The SaccharomycescerevisiaeVMA10 Is an Intron-containing Gene Encoding a Novel 13-kDa Subunit of Vacuolar H-ATPase

doi:10.1074/jbc.270.23.13726

The SaccharomycescerevisiaeVMA10 Is an Intron-containing Gene Encoding a Novel 13-kDa Subunit of Vacuolar H-ATPase (*)

From the (1) Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110

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Abstract

The vacuolar H-ATPase (V-ATPase) functions as a primary proton pump that generates an electrochemical gradient of protons across the membranes of several internal organelles. It is composed of distinct catalytic and membrane sectors, each containing several subunits. We identified a protein (M16) that copurifies with the V-ATPase complex from Saccharomyces cerevisiae and appears to be present at multiple copies/enzyme. Amino acid sequencing of its proteolytic products yielded three nonoverlapping peptide sequences matching an unidentified reading frame located on chromosome VIII. Sequence analysis of cDNA encoding M16 revealed that the gene encoding this protein (VMA10) is interrupted by a 162-nucleotide intron that begins after the ATG codon of the initiator methionine. The cDNA encodes an hydrophilic protein of 12,713 Da with a basic isoelectric point of pH 9. A Δvma10::URA3 null mutant exhibited growth characteristics typical of other vma disruptant mutants in genes encoding subunits of V-ATPase. The null mutant does not grow on medium buffered at pH 7.5. It fails to accumulate quinacrine into its vacuole, and subunits of the catalytic sector are not assembled onto the vacuolar membrane in the absence of M16. A cold inactivation experiment demonstrated that M16 is a subunit of the membrane sector of V-ATPase. M16 exhibits a significant sequence homology with subunit b of F-ATPase membrane sector.

Footnotes

↵* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) U21240.
↵¹ The abbreviations used are:

V-ATPase

vacuolar H-ATPase

PCR

polymerase chain reaction

Mes

4-morpholineethanesulfonic acid

Mops

4-morpholinepropanesulfonic acid

HPLC

high performance liquid chromatography.
↵²L. Supekova, M. Sbia, F. Supek, and N. Nelson, unpublished results.