concanamycin A required for inhibition of 50% of proton transport for both Vph1p and Stv1p-containing complexes was ~0.1 nM. A small residual concanamycin-resistant component of ATP-dependent fluorescence quenching in Stv1p-containing vacuoles was again observed. Nevertheless, the results suggest that Vph1p- and Stv1p-containing complexes have nearly the same affinity for concanamycin A. 


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 4.   Inhibitory effect of concanamycin A on ATP-dependent proton transport in vacuoles containing Vph1p or Stv1p. Vacuolar membranes were isolated from the vph1Delta stv1Delta strain MM112 transformed with Vph1p in pRS316 or Stv1p in YEp352 (both untagged), and ATP-dependent proton transport was measured at the indicated concentrations of concanamycin A by fluorescence (F) quenching using the fluorescence dye ACMA as described under "Experimental Procedures." For Vph1p-containing membranes, 1 µg of protein was employed, whereas for Stv1p-containing membranes, 5 µg of protein was used. Activities are expressed as the initial rate of fluorescence change after the addition of ATP.

Reversible dissociation of the V-ATPase complex has been shown to occur in response to glucose depletion in yeast and has been proposed to represent an important mechanism of regulating V-ATPase activity in vivo (35). To compare glucose-dependent dissociation of V-ATPase complexes containing Vph1p and Stv1p, spheroplasts were prepared from the Vph1p- and Stv1p-expressing strains, incubated in the presence or absence glucose, and solubilized with C12E9, and immunoprecipitation was carried out using the antibody 8B1-F3 directed against the A-subunit of the V1 domain. After SDS-PAGE, Western blotting was performed using either 8B1-F3 to detect the A-subunit or the anti-HA antibody to detect Vph1p or Stv1p in the V0 domain. Dissociation of the V-ATPase is reflected as a decrease in the amount of Vph1p or Stv1p immunoprecipitated using the antibody against subunit A. As shown in Fig. 5a, glucose depletion led to a decrease in the amount of Vph1p immunoprecipitated using the anti-A-subunit antibody (first and second lanes), indicating glucose-dependent dissociation of Vph1p-containing complexes. Because of the lower level of Stv1p expressed using the pRS316 vector, assembled V-ATPase complexes could not be detected at this exposure (Fig. 5, panel a, third and fourth lanes). A longer exposure, however, revealed that Stv1p-containing complexes do not show dissociation on removal of glucose but instead show some increase in assembly (Fig. 5, panel b, third and fourth lanes). By contrast, when Stv1p was expressed at higher levels (under which conditions a significant amount of Stv1p appears in the vacuole), V-ATPase complexes containing Stv1p showed glucose-dependent dissociation (Fig. 5, panel a, fifth and sixth lanes). By comparing the total expression levels of Vph1p and Stv1p in whole cell lysates (Fig. 5, panel c) with the amounts of Vph1p and Stv1p immunoprecipitated with the anti-A-subunit antibody (Fig. 5, panel b), it is clear that a smaller fraction of Stv1p is assembled with V1, even when expressed at low levels and in the presence of glucose than for Vph1p. This result is consistent with the data shown in Fig. 2. Quantitation of the results from five independent experiments by densitometry (Fig. 5d) indicates that glucose removal leads to 65% dissociation of complexes containing Stv1p expressed at high levels as compared with 45% dissociation of complexes containing Vph1p. By contrast, V-ATPase complexes formed from Stv1p expressed at low levels show no significant dissociation upon removal of glucose. These results suggest that the membrane environment plays a crucial role in determining whether the V-ATPase undergoes dissociation in response to glucose depletion, with V-ATPase present in the central vacuole undergoing dissociation and V-ATPase present in the Golgi/endosomes remaining assembled.


