Mutational Analysis of the Non-homologous Region of Subunit A of the Yeast V-ATPase*

Elim Shao, Tsuyoshi NishiDagger, Shoko Kawasaki-Nishi, and Michael Forgac§

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

Received for publication, November 27, 2002, and in revised form, January 22, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Subunit A is the catalytic nucleotide binding subunit of the vacuolar proton-translocating ATPase (or V-ATPase) and is homologous to subunit beta  of the F1F0 ATP synthase (or F-ATPase). Amino acid sequence alignment of these subunits reveals a 90-amino acid insert in subunit A (termed the non-homologous region) that is absent from subunit beta . To investigate the functional role of this region, site-directed mutagenesis has been performed on the VMA1 gene that encodes subunit A in yeast. Substitutions were performed on 13 amino acid residues within this region that are conserved in all available A subunit sequences. Most of the 18 mutations introduced showed normal assembly of the V-ATPase. Of these, one (R219K) greatly reduced both proton transport and ATPase activity. By contrast, the P217V mutant showed significantly reduced ATPase activity but higher than normal levels of proton transport, suggesting an increase in coupling efficiency. Two other mutations in the same region (P223V and P233V) showed decreased coupling efficiency, suggesting that changes in the non-homologous region can alter coupling of proton transport and ATP hydrolysis. It was previously shown that the V-ATPase must possess at least 5-10% activity relative to wild type to undergo in vivo dissociation in response to glucose withdrawal. However, four of the mutations studied (G150A, D157E, P177V, and P223V) were partially or completely blocked in dissociation despite having greater than 30% of wild type levels of activity. These results suggest that changes in the non-homologous region can also alter in vivo dissociation of the V-ATPase independent of effects on activity.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The vacuolar proton-translocating ATPases (or V-ATPases)1 are ATP-driven proton pumps present in both intracellular compartments and the plasma membrane of eukaryotic cells (1-8). They couple the energy released upon ATP hydrolysis to the active transport of protons from the cytoplasm to either the lumen of various intracellular compartments or to the extracellular environment. Acidification of intracellular compartments is important for such processes as receptor-mediated endocytosis, intracellular trafficking of lysosomal enzymes, degradation of macromolecules, uptake of neurotransmitters, and the entry of various envelope viruses and toxins (1-8). Plasma membrane V-ATPases have also been implicated in many normal and disease processes, including bone resorption, renal acidification, pH homeostasis, and tumor metastasis (9-13). Defects in specific V-ATPase subunits have been shown to be responsible for a number of human genetic diseases, including autosomal recessive osteopetrosis and renal tubular acidosis (9, 14-16).

The V-ATPases are multi-subunit complexes composed of two functional domains (1-8). The 640-kDa peripheral V1 domain is responsible of ATP hydrolysis and consists of 8 different subunits (subunits A-H) with molecular masses of 70-14 kDa. The V0 domain is a 260-kDa integral complex composed of five different subunits (subunits a, d, c'', c', and c with molecular masses of 100-17 kDa) that function in proton translocation. The V-ATPases are structurally related to the F-ATPases, which function as proton-driven ATP synthases in mitochondria, chloroplasts, and bacteria (17-22). Both the A and B subunits of the V-ATPases participate in nucleotide binding, with the catalytic nucleotide binding sites located on the A subunit and so called "non-catalytic" nucleotide binding sites located on the B subunit (1-8). The A and B subunits are homologous to the beta  and alpha  subunits of the F-ATPases, respectively (23, 24).

High resolution structural data of F1 reveal a hexameric arrangement of alpha  and beta  subunits surrounding a central cavity containing the gamma  subunit (25-27). The gamma  and epsilon  subunits form a central stalk that connects to a ring of proteolipid c subunits in the F0 domain (27, 28). F1 and F0 are also connected via a peripheral stalk (composed of the delta  and b subunits) that links the alpha 3beta 3 head to the a subunit in F0 (29, 30). The F-ATPases are believed to operate via a rotary mechanism (31, 32) in which ATP hydrolysis in F1 drives rotation of the central gamma  subunit (33-35), which in turn drives rotation of the ring of c subunits relative to subunit a in the F0 domain (36-38). Subunit a is held fixed relative to the hexameric head by the peripheral stalk (29, 30). Rotation of the c subunit ring relative to subunit a leads to active proton translocation across the membrane (31, 32).

