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J. Biol. Chem., Vol. 279, Issue 17, 17361-17365, April 23, 2004
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From the Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, Syracuse, New York 13210
Received for publication, December 23, 2003 , and in revised form, January 30, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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mutant was identified in a screen for yeast mutants exhibiting a Vma- phenotype suggestive of loss of vacuolar proton-translocating ATPase (V-ATPase) activity. The mutation disrupts two genes, CWH36 and a recently identified open reading frame on the opposite strand, YCL005W-A. We demonstrate that disruption of YCL005W-A is entirely responsible for the Vma- growth phenotype of the cwh36 mutant. YCL005W-A encodes a homolog of proteins associated with the Manduca sexta and bovine chromaffin granule V-ATPase. The functional significance of these proteins for V-ATPase activity had not been tested, but we show that the protein encoded by YCL005W-A, which we call Vma9p, is essential for V-ATPase activity in yeast. Vma9p is localized to the vacuole but fails to reach the vacuole in a mutant lacking one of the integral membrane subunits of the V-ATPase. Vma9p is associated with the yeast V-ATPase complex in vacuolar membranes, as demonstrated by co-immunoprecipitation with known V-ATPase subunits and glycerol gradient fractionation of solubilized vacuolar membranes. Based on this evidence, we propose that Vma9p is a genuine subunit of the yeast V-ATPase and that e subunits may be a functionally essential part of all eukaryotic V-ATPases. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Although V-ATPase subunit composition is very similar among eukaryotes, highly purified preparations of the Manduca sexta and bovine chromaffin granule V-ATPases both contain membrane proteins of 9–10 kDa that had not been observed in other systems, including yeast (8, 9). This protein associated tightly with the V0 sector in both preparations and appeared to be present at a comparable stoichiometry with the other V-ATPase subunits, prompting the investigators to name it the "e" subunit. However, it was impossible to determine whether these proteins played a critical role in V-ATPase function or were "accessory" proteins involved in assembly or regulation in an organelle- and/or species-specific manner. In fact, the bovine protein (8) showed limited homology to the yeast Vma21 protein, which is an endoplasmic reticulum protein essential for V-ATPase assembly (10). This suggested that higher eukaryotes had evolved a version of Vma21p that might play a similar role in assembly but remained attached to the assembled enzyme.
We screened a library of yeast deletion strains for mutants exhibiting the Vma- phenotype characteristic of loss of V-ATPase activity.2 One mutant, cwh36, exhibited a very strong Vma- phenotype and no vacuolar acidification. Similar results were reported recently by Davis-Kaplan et al. (11) as the result of a genome-wide screen for mutants defective in iron-dependent growth. Here we provide evidence that the cwh36 mutation disrupts an open reading frame encoding a yeast homolog of the M. sexta and bovine e subunits. This protein is essential for V-ATPase activity and copurifies with the V-ATPase from vacuolar membranes. Based on these results, this protein, which we propose to call Vma9p, appears to be an essential subunit of the V-ATPase in yeast cells.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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::KanMX strain (MAT a his3-1 leu2-0 met15-0 ura3-0 cwh36::KanMX) was from the collection of haploid yeast strains each deleted in a single nonessential gene as described by Shoemaker et al. (12) and purchased from Invitrogen. PCR amplifications were carried out using LA Taq polymerase purchased from Panvera. Restriction enzymes were purchased from New England Biolabs. DNA sequencing was done by the State University of New York Upstate DNA sequencing facility. Concanamycin A was obtained from Wako Biochemicals, and octyl-
-D-glucopyranoside was from Calbiochem. Mouse anti-HA monoclonal antibody was purchased from Covance, and mouse anti-yeast alkaline phosphatase antibody was purchased from Molecular Probes. All other reagents were from Sigma. Yeast strains were maintained on yeast extract/peptone/dextrose (YEPD) or supplemented minimal medium prepared as described previously (13). Where indicated, YEPD medium was buffered to pH 5 or 7.5 with 50 mM sodium phosphate, 50 mM sodium or buffered to pH 7.5 with 50 mM MES, 50 mM MOPS when CaCl2 was added.