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 5.   In vivo dissociation of V-ATPase complexes containing Vph1p or Stv1p after glucose depletion. a, The vph1Delta stv1Delta strain MM112 transformed with HA-tagged Vph1p in pRS316, HA-tagged Stv1p in pRS316, or HA-tagged Stv1p in YEp352 were grown in selective media overnight and converted to spheroplasts. The spheroplasts were incubated for 40 min in YEP media with or without 2% glucose. The V-ATPase was then solubilized with C12E9 and immunoprecipitated using the monoclonal antibody 8B1-F3 against the A-subunit. Samples were then subjected to SDS-PAGE on 8% acrylamide gels followed by transfer to nitrocellulose and Western blot analysis using the HRP-conjugated monoclonal antibody 3F10 against HA or the monoclonal antibody 8B1-F3 against the 69-kDa A-subunit followed by an HRP-conjugated secondary antibody. Dissociation of the V-ATPase complex is reflected as a decrease in the amount of Vph1p or Stv1p immunoprecipitated using the antibody against subunit A. b, a longer exposure of the film shown in panel a showing the degree of glucose-dependent dissociation for Stv1p expressed at lower levels. c, Western blot analysis was performed on an aliquot of the whole cell lysate derived from each of the strains described in part a using antibodies against HA or subunit A. d, quantitation of the data from five independent determinations was performed by densitometric analysis, and the results are expressed as the amount of assembled V-ATPase observed upon glucose depletion relative to that observed in the presence of glucose.

Dissociation of Vph1p-containing complexes in response to glucose depletion has previously been shown to be reversible upon the readdition of glucose to the media (35). To test whether dissociation of Stv1p-containing complexes that have been targeted to the vacuole is also reversible, spheroplasts were prepared from cells expressing Stv1p from the high copy plasmid, incubated in the presence or absence of glucose for 20 min, or incubated in the absence of glucose for 20 min followed by the addition of glucose and incubation for an additional 20 min. After these incubations, spheroplasts were solubilized with C12E9, and immunoprecipitation was carried out using the anti-A-subunit antibody followed by SDS-PAGE and Western blotting using antibodies against both HA and subunit A, as described above. As can be seen from the data in Fig. 6, dissociation of Stv1p-containing complexes targeted to the vacuole in response to glucose depletion is also reversible upon the readdition of glucose to the media.


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 6.   Reversal of dissociation of Stv1p-containing complexes present in the vacuolar membrane upon the readdition of glucose to the medium. The vph1Delta stv1Delta strain MM112 transformed with HA-tagged Stv1p in YEp352 was grown in selective media overnight and converted to spheroplasts. The spheroplasts were incubated for 20 min in YEP media with 2% glucose (+), for 20 min in YEP media without glucose (-), or for 20 min in YEP media without glucose followed by the addition of 2% glucose and incubation for an additional 20 min (- right-arrow +). The V-ATPase was then solubilized with C12E9 and immunoprecipitated using the anti-A-subunit antibody followed by SDS-PAGE and Western blot analysis using the anti-HA and anti-A-subunit antibodies as described in the legend to Fig. 5.

To further test the importance of the membrane environment in affecting in vivo dissociation of the V-ATPase complex, glucose-dependent dissociation was examined in two yeast strains that are disrupted in different steps of the membrane traffic pathway from the Golgi to the vacuole. In vps21Delta mutants, Vph1p is found in Golgi-derived vesicles that accumulate as a result of a block in fusion with the prevacuolar compartment, whereas in the vps27Delta mutants, Vph1p appears in an exaggerated form of the prevacuolar compartment (36). Glucose-dependent dissociation of Vph1p-containing complexes was examined in these strains relative to the parent strain using an antibody specific for Vph1p. As shown in Fig. 7, glucose depletion appeared to lead to more complete dissociation of Vph1p-containing complexes in the wild-type strain (87%) than was observed for Vph1p-containing complexes in the vph1Delta stv1pDelta strain described in Fig. 5. When the experiment in Fig. 5 was repeated using the antibody against Vph1p, approximately the same level of dissociation was observed upon glucose depletion (90%) as was seen in Fig. 7. This indicates that the apparent difference in dissociation observed between Fig. 5 and Fig. 7 is due to differences in the ability of the anti-HA and anti-Vph1p antibodies to detect low levels of the 100-kDa proteins. However, it was necessary to use the anti-HA antibody for the experiments described in Fig. 5 because of the absence of antibodies specific for Stv1p.