Sequence alignment of the A subunit of V1 and the beta  subunit of F1 reveals the existence of a 90-amino acid insert (termed the "non-homologous" region), which is conserved among A subunit sequences but that is absent from the beta  subunit sequence (23, 39-41). To address the function of the non-homologous region, site-directed mutations have been introduced at conserved amino acid residues in the yeast V-ATPase A subunit (Vma1p). The results obtained suggest that changes in the non-homologous region can alter both coupling of proton transport and ATP hydrolysis and dissociation of the V-ATPase complex in vivo. Reversible dissociation has been shown to play an important role in regulation of V-ATPase activity in cells (42, 43).

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials and Strains-- Escherichia coli and yeast culture media were purchased from Difco. Restriction endonucleases, T4 DNA ligase, and other molecular biology reagents were from Invitrogen. Zymolyase 100T was purchased from Sekagaku America, Inc. Protease inhibitors were purchased from Roche Molecular Biochemicals. Concanamycin A was purchased from Fluka Chemical Corp. 9-Amino-6-chloro-2-methoxyacridine was purchased from Molecular Probes, Inc. SDS, nitrocellulose membrane (0.45 µm pore size), Tween 20, horseradish peroxidase-conjugated goat anti-rabbit IgG, and horse radish peroxidase-conjugated goat anti-mouse IgG were from Bio-Rad. The chemiluminescence substrate for horseradish peroxidase was from Kirkegaard & Perry Laboratories. Protein A-Sepharose, ATP, polyoxyethylene 9 lauryl ether, and most other chemicals were obtained from Sigma.

Mouse monoclonal antibodies 8B1-F3 against Vma1p, 13D11-B2 against Vma2p, and 10D7 against Vph1p were purchased from Molecular Probes, Inc. Rabbit polyclonal antibodies against Vma6p, Vma7p, Vma8p, Vma10p, and Vma13p were generous gifts from Dr. Tom Stevens (University of Oregon, Eugene, OR). The rabbit polyclonal antibody against Vma4p was kindly provided by Dr. Daniel Klionsky (University of Michigan, Ann Arbor, MI). The mouse monoclonal antibody 11E6 against Vma1p and a mouse monoclonal antibody against Vma5p were generous gifts from Dr. Patricia Kane (State University of New York Upstate Medical University, Syracuse).

Yeast strain SF838-5Aa vma1D-8 (MATa, leu2-3, 112, ura3-52, ade6, vma1D::LEU2) was used for integration and subsequent biochemical characterization. Yeast cells were grown in YPD media containing dextrose or YEP media without dextrose.

