Mutational analysis of the subunit C (Vma5p) of the yeast vacuolar H+-ATPase.
Subunit C is a V(1) sector subunit found in all vacuolar H(+)-ATPases (V-ATPases) that may be part of the peripheral stalk connecting the peripheral V(1) sector with the membrane-bound V(0) sector of the enzyme (Wilkens, S., Vasilyeva, E., and Forgac, M. (1999) J. Biol. Chem. 274, 31804--31810). To elucidate subunit C function, we performed random and site-directed mutagenesis of the yeast VMA5 gene. Site-directed mutations in the most highly conserved region of Vma5p, residues 305--325, decreased catalytic activity of the V-ATPase by up to 48% without affecting assembly. A truncation mutant (K360stop) identified by random mutagenesis suggested a small region near the C terminus of the protein (amino acids 382--388) might be important for subunit stability. Site-directed mutagenesis revealed that three aromatic amino acids in this region (Tyr-382, Phe-385, and Tyr-388) in addition to four other conserved aromatic amino acids (Phe-260, Tyr-262, Phe-296, Phe-300) are essential for stable assembly of V(1) with V(0), although alanine substitutions at these positions support some activity in vivo. Surprisingly, three mutations (F260A, Y262A, and F385A) greatly decrease the stability of the V-ATPase in vitro but increase its k(cat) for ATP hydrolysis and proton transport by at least 3-fold. The peripheral stalk of V-ATPases must balance the stability essential for productive catalysis with the dynamic instability involved in regulation; these three mutations may perturb that balance.
Curtis
Kelly Keenan
KK
Department of Biochemistry, State University of New York, Upstate Medical University, Syracuse, New York 13210, USA.
Francis
Smitha A
SA
Oluwatosin
Yemisi
Y
Kane
Patricia M
PM
D000595:Amino Acid Sequence; D017124:Conserved Sequence; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D021122:Protein Subunits; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000502:physiology
D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; C447616:VMA5 protein, S cerevisiae; D025262:Vacuolar Proton-Translocating ATPases