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 7.   In vivo dissociation of V-ATPase complexes containing Vph1p in the wild-type strain and vps21Delta and vps27Delta strains disrupted in vacuolar targeting. a, yeast strain SF838-1D (wild type), SGY79 (vps21Delta ), and SGY73 (vps27Delta ) were grown in selective media overnight and converted to spheroplasts. The spheroplasts were incubated for 40 min in YEP media with or without 2% glucose. The V-ATPase was then solubilized with C12E9 and immunoprecipitated using the monoclonal antibody 8B1-F3 against the A-subunit. Samples were then subjected to SDS-PAGE on 8% acrylamide gels followed by transfer to nitrocellulose, and Western blot analysis was performed using the monoclonal antibody 10D7-A7 against Vph1p or the antibody 8B1-F3 against subunit A. Also shown are the growth phenotypes of each strain on YPD plates buffered to pH 7.5. b, Western blot analysis was performed on an aliquot of the whole cell lysate derived from each of the strains described in part a using antibodies against Vph1p or subunit A. c, quantitation of the data from three independent determinations was performed by densitometric analysis, and the results are expressed as the amount of assembled V-ATPase observed upon glucose depletion relative to that observed in the presence of glucose.

In both the vps21Delta and vps27Delta strains, dissociation of the V-ATPase in response to glucose depletion was observed but was less complete than for the wild-type strain. Quantitation of three independent experiments (Fig. 7c) revealed dissociation levels of 63 and 76% in the vps21Delta and vps27Delta strains, respectively, as compared with the 87% dissociation observed in the parental wild-type strain. Western blot analysis of whole cell lysates indicated a slight reduction in the level of Vph1p in the vps21Delta strain (Fig. 7b). Glucose-dependent dissociation of the V-ATPase was shown to be reversible upon the readdition of glucose to the media for the wild-type, vps21Delta , and vps27Delta strains (Fig. 8). Because Vph1p-containing complexes are prevented from reaching the vacuole in the vps21Delta and vps27Delta strains, the results indicate that complexes containing Vph1p that are present in other intracellular compartments are still able to undergo glucose-dependent dissociation, although to different degrees. It should be noted that because the compartments that accumulate in the vps21Delta and vps27Delta strains are aberrant (36), it is possible that the reduction in dissociation observed in these strains may be due to some altered property of these compartments. For example, the multilamellar structure of the prevacuolar compartment that accumulates in the vps27Delta strain may prevent some essential cytoplasmic signal from reaching a significant fraction of the V-ATPase present in this compartment. Nevertheless, together with the experiments described above, these results suggest that in vivo dissociation of the V-ATPase complex is affected by both the a-subunit isoform present and the intracellular environment in which the complex resides.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 8.   Reversal of dissociation of V-ATPase in wild type and vps21Delta and vps27Delta strains upon the readdition of glucose to the medium. Yeast strain SF838-1D (wild type), SGY79 (vps21Delta ), and SGY73 (vps27Delta ) were grown in selective media overnight and converted to spheroplasts. The spheroplasts were incubated for 20 min in YEP media with 2% glucose (+), for 20 min in YEP media without glucose (-), or for 20 min in YEP media without glucose followed by the addition of 2% glucose and incubation for an additional 20 min (- right-arrow +). The V-ATPase was then solubilized with C12E9 and immunoprecipitated using the anti-A-subunit antibody followed by SDS-PAGE and Western blot analysis using the anti-Vph1p and anti-A-subunit antibodies as described in the legend to Fig. 7.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