Construction of Mutants-- Mutations in the non-homologous region of subunit A were made using the Altered Sites II in vitro mutagenesis system (Promega) following the manufacturer's protocol. The full-length VMA1 gene without the spacer region (44) was cloned into pAlter-1 using BamH1 and SalI sites. The mutagenesis oligonucleotides were as follows with the substitution sites underlined: G150A, 5'-GGAAAGTTTCAAGTCGCCGATCATATTTCCGGT-3'; G156V, 5'-GATCATATTTCCGGTGTTGATATTTACGGTTCC-3'; D157E, 5'-CATATTTCCGGTGGTGAAATTTACGGTTCCGTT-3'; D157A, 5'-CATATTTCCGGTGGTGCTATTTACGGTTCCGTT-3'; V162L, 5'-GATATTTACGGTTCCCTTTTTGAGAATTCGCTA-3'; V162A, 5'-GATATTTACGGTTCCGCTTTTGAGAATTCGCTA-3'; E164D, 5'-ATTTACGGTTCCGTTTTTGACAATTCGCTAATTTCA-3'; E164A, 5'-ATTTACGGTTCCGTTTTTGCGAATTCGCTAATTTCA-3'; P177V, 5'-AGCCATAAGATTCTTTTGCCAGTAAGATCAAGAGGTACAATC-3'; G181A, 5'-CCACCAAGATCAAGAGCTACAATCACTTGGATT-3'; G205V, 5'-GAAGTTGAATTTGATGTCAAGAAGTCTGATTTC-3'; W216F, 5'-GATTTCACTCTTTACCATACTTTCCCTGTTCGTGTTCCAAGA-3'; W216A, 5'-GATTTCACTCTTTACCATACTGCGCCTGTTCGTGTTCCAAGA-3'; P217V, 5'-TTCACTCTTTACCATACTTGGGTTGTTCGTGTTCCAAGA-3'; R219K, 5'-CTTTACCATACTTGGCCTGTTAAAGTTCCAAGACCAGTTACT-3'; R219A, 5'-CTTTACCATACTTGGCCTGTTGCTGTTCCAAGACCAGTTACT-3'; P223V, 5'-GTTCGTGTTCCAAGAGTAGTTACTGAAAAGTTATCT-3'; P233V, 5'-AAGTTATCTGCTGACTATGTTTTGTTAACAGGTCAA-3'. All mutants were confirmed by DNA sequencing using an automated sequencer from Applied Biosystems and subcloned into the YIp5 vector using BamHI and SalI sites along with wild type VMA1. No other mutations were detected in the final product.

The wild type plasmid (YIp5-VMA1), vma1 mutant plasmids, or YIp5 vector alone were linearized with ApaI to target integration of the constructs to the URA3 locus. Transformation of yeast cells (SF838-5Aalpha vma1Delta -8) and selection of transformants on Ura- plates were done as described previously (45, 46). Chromosomal DNA was isolated from the transformed yeast cells, and the VMA1 gene was amplified by a polymerase chain reaction. The presence of the site-directed mutations was confirmed by sequencing the polymerase chain reaction products. Growth phenotypes of the mutants were assessed on YPD plates buffered with 50 mM KH2PO4 or 50 mM succinic acid to either pH 5.5 or pH 7.5.

Isolation of Vacuolar Membrane Vesicles-- Vacuolar membrane vesicles were isolated using a modified protocol described by Uchida et al. (47). The yeast integrated with the wild type plasmid (YIp5-VMA1), mutants, or the vector YIp5 alone were cultured overnight in 1 liter of YPD (pH 5.5) to log phase. 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, 100 mM MES-Tris, pH 7.5, and 2 mg of zymolyase 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 determined by Lowry assay (48).

Immunoblot Analysis-- Whole cell lysates (prepared as described previously (49)) and isolated vacuolar membrane vesicles were treated with 50 mM Tris-HCl, pH 6.8, 8 M urea, 5% SDS, 1 mM EDTA, and 5% beta  -mercaptoethanol followed by SDS-PAGE and transferred to nitrocellulose. Blots were then incubated with antibodies against subunits of the yeast V-ATPase followed by horseradish peroxidase-conjugated secondary antibodies as previously described (50). Immunoblots were developed using a chemiluminescent detection method from Kirkegaard & Perry Laboratories.

Biochemical Characterization-- ATPase activity was measured using a coupled spectrophotometric method as described previously (51) with some modification. Isolated vacuolar membrane vesicles 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 initiated by the addition of 0.5 mM ATP, and the absorbance at 341 nm was measured continuously using a Kontron UV-visible spectrophotometer. ATP-dependent proton transport was measured as previously described (52) using 10 µg of vacuolar protein in 50 mM NaCl, 30 mM KCl, 20 mM HEPES-NaOH, pH 7.0, 0.2 mM EGTA, 10% glycerol, except that the initial rate of fluorescence quenching (rather than the net fluorescence change) after the addition of 0.5 mM ATP and 1.0 mM MgCl2 was measured using the fluorescence probe 9-amino-6-chloro-2-methoxyacridine in the presence or absence of 1 µM concanamycin A using a PerkinElmer Life Sciences LS50B spectrofluorometer.

