The First Putative Transmembrane Segment of Subunit c" (Vma16p) of the Yeast V-ATPase Is Not Necessary for Function

• Published in: The Journal of biological chemistry Vol. 278; no. 8; pp. 5821 - 5827
• Main Authors: Nishi, T, Kawasaki-Nishi, S, gac, M
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 21.02.2003
• Subjects: Amino Acid Sequence; Amino Acid Substitution; Molecular Sequence Data; Mutagenesis, Site-Directed; Protein Subunits - chemistry; Protein Subunits - metabolism; Proteolipids - chemistry; Proteolipids - metabolism; Proton-Translocating ATPases - chemistry; Proton-Translocating ATPases - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - metabolism; Sequence Alignment; Sequence Homology, Amino Acid; Vacuolar Proton-Translocating ATPases - chemistry; Vacuolar Proton-Translocating ATPases - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M209875200

The yeast vacuolar ATPase (V-ATPase) contains three proteolipid subunits: c (Vma3p), c′ (Vma11p), and c" (Vma16p). Each subunit contains a buried glutamate residue that is essential for function, and these subunits are not able to substitute for each other in supporting activity. Subunits c and c′ each contain four putative transmembrane segments (TM1–4), whereas subunit c" is predicted to contain five. To determine whether TM1 of subunit c" serves an essential function, a deletion mutant of Vma16p was constructed lacking TM1 (Vma16p-ΔTM1). Although this construct does not complement the loss of Vma3p or Vma11p, it does complement the loss of full-length Vma16p. Vacuoles isolated from the strain expressing Vma16p-ΔTM1 showed V-ATPase activity and proton transport greater than 80% relative to wild type and displayed wild type levels of subunits A and a, suggesting normal assembly of the V-ATPase complex. These results suggest that TM1 of Vma16p is dispensable for both activity and assembly of the V-ATPase. To obtain information about the topology of Vma16p, labeling of single cysteine-containing mutants using the membrane-permeable reagent 3-( N -maleimidylpropionyl)biocytin (MPB) and the -impermeable reagent 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid (AMS) was tested. Both the Cys-less form of Vma16p and eight single cysteine-containing mutants retained greater than 80% of wild type levels of activity. Of the eight mutants tested, two (S5C and S178C) were labeled by MPB. MPB-labeling of S5C was blocked by AMS in intact vacuoles, whereas S178C was blocked by AMS only in the presence of permeabilizing concentrations of detergent. In addition, a hemagglutinin epitope tag introduced into the C terminus of Vma16p was recognized by an anti-hemagglutinin antibody in intact vacuolar membranes, suggesting a cytoplasmic orientation for the C terminus. These results suggest that subunit c" contains four rather than five transmembrane segments with both the N and C terminus on the cytoplasmic side of the membrane.