V-ATPases have been shown to reside in a large number of different intracellular compartments in eukaryotic cells (1-8). These include clathrin-coated vesicles, endosomes, lysosomes, Golgi-derived vesicles and secretory vesicles such as chromaffin granules and synaptic vesicles. V-ATPases have also been identified in the plasma membrane of numerous animal cell types, including osteoclasts, renal intercalated cells, macrophages, neutrophils, insect goblet cells, and certain tumor cells (9-11, 37, 38). An important question is how V-ATPases are targeted to their correct cellular destination. Recent results suggest that the 100-kDa a-subunit may play a crucial role in this targeting process. Thus, three unique isoforms of the a-subunit have been identified in mouse (a1, a2, and a3) (39, 40). In osteoclasts, the a1 isoform has been shown to localize to intracellular compartments, whereas the a3 isoform localizes to the plasma membrane (40). Consistent with this observation, it has been shown that disruption of the a3 gene in mouse leads to defective bone resorption (41). In yeast, the a-subunit is also encoded by multiple genes whose protein products show distinct cellular localization. Vph1p is localized to the central vacuole, whereas Stv1p is localized to some other intracellular compartment, possibly Golgi or endosomes (24). An important but unanswered question is how the properties of V-ATPases containing unique isoforms of the 100-kDa a-subunit compare.

To address this question, we have overexpressed Stv1p in a strain disrupted in both VPH1 and STV1, under which circumstances a significant amount of Stv1p becomes localized to the vacuole (24). This allowed a direct comparison of the properties of V-ATPase complexes present in the vacuolar membrane that differed only in the a-subunit isoform present. The results suggest that many of the kinetic properties of V-ATPases containing Vph1p or Stv1p are very similar. Thus, these enzymes exhibit the same affinity for ATP and the same (or very similar) inhibitor sensitivity. By contrast, V-ATPases containing the two a-subunit isoforms differ markedly in three properties. First, they show a significant (4-5-fold) difference in the ratio of proton transport to ATP hydrolysis, with Stv1p-containing complexes exhibiting a much lower efficiency of coupling. Changes in coupling efficiency have been proposed to play a role in controlling V-ATPase activity in vivo (42, 43), but this is the first comparison of coupling for two V-ATPases expressed in the same cell. This result also suggests that the V1 and V0 domains may be more loosely connected for Stv1p-containing complexes than for those containing Vph1p.

Consistent with this idea is the second major difference between the two isoforms, namely the degree of assembly of V1 and V0. Thus, V0 complexes containing Stv1p showed a lower degree of assembly with V1 than did V0 complexes containing Vph1p. This may be related to the observation that Stv1p present in the vacuole also shows a lower association with subunit d, which is normally an essential component of the V0 domain (44). Thus, the ratio of subunit d to subunit A in vacuoles derived from the strain overexpressing Stv1p is the same as that observed for vacuoles derived from the strain expressing Vph1p (Fig. 3). The absence of subunit d has been shown to lead to the loss of assembly of V1 subunits with the vacuolar membrane (44), and this may account for the reduced assembly of Stv1p-containing complexes. Nevertheless, it should be noted that Stv1p expressed at high levels appears to be stable in the vacuolar membrane (Figs. 2 and 3), suggesting that assembly of the entire V0 domain is not defective.

It is possible that these differences between Vph1p and Stv1p may in part be due to the presence of Stv1p in an intracellular compartment in which it does not normally reside. However, it is also possible that V-ATPase complexes in the Golgi (which normally contain Stv1p) may not need to be as active or as tightly coupled as V-ATPases in the central vacuole (which normally contains Vph1p). In support of this idea is the observation that in animal cells the pH of the Golgi is generally maintained in the range of 6.0-6.5 (45, 46), whereas the pH of lysosomes (the compartment analogous to the vacuole in higher eukaryotes) is typically 4.0-5.0 (47). Moreover, the lower degree of assembly of V1 and V0 was also observed for Stv1p present in its normal cellular location (Fig. 5).