In Vivo Dissociation and Reassembly of the V-ATPase in Response to Glucose Depletion and Readdition-- Dissociation of the V-ATPase in response to glucose depletion and reassembly in response to glucose readdition were measured as described previously (50) with some modifications. The yeast integrated with the wild type plasmid (YIp5-VMA1) or the mutants were grown in YPD media, pH 5.5, overnight to an absorbance at 600 nm of <1.0. The cells were converted to spheroplasts by treatment with zymolyase 100T and incubated in YEP media with or without 2% glucose for 40 min at 30 °C. To observe reversal of dissociation of the V-ATPase upon glucose readdition, spheroplasts were incubated in YEP media with or without 2% glucose for 20 min. An aliquot of the spheroplasts incubated in the absence of glucose was then incubated in YEP media with 2% glucose for an additional 20 min. Spheroplasts were pelleted and lysed in phosphate-buffered saline containing 1% polyoxyethylene 9 lauryl ether, protease inhibitors (2 µg/ml aprotinin, 0.7 µg/ml pepstatin, 5 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride), and 1 mM dithiobis(succinimidyl propionate). The V-ATPase complexes were immunoprecipitated using monoclonal antibody 13D11 against the B subunit and protein A-Sepharose followed by separation on 8% acrylamide gels and transfer to nitrocellulose. Western blotting was then performed using antibody 8B1-F3 against subunit A and antibody 13D11 against subunit B to detect the V1 domain and antibody 10D7-A7 against Vph1p to detect the V0 domain followed by incubation with horseradish peroxidase-conjugated secondary antibody. Dissociation of the V-ATPase complex is reflected as a reduction in the amount of the V0 subunit Vph1p, immunoprecipitated using the antibody directed against subunit B of the V1 domain.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Effects of Mutations in the Non-homologous Region of Subunit A on Growth Phenotype and Assembly of the V-ATPase Complex-- To investigate the function of the non-homologous region of subunit A of the V-ATPase, site-directed mutagenesis of the VMA1 gene encoding subunit A in yeast was performed. Alignment of the sequence of the non-homologous region of subunit A from 8 species is shown in Fig. 1. Of the 90 residues in this region, 13 are conserved in all eukaryotic A subunit sequences available. A total of 18 site-directed mutations were constructed at these 13 sites, with in some cases both conservative and non-conservative substitutions tested. Each of the four conserved proline residues was replaced by valine, whereas the four conserved glycine residues were replaced with either alanine or valine. The three charged residues (Asp-157, Glu-164, and Arg-219) were replaced with either alanine or a residue that conserved the original charge. Finally, the two hydrophobic residues (Val-162 and Trp-216) were replaced with either alanine or a similarly bulky, hydrophobic amino acid to the original.


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Fig. 1.   Amino acid alignment of the non-homologous region of subunit A of the V-ATPases. Sequence alignment of eight eukaryotic V-ATPase A subunit sequences are shown. Amino acid residues identical in all known A subunit sequences are shown shaded. The numbers shown above the sequence correspond to the residue number in the yeast Vma1p sequence. S. cerevisiae, Saccharomyces cerevisiae; N. Crassa, Neurospora crassa; D. carota, Daucus carota.

We first determined the effect of the introduced mutations on the growth phenotype of the yeast strain expressing them. Deletion of genes encoding subunits of the V-ATPase leads to a conditional lethal phenotype (vma-), in which yeast cells are not able to grow in media buffered to pH 7.5 (40, 44, 53). Point mutations that cause a loss of greater than 80% of wild type activity also result in a vma- phenotype (54, 55). As can be seen in Fig. 2, four of the mutations (G156V, W216A, R219K, and R219A) led to a full vma- phenotype, three mutations (G181A, W216F, and P223V) led to a very substantial reduction in growth at pH 7.5, and four mutations (E164D, E164A, P177V, and G205V) resulted in a mild growth defect at neutral pH. To determine whether the observed growth defects were the result of defective assembly of the V-ATPase, vacuolar membranes isolated from each of the mutant strains were subjected to SDS-PAGE and analyzed by Western blot using antibodies against 10 of the 13 V-ATPase subunits. Defects in V-ATPase assembly are manifested as a reduction of one or more V-ATPase subunits from the vacuolar membrane (56, 57). As seen in Fig. 2, three mutations (G156V, G181A, and W216A) led to severely defective assembly, whereas the W216F mutation led to reductions in the levels of several V-ATPase subunits on the vacuolar membrane. The remaining mutants appear to assemble normally on the vacuolar membrane.


