J. Biol. Chem., Vol. 263, Issue 28, 14134-14139, Oct, 1988

Mutations of the yeast plasma membrane H+-ATPase which cause thermosensitivity and altered regulation of the enzyme

A Cid and R Serrano
European Molecular Biology Laboratory, Heidelberg, West Germany.

Four random mutations of the plasma membrane H+-ATPase of Saccharomyces cerevisiae which result in thermosensitive growth have been sequenced. All of the mutations map in regions conserved by the family of ATPases which form a phosphorylated intermediate. The Gly254----Ser mutation affects a glycine residue conserved in all of the sequenced ATPases. The Thr212----Ile and Ala547----Val mutations do not affect conserved amino acids, but their replacements are not found in any of the sequenced ATPases. Thr212 and Gly254 occur in the proposed phosphatase domain, whereas Ala547 is located within the putative ATP-binding site. The other mutation is a double change (Asp91----Tyr and Glu92----Lys) in the N-terminal domain, in which the altered glutamate is conserved in fungal and protozoan H+-ATPases. Proton efflux from whole cells and ATP hydrolysis by purified plasma membranes are more thermolabile in cells carrying the ATPase mutations than in wild-type yeast. Therefore, the defects in growth and proton transport at the nonpermissive temperature can be attributed to impairment of ATPase activity. Incubation of wild-type yeast cells with glucose before homogenization induces changes in the specific activity, Km, pH optimum, and vanadate sensitivity of the plasma membrane ATPase. The Ala547----Val mutation results in an enzyme from starved cells with the kinetic parameters of the glucose-activated wild-type ATPase. Therefore, a single amino acid change mimics the poorly understood regulatory mechanism triggered by glucose.
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