The third property in which V-ATPase complexes containing Vph1p and Stv1p differ is their degree of dissociation in response to glucose deprivation. The Kane laboratory (35) demonstrates that the V-ATPase in yeast undergoes rapid and reversible dissociation in response to removal of glucose from the medium and has proposed that this process represents an important mechanism of regulating V-ATPase activity in vivo. They have shown that many of the signal transduction pathways activated by glucose starvation, including the Ras-cyclic AMP and protein kinase C-dependent pathways, are not involved in this response (48). Dissociation of the V1 and V0 domains has also been shown to occur in insects during molting, under which conditions the need for an active V-ATPase at the apical membrane of goblet cells lining the midgut may be dramatically reduced (49). Pools of free V1 and V0 domains have also been identified in Madin-Darby bovine kidney cells (50), but rapid and reversible dissociation of the V-ATPase in mammalian cells has not yet been reported.

In the present report it is shown that dissociation of V-ATPase complexes in response to glucose depletion appears to be controlled by both the a-subunit isoform present and the intracellular membrane in which the complex resides. Thus, Stv1p-containing complexes show no glucose-dependent dissociation when localized to their normal intracellular site (Golgi/endosomes) but do dissociate when targeted to the vacuole. By contrast, Vph1p-containing complexes appear to undergo dissociation both in their normal cellular environment (the vacuole) and when retained in Golgi or prevacuolar compartments. Nevertheless, dissociation of Vph1p-containing complexes is at least partly affected by the cellular environment in which these complexes reside, although altered properties of the compartments that accumulate in the vps mutants may be partly responsible for the reduced glucose-dependent dissociation. These results suggest that there are signals present both in the sequence of the a-subunit and in distinct intracellular sites that control dissociation of the V-ATPase complex in vivo. One possible reason that V-ATPase complexes present in the vacuole show more complete dissociation in response to glucose depletion than complexes present in other intracellular compartments (such as the Golgi) is that there may be a larger reserve of active V-ATPase in the central vacuole that the cell is able to survive without. Thus, the activity remaining after dissociation may be sufficient to keep the central vacuole relatively acidic while conserving significant amounts of cellular ATP. By contrast, V-ATPases present in the Golgi (or other prevacuolar compartments) appear to be less abundant but may therefore be less dispensable for cell viability. It should be noted, however, that because Stv1p-containing complexes show a lower degree of assembly with V1 than Vph1p-containing complexes, even in the presence of glucose, glucose withdrawal may not be able to affect any greater degree of dissociation of these complexes.

    ACKNOWLEDGEMENTS

We thank Dr. Tom Stevens, Institute of Molecular Biology, University of Oregon, for the gift of antibody against subunit d and yeast strains SGY73 and SGY79 disrupted in VPS27 and VPS21, respectively.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant GM34478 (to M. F.) and a Charles A. King Trust Fellowship Award (to T. N.).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.

Dagger These authors contributed equally to this work.

§ To whom correspondence should be addressed: Dept. of Physiology, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111. Tel.: 617-636-6939; Fax: 617-636-0445; E-mail address: michael.forgac@tufts.edu.

Published, JBC Papers in Press, March 2, 2001, DOI 10.1074/jbc.M010790200

    ABBREVIATIONS

The abbreviations used are: V-ATPase, vacuolar proton-translocating adenosine triphosphatase; ACMA, 9-amino-6-chloro-2-methoxyacridine; HA, influenza hemagglutinin; MES, 2-(N-morpholino)ethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; SD medium, synthetic dropout medium; YPD medium, yeast extract-peptone-dextrose medium; YEP medium, yeast extract-peptone medium; HRP, horseradish peroxidase, C12E9, polyoxyethylene-9-lauryl ether.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Forgac, M. (1999) J. Biol. Chem. 274, 12951-12954[Free Full Text]
2. Stevens, T. H., and Forgac, M. (1997) Annu. Rev. Cell Dev. Biol. 13, 779-808[CrossRef][Medline] [Order article via Infotrieve]
3. Margolles-Clark, E., Tenney, K., Bowman, E. J., and Bowman, B. J. (1999) J. Bioenerg. Biomembr. 31, 29-38[CrossRef][Medline] [Order article via Infotrieve]
4. Kane, P. M. (1999) J. Bioenerg. Biomembr. 31, 49-56[CrossRef][Medline] [Order article via Infotrieve]
5. Nelson, N., and Harvey, W. R. (1999) Physiol. Rev. 79, 361-385[Abstract/Free Full Text]
6. Moriyama, Y., Yamamoto, A., Yamada, H., Tashiro, Y., and Futai, M. (1996) Biol. Chem. Hoppe-Seyler 377, 155-165[Medline] [Order article via Infotrieve]
7. Anraku, Y., Umemoto, N., Hirata, R., and Ohya, Y. (1992) J. Bioenerg. Biomembr. 24, 395-405[Medline] [Order article via Infotrieve]
8. Sze, H., Ward, J. M., and Lai, S. (1992) J. Bioenerg. Biomembr. 21, 371-382
9. Chatterjee, D., Chakraborty, M., Leit, M., Neff, L., Jamsa-Kellokumpu, S., Fuchs, R., and Baron, R. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 6257-6261[Abstract]
10. Gluck, S. L. (1992) J. Bioenerg. Biomembr. 21, 351-360
11. Nanda, A., Brumell, J. H., Nordstrom, T., Kjeldsen, L., Sengelov, H., Borregaard, N., Rotstein, O. D., and Grinstein, S. (1996) J. Biol. Chem. 271, 15963-15970[Abstract/Free Full Text]
12. Weber, J., and Senior, A. (1997) Biochim. Biophys. Acta 1319, 19-58[Medline] [Order article via Infotrieve]
13. Fillingame, R. H. (2000) J. Exp. Biol. 203, 9-17[Abstract]
14. Cross, R. L., and Duncan, T. M. (1996) J. Bioenerg. Biomembr. 28, 403-408[Medline] [Order article via Infotrieve]
15. Capaldi, R. A., Schulenberg, B., Murray, J., and Aggler, R. (2000) J. Exp. Biol. 203, 29-33[Abstract]
16. Pedersen, P. L. (1996) J. Bioenerg. Biomembr. 28, 389-395[Medline] [Order article via Infotrieve]
17. Futai, M., and Omote, H. (1996) J. Bioenerg. Biomembr. 28, 409-414[Medline] [Order article via Infotrieve]
18. Zimniak, L., Dittrich, P., Gogarten, J. P., Kibak, H., and Taiz, L. (1988) J. Biol. Chem. 263, 9102-9112[Abstract/Free Full Text]
19. Bowman, B. J., Allen, R., Wechser, M. A., and Bowman, E. J. (1988) J. Biol. Chem. 263, 14002-14007[Abstract/Free Full Text]
20. Mandel, M., Moriyama, Y., Hulmes, J. D., Pan, Y. C., Nelson, H., and Nelson, N. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 5521-5524[Abstract]
21. Hirata, R., Graham, L. A., Takatsuki, A., Stevens, T. H., and Anraku, Y. (1997) J. Biol. Chem. 272, 4795-4803[Abstract/Free Full Text]
22. Hunt, I. E., and Bowman, B. J. (1997) J. Bioenerg. Biomembr. 29, 533-540[CrossRef][Medline] [Order article via Infotrieve]
23. Manolson, M. F., Proteau, D., Preston, R. A., Stenbit, A., Roberts, B. T., Hoyt, M., Preuss, D., Mulholland, J., Botstein, D., and Jones, E. W. (1992) J. Biol. Chem. 267, 14294-14303[Abstract/Free Full Text]
