The first putative transmembrane segment of subunit c" (Vma16p) of the yeast V-ATPase is not necessary for function.
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-Delta TM1). 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-Delta TM1 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.
Nishi
Tsuyoshi
T
Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
Kawasaki-Nishi
Shoko
S
Forgac
Michael
M
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D011510:Proteolipids / Q000737:chemistry / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism*
D021122:Protein Subunits; D011510:Proteolipids; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C072100:VMA11 protein, S cerevisiae; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases