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<b>Abstract</b>— <p>The M6 membrane segment of the yeast PMA1 ATPase is crucial for the enzyme structure–function relationships. We have previously found that the Ala/Ser substitution of amino acid residues in M6 resulted in emergence of inactive mutant ATPases expressed under heat shock conditions in half of the studied cases. Since heat shock is an integral part of the secretory vesicle isolation, elevated temperature can be considered a cause for impaired biogenesis and functioning of the enzyme. The corresponding Ala substitutions of amino acid residues in M6 were integrated into the chromosomal <i>PMA1</i> gene for expression in plasma membranes in the absence of heat shock. Of 10 substitutions, only the F728A mutant proved viable. This mutant ATPase was studied in greater detail, by measuring its phosphohydrolazing activity at different pH and temperatures. The F728A mutation had no effect on basal activity in starved cells at pH 5.70 and pH 6.25, and at 30 and 37°C compared to the wild-type. However, it significantly affected the enzyme activity in the glucose-metabolizing cells, decreasing it and reducing the degree of ATPase activation. A temperature of 37°C led to increased activity and partial ATPase activation in starved cells, mimicking glucose-dependent activation of the enzyme.</p>

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The M6 Membrane Segment in the Structure-Function Organization of the Saccharomyces cerevisiae PMA1 H+-ATPase: Impact of the F728A Mutation

  • 期刊论文

  • 发表日期：

    2026年7月17日

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  • V. V. Petrov

摘要

Abstract—

The M6 membrane segment of the yeast PMA1 ATPase is crucial for the enzyme structure–function relationships. We have previously found that the Ala/Ser substitution of amino acid residues in M6 resulted in emergence of inactive mutant ATPases expressed under heat shock conditions in half of the studied cases. Since heat shock is an integral part of the secretory vesicle isolation, elevated temperature can be considered a cause for impaired biogenesis and functioning of the enzyme. The corresponding Ala substitutions of amino acid residues in M6 were integrated into the chromosomal PMA1 gene for expression in plasma membranes in the absence of heat shock. Of 10 substitutions, only the F728A mutant proved viable. This mutant ATPase was studied in greater detail, by measuring its phosphohydrolazing activity at different pH and temperatures. The F728A mutation had no effect on basal activity in starved cells at pH 5.70 and pH 6.25, and at 30 and 37°C compared to the wild-type. However, it significantly affected the enzyme activity in the glucose-metabolizing cells, decreasing it and reducing the degree of ATPase activation. A temperature of 37°C led to increased activity and partial ATPase activation in starved cells, mimicking glucose-dependent activation of the enzyme.