24. Manolson, M. F., Wu, B., Proteau, D., Taillon, B. E., Roberts, B. T., Hoyt, M. A., and Jones, E. W. (1994) J. Biol. Chem. 269, 14064-14074[Abstract/Free Full Text]
25. Perin, M. S., Fried, V. A., Stone, D. K., Xie, X. S., and Sudhof, T. C. (1991) J. Biol. Chem. 266, 3877-3881[Abstract/Free Full Text]
26. Kane, P. M., Yamashiro, C. T., and Stevens, T. H. (1989) J. Biol. Chem. 264, 19236-19244[Abstract/Free Full Text]
27. Kane, P. M., Kuehn, M. C., Howald-Stevenson, I., and Stevens, T. (1992) J. Biol. Chem. 267, 447-454[Abstract/Free Full Text]
28. Sherman, F. (1991) Methods Enzymol. 194, 3-21[Medline] [Order article via Infotrieve]
29. Gietz, D., St. Jean, A., Woods, R. A., and Schiestl, R. H. (1992) Nucleic Acids Res. 20, 1425[Medline] [Order article via Infotrieve]
30. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (eds) (1992) Short Protocols in Molecular Biology , John Wiley & Sons, Inc., New York
31. Uchida, E., Ohsumi, Y., and Anraku, Y. (1985) J. Biol. Chem. 260, 1090-1095[Abstract/Free Full Text]
32. Leng, X. H., Manolson, M., Liu, Q., and Forgac, M. (1996) J. Biol. Chem. 271, 22487-22493[Abstract/Free Full Text]
33. Forgac, M., Cantley, L., Wiedenmann, B., Altstiel, L., and Branton, D. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 1300-1303[Abstract]
34. Feng, Y., and Forgac, M. (1992) J. Biol. Chem. 267, 5817-5822[Abstract/Free Full Text]
35. Kane, P. M. (1995) J. Biol. Chem. 270, 17025-17032[Abstract/Free Full Text]
36. Gerard, S. R., Bryant, N. J., and Stevens, T. H. (2000) Mol. Biol. Cell 11, 613-626[Abstract/Free Full Text]
37. Wieczorek, H., Putzenlechner, M., Zeiske, W., and Klein, U. (1991) J. Biol. Chem. 266, 15340-15347[Abstract/Free Full Text]
38. Martinez-Zaguilan, R., Lynch, R., Martinez, G., and Gillies, R. (1993) Am. J. Physiol. 265, C1015-C1029[Abstract/Free Full Text]
39. Nishi, T., and Forgac, M. (2000) J. Biol. Chem. 275, 6824-6830[Abstract/Free Full Text]
40. Toyomura, T., Oka, T., Yamaguchi, C., Wada, Y., and Futai, M. (2000) J. Biol. Chem. 275, 8760-8765[Abstract/Free Full Text]
41. Li, Y. P., Chen, W., Liang, Y., Li, E., and Stashenko, P. (1999) Nat. Genet. 23, 447-451[CrossRef][Medline] [Order article via Infotrieve]
42. Nelson, N. (1992) J. Bioenerg. Biomembr. 24, 407-414[Medline] [Order article via Infotrieve]
43. Arai, H., Pink, S., and Forgac, M. (1989) Biochemistry 28, 3075-3082[Medline] [Order article via Infotrieve]
44. Bauerle, C., Ho, M. N., Lindorfer, M. A., and Stevens, T. F. (1993) J. Biol. Chem. 268, 12749-12757[Abstract/Free Full Text]
45. Kim, J. H., Lingwood, C. A., Williams, D. B., Furuya, W., Manolson, M. F., and Grinstein, S. (1996) J. Cell Biol. 134, 1387-1399[Abstract]
46. Seksek, O., Biwersi, J., and Verkman, A. S. (1995) J. Biol. Chem. 270, 4967-4970[Abstract/Free Full Text]
47. Ohkuma, S., and Poole, B. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 3327-3331[Abstract]
48. Parra, K. J., and Kane, P. M. (1998) Mol. Cell. Biol. 18, 7064-7074[Abstract/Free Full Text]
49. Sumner, J. P., Dow, J. A., Early, F. G., Klein, U., Jager, D., and Wieczorek, H. (1995) J. Biol. Chem. 270, 5649-5653[Abstract/Free Full Text]
50. Myers, M., and Forgac, M. (1993) J. Cell. Physiol. 156, 35-42[Medline] [Order article via Infotrieve]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.