Plasmid and Strain Constructions—A fragment of genomic DNA containing the CWH36 and YCL005W-A genes was amplified by PCR from genomic DNA using oligonucleotides 5'-CGGTGAACCACATAAGTGTATGTGCACACGCG-3' (5'-flanking primer) and 5'-GAGCCAATTGACCACCGCCTC-3' (3'-flanking primer). The PCR fragment was cleaved with BglII and HindIII and inserted into the BamHI and HindIII sites of pRS315 (14). Premature stop sites were introduced into the CWH36 and YCL005W-A open reading frames by fusion PCR. To generate the early stop site of Cwh36p, two fragments were amplified using the following oligonucleotides (mutations are underlined): 1) 5'-GGCCCACATTAAAAACATCATGGCCAATGTC-3' and the 3'-flanking primer and 2) 5'-GGCCATGATGTTTTTAATGTGGGCCATTACG-3' and the 5'-flanking primer. The two fragments were then purified and combined, and the mutated fragment was obtained by fusion PCR using the two flanking primers. The premature stop codon in YCL005W-A was introduced by the same method except that the primer pairs for the first PCR reaction were the following: 1) 5'-GCTTGGTTGTTCTATGGAGCCATTATCCAGAAC-3' and the 3'-flanking primer and 2) 5'-GGATAATGGCTCCATAGAACAACCAAGCGTATG-3' and the 5'-flanking primer. Both mutations were confirmed by DNA sequencing and cloned into vector pRS315 as described above.
The 3HA epitope tag was inserted at the C terminus of Vma9p as described by Wach (15). The following three fragments were amplified by PCR. 1) The sequence encoding the VMA9 ORF was amplified from genomic DNA using oligonucleotides 5'-GAGAAGGCGCTGGTGTTTCG-3' (VMA9 5'-flanking primer) and 5'-CCCGGGGATCCGTTCGTCAAATTCAGGTCCAAATC-3', 2) a fragment containing the 3HA epitope and a KanMX genetic marker was amplified from plasmid pFA6a-3HA-KanMX6 (16) using oligonucleotides 5'-CGGATCCCCGGGTTAATTAA-3' (F2) and 5'-GAATTCGAGCTCGTTTAAAC-3' (R1), and 3) the sequence just downstream of the VMA9 stop codon was amplified from genomic DNA using oligonucleotides 5'-GAGCTCGAATTCAGCGTCGAGTATATCAAGCCAAG-3' and the CWH36 5'-flanking primer described above. The underlined portions of fragments 1 and 3 are complementary to portions of fragment 2, and these sequences supported fusion of the three purified fragments after they were combined and PCR-amplified in the presence of the two flanking primers. The product of the fusion PCR was used to transform SF838-1D
cells (MAT leu2-112 ura3-52 his4-519 ade6 gal2 pep4-3) (17). Transformants were selected by their growth on YEPD plates containing 200 µg/ml G418. Insertion of the 3HA tag immediately before the VMA9 stop codon at the VMA9 genomic locus was confirmed by PCR from genomic DNA of the transformants and DNA sequencing. The vma2::LEU2 and vma3::URA3 alleles were introduced by PCR amplification of the mutant alleles followed by transformation into the epitope-tagged strain as described previously (18).