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Fig. 2.   Effect of mutations in the non-homologous region on assembly of the V-ATPase complex. Vacuolar membrane vesicles were isolated from the vma1Delta strain transformed with YIp5 alone (Plasmid only), wild type VMA1 in YIp5 (WT), or VMA1 containing the indicated mutations in YIp5. The proteins were separated by SDS-PAGE and transferred to nitrocellulose, and Western blotting was performed using the indicated subunit-specific antibodies as described under "Experimental Procedures." For subunits a, A, B, and H, 1 µg of protein was used; for subunits C, d, and E, 10 µg of protein was used; for subunits D, F, and G. 20 µg of protein was used. Also shown is the growth phenotype of each strain on YPD agar plates buffered to pH 7.5. +++ indicates wild type growth, ++ indicates partially defective growth, + indicates substantially defective growth, and - indicates almost no growth. Growth was assessed by colony size. Panel a, mutants G150A to P177V. Panel b, mutants G181A to P233V.

Effects of Mutations in the Non-homologous Region on Proton Transport and ATP Hydrolysis by the V-ATPase-- We next tested the effects of the mutations on the activity of the V-ATPase. Both concanamycin-sensitive ATPase activity and ATP-dependent proton transport (as assessed by fluorescence quenching using the probe 9-amino-6-chloro-2-methoxyacridine) were measured in isolated vacuolar membrane vesicles at an ATP concentration of 0.5 mM. As can be seen in Fig. 3, three of the mutations (G156V, W216A, and R219K) resulted in loss of greater than 80% of both proton transport and ATPase activity. Of these, both G156V and W216A showed severe defects in assembly that likely account for the observed loss of activity. By contrast, the R219K mutant, which had less than 10% wild type activity, showed normal assembly on vacuolar membranes. These results suggest that the R219K mutation directly affects V-ATPase activity.


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Fig. 3.   Effect of mutations in the non-homologous region on concanamycin A-sensitive ATPase activity and ATP-dependent proton transport in isolated vacuolar membrane vesicles. Vacuolar membrane vesicles (10 µg of protein) were isolated from cells expressing wild type or mutant forms of subunit A and assayed for both concanamycin A-sensitive ATPase activity (shaded bars) and concanamycin A-sensitive, ATP-dependent proton transport (open bars), as described under "Experimental Procedures." ATPase activity and proton transport are measured at 0.5 mM ATP and are expressed relative to values obtained for vacuoles isolated from the strain expressing wild type subunit A (100%), with each value the average of measurements on two or three independent vacuole preparations (error bars correspond to the S.E.). The specific activity of the concanamycin A-sensitive ATPase activity of vacuoles containing the wild type subunit A is 0.13 µmol of ATP/min/mg of protein at 0.5 mM ATP and 23 °C, which is similar to values previously reported for this strain (55). Panel a, mutants G150A to P177V. Panel b, mutants G181A to P233V. Ywt, wild type.

Four mutations (E164D, G181A, W216F, and R219A) reduce both proton transport and ATPase activity in parallel by 50-80% relative to wild type. Both G181A and W216F show partial or complete defects in assembly, whereas E164D and R219A assemble normally. Interestingly, the R219A mutant shows a severe growth phenotype (Fig. 2) despite possessing at least 40% of wild type levels of activity in vitro.

Three mutations (P217V, P223V, and P233V) have significantly different effects on ATPase activity and proton transport. Both P223V and P233V show significant reductions in proton transport while retaining wild type levels of ATPase activity, suggesting a decrease in coupling between proton transport and ATP hydrolysis. By contrast, the P217V mutant possesses only about 34% of wild type ATPase activity but 140% of wild type proton pumping activity. These results suggest that changes in the non-homologous region of the A subunit (and particularly the region between residues 217 and 233) can result in significant changes in coupling of proton transport and ATP hydrolysis by the V-ATPase. It should be noted, however, that because the activities reported in Fig. 3 correspond to measurements done at a single ATP concentration (0.5 mM), which is below typical cytoplasmic levels of ATP, the observed differences in coupling would not necessarily occur under in vivo conditions. Also, because this ATP concentration is below the Km measured for the wild type enzyme (0.75 mM ATP), the observed differences do not necessarily reflect differences in maximal velocity.

In Vivo Glucose-dependent Dissociation of the V-ATPase-- In yeast, dissociation of the V1 and V0 domains occurs in response to glucose depletion and represents an important mechanism of regulating V-ATPase activity in vivo (42). It has previously been demonstrated that in vivo dissociation of the V-ATPase requires catalytic activity (58, 59), although mutants of subunit D possessing as little as 5-10% of wild type activity are still able to undergo dissociation (60). To evaluate the effects of mutations in the non-homologous region on in vivo dissociation of the V-ATPase, spheroplasts were incubated in the presence or absence of glucose and then lysed with detergent, and the V-ATPase was immunoprecipitated using the antibody 13D11 against subunit B of the V1 domain. After separation of the immunoprecipitated polypeptides by SDS-PAGE, Western blot analysis was performed using antibodies against subunits A and B of V1 and subunit a of V0. Dissociation of the V-ATPase appears as a reduction in the amount of subunit a (and hence, V0) precipitated using an antibody that recognizes V1, as previously described (50, 60). The four mutants defective in normal assembly of the V-ATPase complex were not tested for glucose-dependent dissociation. The remaining mutants fell into one of five categories (Fig. 4). The first class is mutants possessing greater than 30% wild type activity in vitro that show near normal glucose-dependent dissociation and includes D157A, V162L, V162A, E164A, and G205V. The second class (represented by R219K) is inactive and, as predicted from previous results (50, 60), does not undergo dissociation. The third class (that includes only P217V) shows normal in vivo dissociation while displaying higher than normal proton transport and somewhat reduced ATPase activity in vitro. The fourth class, which includes E164D, R219A, and P233V, possesses greater than 30% wild type activity and is partially blocked in glucose-dependent dissociation but also show some defect in assembly even in the presence of glucose. The fifth class, which includes G150A, D157E, P177V, and P223V, possesses greater than 30% wild type activity and shows normal assembly in the presence of glucose but is completely blocked in glucose-dependent dissociation. These results suggest that changes in the non-homologous region can alter in vivo dissociation of the V-ATPase independent of effects on catalytic activity.


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Fig. 4.   Effect of mutations in the non-homologous region on in vivo dissociation of the V-ATPase in response to glucose withdrawal. The vma1Delta strain expressing the wild type (WT) or mutant forms of the Vma1p were grown overnight in YPD media and converted to spheroplasts by treatment with zymolyase 100T. Spheroplasts were incubated for 40 min in YEP media with or without 2% glucose at 30 °C (as indicated) and then lysed in phosphate-buffered saline containing 1% polyoxyethylene 9 lauryl ether, protease inhibitors, and 1 mM dithiobis(succinimidyl propionate). The V-ATPase complexes were immunoprecipitated using the monoclonal antibody 13D11 against subunit B and protein A-Sepharose. The proteins were separated by SDS-PAGE on 8% acrylamide gels followed by transfer to nitrocellulose. Western blot analysis was performed using the monoclonal antibodies 10D7-A7 against subunit a (Vph1p), 13D11 against subunit B, or 8B1-F3 against subunit A, as described under "Experimental Procedures." Dissociation of the V1 and V0 domains is reflected as a decrease in the amount of subunit a (part of the V0 domain) immunoprecipitated using the antibody against subunit B (part of the V1 domain).

