The Impact of Replacing Amino Acid Residues Ser-911 and Thr-912 in the Yeast Saccharomyces cerevisiae Plasma Membrane H⁺-ATPase on Its Activity and Polyphosphate Distribution

The vital enzyme of yeast metabolism, the H⁺-ATPase of the plasma membrane (PMA1), is phosphorylated during folding and functioning. The main phosphorylation sites are Ser-911 and Thr-912 in the C-terminal regulatory domain. In this study, wild-type and mutant forms of the enzyme with substitutions of these amino acid residues by Ala or Asp were used to determine their role in the ATPase function and the distribution of polyphosphates (PolyP) among fractions. The growth parameters, ATP content, and in situ distribution of PolyP were determined in whole cells. Plasma membranes containing the wild-type and mutant forms of the enzyme were isolated to determine in vitro ATPase activity. Mutants S911D, T912D, and S911D/T912A exhibited ATPase activity similar to that of the wild type, while mutant S911A showed increased activity. The growth rate of the S911D, T912D, and S911D/T912A strains was 2.0−3.0 times lower than that of the wild type, while the growth rate of S911A was similar to that of the wild type. Mutations S911D and S911D/T912A caused a 2.0–2.5 fold decrease in ATP content. All substitutions affected the distribution of PolyP into fractions. The effect depended on the chemical nature of the substitution: in the case of substitution with Asp, which changes the type of phosphosite, there was a decrease in the PolyP1 fraction and an increase in the PolyP2 fraction; in the case of substitution with Ala, which removes the phosphosite, the effect was opposite. The content of the PolyP3 fraction was increased in all mutants. The data indicate that residues Ser-911 and Thr-912 are essential not only for the normal functioning of the PMA1 H⁺-ATPase but also for the regulation of phosphorus and energy metabolism.