Biochemical and Immunological Methods—Vacuoles were purified by Ficoll flotation as described by Roberts et al. (19) and resuspended in 15 mM MES, pH 7, 4.8% glycerol buffer. Concanamycin A-sensitive ATPase activity was assayed by a coupled enzyme assay (20) performed in the absence and presence of 200 nM concanamycin A. Protein concentrations were determined by Lowry assay (21). Western blot analysis of vacuolar vesicles and whole cell lysates was performed as described previously (22) using mouse monoclonal antibodies 8B1, 13D11, and 10D7, monoclonal antibodies against the A, B, and a subunits, respectively, of the V-ATPase; monoclonal antibody 1D3 against yeast alkaline phosphatase; and monoclonal antibody HA.11 against the 3HA epitope. Immunoprecipitation of the V-ATPase complex was performed as described by Parra and Kane (23) with the following exceptions. Vacuoles were solubilized in 2% octyl--D-glucopyranoside 0.35% of the same detergent was included throughout the immunoprecipitation, and V-ATPase complexes were immunoprecipitated with the anti-B subunit antibody (13D11). Glycerol gradient purification of the V-ATPase was performed as described previously (24) with the following modifications. 1) Washed vacuolar vesicles were resuspended at a concentration of 5 mg/ml protein and solubilized in 2% octyl--D-glucopyranoside. 2) A 7.8-ml glycerol step gradient, consisting of equal volume steps varying by 5% glycerol and covering a range of 50–20% glycerol, was prepared in a 16 x 60-mm Beckman Ultracentrifuge tube. All of the glycerol solutions contained 10 mM Tris, pH 7.5, 1 mM EDTA, and 0.35% octyl--D-glucopyranoside. The solubilized vesicles were loaded on top of the gradient and then spun at 42,000 rpm for 7 h in a Beckman 70.1 Ti rotor. Fractions were collected from the bottom of the tube, precipitated by addition of trichloroacetic acid to a 10% final concentration, and then solubilized and visualized by immunoblotting as described.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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(YCL007c). Vacuoles isolated from this deletion strain had a very low V-ATPase activity, suggesting a role for this gene in V-ATPase function.2 Similar observations were also made by Davis-Kaplan et al. (11) starting from a genomic screen for yeast mutants defective in irondependent growth (11).
To investigate the role of this protein in V-ATPase function, C-terminal HA, Myc, and green fluorescent protein epitope tags were fused to the CWH36 ORF (15, 16). However, no tagged protein was expressed in any of the isolates screened. Kellis et al. (25) suggested that the CWH36 ORF overlapped with another, previously unidentified gene on the complement strand (YCL005W-A). The cwh36 mutation would also delete most of this gene, and we hypothesized that deletion of this gene may have led to Vma- phenotype of the cwh36 strain. Fig. 1A shows the genome position of the two genes.
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To clarify the role that Cwh36p or YCL005W-A protein may have in V-ATPase function, a complementation assay was performed. A fragment encompassing both genes was cloned into a low copy number plasmid and shown to complement the cwh36 mutation as demonstrated by restored growth of the transformed cells on YEPD, pH 7.5 + CaCl2 medium (Fig. 1C). Premature stop codons were then introduced into both reading frames, with the stop codon chosen to give no change in amino acid sequence for the gene on the opposite strand. (The positions of the stop codons are shown in Fig. 1A). The mutant plasmids were transformed into a cwh36 strain. The plasmid with the mutation predicted to truncate Cwh36p still permits growth of the mutant on YEPD, pH 7.5 and YEPD, pH 7.5 + CaCl2 medium, indicating that loss of Cwh36p does not affect function (Fig. 1C). The plasmid with the mutation predicted to truncate the YCL005W-A ORF does not complement the cwh36 mutant phenotype. These data indicate that the Vma- mutant phenotype of the cwh36 mutant arises from deletion of YCL005W-A, not CWH36. Based on this information and data described below we propose to rename YCL005W-A as VMA9.
To track this putative subunit and compare the effects of a V1 or V0 deletion on subunit localization, the gene was epitopetagged at the C terminus with three copies of the influenza hemagglutinin epitope in a vacuolar protease-deficient strain (pep4 mutant; Ref. 27). (We were unable to detect the tag when it was introduced into a PEP4 wild-type strain.) The strain containing the epitope-tagged Vma9p-3HA grew as well as the wild-type strain on YEPD, pH 7.5 + CaCl2 medium (data not shown). Vacuolar vesicles from the tagged strain had a concanamycin A-sensitive ATPase activity of 0.40 ± 0.07 µmol min-1mg-1 (n = 2), 53% of the activity obtained from the congenic wild-type strain. Western blots of the vacuolar vesicles were probed with antibodies to the HA epitope (Fig. 2A). Vacuoles from the VMA9-3HA-containing strain contain a cross-reacting band of 
15 kDa that is not found in the untagged strain. (The predicted size of Vma9p from the sequence is 8.4 kDa, and the 3HA tag has a predicted molecular mass of 4.8 kDa.) These data indicate that introduction of the 3HA tag into VMA9 does not prevent V-ATPase function and that Vma9p is found at the vacuole.
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mutation, which deletes the V1 B subunit, or a vma3 mutation, which deletes the V0 c subunit, into the VMA9-3HA strain. Isolated vacuoles from these strains had no V-ATPase activity, characteristic of these deletion strains (data not shown). Western analysis (Fig. 2A) confirmed that in the absence of an intact V1 (vma2 strain), the HA-tagged protein was still present at the vacuole. However, in the absence of V0 (vma3 strain), there was a clear decrease in this protein. In all strains, relative concentrations of the vacuolar protein alkaline phosphatase were the same (Fig. 2A). Western analysis of whole cell lysates showed little difference in relative subunit concentrations between strains (Fig. 2B), except that the V0 a subunit is at very low levels in the vma3 strain (22).
The results described above demonstrate that Vma9p is a vacuolar protein and that its presence in the vacuole is dependent on the presence of an intact V0 sector. To test whether Vma9p is actually a subunit of the V-ATPase, we isolated V-ATPase complexes by two different methods. First, we solubilized vacuolar membranes and immunoprecipitated assembled V-ATPase complexes using an antibody to the V1 subunit B. Immunoprecipitates from the Vma9p-3HA strain contain the 15-kDa HA-tagged Vma9p, in addition to the 100-kDa V0 subunit a and the 69-kDa V1 subunit A, but immunoprecipitates from the untagged strain or from a mock precipitation lacking any vacuolar membranes contain little or no HA-tagged protein (Fig. 3A). Vacuoles from a vma2 strain contain assembled V0 sectors even though they lack V1 subunits (23). To confirm that Vma9p is associated with the V0 sector of the V-ATPase, we performed a similar immunoprecipitation from the vma2 vacuoles containing HA-tagged Vma9p, in this case using an antibody to V0 subunit a that is capable of immunoprecipitating free V0 sectors (23). As shown in Fig. 3B, the anti-a subunit antibody coprecipitates Vma9p-3HA from these vacuoles. In addition, glycerol gradient analysis of solubilized vacuoles was used to determine whether the tagged subunit fractionated with the assembled V-ATPase complex. Fig. 3C shows an immunoblot of fractions 2–14 of the glycerol gradient. As described previously (24), the V1V0 complex migrated to a glycerol concentration of 35% (fraction 7), and the V1 subunits (A and B) peaked at this position. V0 subunit a is also present in this fraction but shows a somewhat broader distribution through fractions 6–10. Like the V0 a subunit, Vma9p-3HA was present in fraction 7 with the intact V1V0 complexes but was also found in fractions 6–10. All of the subunits were also at the top of the gradient, in fractions 13–14; these fractions may include free V0 sectors as well as disassembled or unsolubilized complexes. Taken together, these data indicate that Vma9p is part of assembled V1V0 complexes and free V0 complexes.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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mutants, but is not part of the final assembled V-ATPase complex (10). Significantly, the VMA21 gene showed some homology to the mammalian e subunit genes (8) but little or no homology to the M. sexta and yeast genes, and it was not grouped with the e subunits by the CDART program (26). The functional data presented here, along with the homology with the 9-kDa V-ATPase-associated proteins from M. sexta and bovine cells (8, 9) and the presence of homologs in many different eukaryotes, suggest that the e subunit may play an essential role in all V-ATPases.