Glucose-dependent Reassembly of the V-ATPase-- Dissociation of the V-ATPase in response to glucose depletion is reversed upon re-addition of glucose (42). To determine whether the glucose-dependent dissociation observed in mutants of the non-homologous region was also reversible, spheroplasts were incubated in the presence or absence of glucose for 20 min or were incubated in the absence of glucose for 20 min followed by incubation in the presence of glucose for an additional 20 min. Solubilization of the V-ATPase, immunoprecipitation, and Western blotting were then performed as described above. As can be seen in Fig. 5, of the six mutants that exhibited glucose-dependent dissociation, all showed reassembly upon re-addition of glucose.


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Fig. 5.   Glucose-dependent reassembly of the V-ATPase after in vivo dissociation. The vma1Delta strain expressing the wild type (WT) or mutant forms of the Vma1p were grown overnight in YPD media and converted to spheroplasts by treatment with zymolyase 100T. The spheroplasts were incubated for 20 min in YEP media with (+) or without (-) 2% glucose. An aliquot of the spheroplasts incubated in the absence of glucose was then incubated in YEP media with 2% glucose for an additional 20 min (- right-arrow +). The V-ATPase was then solubilized with polyoxyethylene 9 lauryl ether and immunoprecipitated using the antibody against subunit B followed by SDS-PAGE and Western blot analysis as described in the legend to Fig 4.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The structural similarity between the V- and F-ATPases has led to the suggestion that both classes of proteins operate via a rotary mechanism (1, 2). In this mechanism, ATP hydrolysis by nucleotide binding subunits in the peripheral domain drives rotation of a central stalk, which in turn causes rotation of a ring of proteolipid subunits in the integral domain (31-38). It is the rotation of this proteolipid ring relative to subunit a in the integral domain that is thought to drive active proton translocation (31, 32). In order for ATP hydrolysis and proton transport to be coupled in this mechanism, subunit a must be held fixed relative to the nucleotide binding subunits or the energy released from ATP hydrolysis would be dissipated in non-productive rotation of the peripheral domain. This is accomplished by the presence of a peripheral stalk, or "stator," that connects subunit a to the nucleotide binding subunits. In the F-ATPases, this peripheral stalk is composed of the delta  subunit and the soluble domain of the b subunit (29, 30). In the V-ATPases, the subunit composition of the peripheral stalk is less certain, but cross-linking and immunoprecipitation studies implicate subunits C, E, G, and H as well as the hydrophilic domain of subunit a in this function (61-64).

Modeling of the nucleotide binding subunits A and B of the V-ATPases based upon sequence homology with the beta  and alpha  subunits of F1 and the available x-ray structural data (25-27) reveals a very similar overall fold to the polypeptide chains (55, 62, 63, 65) despite the existence of only about 25% amino acid sequence identity. The non-homologous region of the V-ATPase A subunit represents a unique protein domain that is present in all V-ATPase A subunits but which is absent from the homologous beta  subunit of F1 (23, 39-41). Because of the close packing of the alpha  and beta  subunits in the hexameric ring of F1 (25-27), the non-homologous region is likely to form a unique domain located on the outer surface of the V1 complex. Such a location suggests the possibility that the non-homologous region may also form part of the peripheral stalk connecting the V1 and V0 domains. Because of the critical role of the peripheral stalk in coupling of ATP hydrolysis to proton transport, it might be imagined that changes in this structure could lead to changes in coupling efficiency.

The results presented in the current study suggest that mutations in the non-homologous region of the A subunit do in fact lead to changes in coupling of proton transport and ATP hydrolysis by the V-ATPases. Regulation of coupling of proton transport and ATP hydrolysis has previously been suggested to function in controlling acidification of intracellular compartments (66, 67). For example, partial uncoupling of the bovine V-ATPase has been reported to occur at ATP concentrations above 0.5 mM, which is in the physiological range (66). Mild proteolysis of the V-ATPase also leads to uncoupling (68), and in the detergent-solubilized state, the bovine V-ATPase is not inhibited by DCCD, suggesting a functional uncoupling of the V1 and V0 domains (69). A 2-fold difference in coupling between the V-ATPase of lemon epicotyl and fruit was reported (70), and recently, a 4-5-fold difference in coupling efficiency of yeast V-ATPase complexes containing two different isoforms of the a subunit has been demonstrated (50). Moreover, mutations have been isolated in several subunits, including subunits a (71) and D (60), which lead to decreased coupling of proton transport and ATPase activity. These mutations are similar to the P223V and P233V mutations described in the current report that cause partial uncoupling of proton transport and ATPase activity. Because, as noted above, higher concentrations of ATP have been reported to lead to uncoupling of the bovine V-ATPase, it is possible that some of the coupling effects observed may be the result of changes in Km for ATP that cause a shift in this optimal ATP concentration for coupling. The P217V mutant of the non-homologous domain represents the first mutational change to lead to a substantial increase in coupling efficiency. These results suggest not only that changes in the non-homologous region can affect coupling efficiency but also that in the wild type complex, ATP hydrolysis and proton transport are not optimally coupled.

Reversible dissociation of the V-ATPase has been demonstrated to occur in both yeast and insect cells (42, 43) and represents an important mechanism of controlling V-ATPase activity in vivo. Dissociation in yeast occurs rapidly and does not involve any of the signal transduction pathways known to be activated by glucose deprivation (58), although an intact microtubular network appears to be required for dissociation (72). In addition, glucose-dependent reassembly (as well as normal assembly) is promoted by a complex termed RAVE that includes as one of its components the ubiquitin ligase subunit Skp1 (73, 74). Dissociation of the V-ATPase has been shown to require catalytic activity (58, 59), although the activity required for dissociation may be as low as 5-10% of the wild type level (60). This dependence may reflect the need to achieve a particular conformational state of the complex that is only reached during turnover of the enzyme. Consistent with these findings, the R219K mutant investigated in the present study was inactive and failed to dissociate upon glucose withdrawal.

The most interesting mutants identified in the present study (and exemplified by the G150A mutant) are those that show significant levels of proton transport and ATPase activity (>30% relative to wild type) but which are at least partially blocked in glucose-dependent dissociation. Two mutations have previously been reported in subunit G (Vma10p) that also show normal ATPase activity and are partly inhibited in dissociation in response to glucose withdrawal (75). Although proton transport was not measured in these mutants, both showed a wild type growth phenotype, suggesting substantially normal function. These mutations were proposed to partially inhibit dissociation through stabilization of the complex, as indicated by the presence of greater than wild type levels of V1 subunits on the vacuolar membrane (72). It is possible that a similar mechanism may be involved for the mutants in the non-homologous region of the A subunit described in the present report, and several of the mutants (including G150A, P177V, and P223V) seem to show higher levels of assembly by immunoprecipitation (Fig. 4), although no increase in V1 subunits associated with the vacuolar membrane was detected (Fig. 2). These results suggest that changes in the non-homologous region can directly affect in vivo dissociation of the V-ATPase independent of effects on activity. Whether this domain normally functions in control of this process and what changes in this region may occur in vivo remain to be determined.

    ACKNOWLEDGEMENTS

We thank Drs. Ting Xu, Yoichiro Arata, and Takao Inoue for many helpful discussions.

    FOOTNOTES

* This work was support in part by National Institutes of Health Grant GM 34478 (to M. F.). E. coli strains were provided through National Institutes of Health Grant DK34928.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 Present address: Division of Biological Sciences, Institute of Scientific and Industrial Research, Osaka University, Osaka 567-0047, Japan.

§ 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: michael.forgac@tufts.edu.

Published, JBC Papers in Press, February 4, 2003, DOI 10.1074/jbc.M212096200

    ABBREVIATIONS

The abbreviations used are: V-ATPase, vacuolar proton-translocating adenosine 5-triphosphatase; F-ATPase, F1F0 ATP synthase; MES, 4-morpholineethanesulfonic acid; YPD, yeast extract-peptone-dextrose; YEP, yeast extract-peptone.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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