These results also predict certain features of this subunit that can be probed in future experiments. Analysis of the predicted amino acid sequence of YCL005W-A (Saccharomyces Genome Database; www.yeastgenome.org) suggests that Vma9p might have two transmembrane domains. This, along with the data indicating that its presence in the vacuole is dependent on the presence of an intact V0 sector but not an intact V1 sector, suggests that it is a V0 sector subunit; coprecipitation of Vma9p-3HA with the V0 sector from vma2 vacuoles (Fig. 3B) provides strong evidence that it is indeed a V0 sector subunit. Although we had never noticed an 8.4-kDa protein in previous purifications of the yeast V-ATPase, V0 sectors immunoprecipitated from vacuolar membranes of a vma2 mutant do contain a small molecular mass protein (23) that might be Vma9p. In addition, Ludwig et al. (8) noted previously that the chromaffin 9.2-kDa protein was difficult to visualize by SDS-PAGE. The apparent sensitivity of the C-terminal 3HA tag to vacuolar proteases in a Pep4+ strain would suggest that the C terminus of the Vma9p might face the vacuolar lumen, and this is consistent with the topology proposed for the M. sexta e subunit, based on glycosylation of this protein at a site near the C terminus of the protein (8, 9). It is also interesting that although Vma9p does not reach the vacuole in the vma3 mutant, it is present at near wild-type levels in whole cell lysates of the mutant (Fig. 2B). In contrast, the a subunit is rapidly degraded when one of the other V0 subunits is missing. This suggests that proper assembly of V0 sectors is not essential for Vma9p stability, but there is still a possibility that the 3HA tag is stabilizing Vma9p in our experiments. Finally, the position of the e subunit in the V0 sector is an intriguing question. The e subunit appears to contain no intramembrane acidic residues, like the three essential c-like subunits of the yeast V0 sector (28), so it is unlikely that it directly participates in proton transport. Placement of the e subunit in the V0 complex may provide further insights into its functional contribution.
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FOOTNOTES |
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To whom correspondence should be addressed. Tel.: 315-464-8742; Fax: 315-464-8750; E-mail: kanepm{at}upstate.edu.
1 The abbreviations used are: V-ATPase, vacuolar proton-translocating ATPase; HA, hemagglutinin; 3HA, three copies of the influenza hemagglutinin epitope; YEPD, yeast extract/peptone/dextrose; MES, 4-morpholineethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid; ORF, open reading frame; CDART, Conserved Domain Architecture Retrieval Tool.
2 M. Sambade, M. Alba, R. W. West, A. M. Smardon, and P. M. Kane, manuscript in preparation.
3 The National Center for Biotechnology Information CDART program identifies this common structural motif in 28 predicted protein sequences in addition to Vma9p. These sequences have the following GenBankTM/EBI accession numbers: CAE74051
[GenBank]
EAA55854
[GenBank]
XP_088142
[GenBank]
, CAE45916
[GenBank]
CAE02898
[GenBank]
XP_313345
[GenBank]
, XP_313344
[GenBank]
, EAA38504
[GenBank]
CAD27522
[GenBank]
NP_649327
[GenBank]
, NP_647882
[GenBank]
, NP_650578
[GenBank]
, BAC05292
[GenBank]
NP_647868
[GenBank]
, AAK67647
[GenBank]
NP_446030
[GenBank]
, NP_598525
[GenBank]
, NP_568823
[GenBank]
, AAL48783
[GenBank]
NP_501636
[GenBank]
, NP_194401
[GenBank]
, AAK28556
[GenBank]
NP_079548
[GenBank]
, AAF71753
[GenBank]
NP_003936
[GenBank]
, CAA06822
[GenBank]
P81103
[GenBank]
, and CAA75570
[GenBank]
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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