Introduction: PMA1 H⁺-ATPase is the key enzyme of yeast metabolism. It belongs to P-ATPases, also including mammalian K⁺,Na⁺-, H⁺, K⁺-, Ca²⁺- and other metal-transporting ATPases. This pump couples ATP hydrolysis to transport H⁺ ions across the plasma membrane, thus generating electrochemical proton gradient. The enzyme is anchored in the lipid bilayer by ten hydrophobic segments (M1-M10), which form a membrane domain that carries H⁺-translocating sites. This work aimed to study the role of M6 amino acid residues in the structure-function relationships in PMA1 ATPase.

Methods: We used Ala-scanning mutagenesis to examine the functional role of amino acid residues throughout M6 of the PMA1 H⁺-ATPase. The yeast strains SY4 and NY13 were employed for the enzyme expression in secretory vesicles (SV) and plasma membranes (PM), respectively. In SV, mutant proteins were expressed from plasmid pma1 gene under the heat shock-inducible promoter at 39^oC and in PM, from the chromosomal PMA1 gene at permissive temperature. SV and PM were isolated to measure the expression and ATPase activity.

Results and Discussion: Nearly half of the SV mutants possessed sufficient activity and expression levels for further investigation. The majority of them exhibited abnormalities in kinetics and/or H⁺-transport. The rest of the mutations led to a loss of activity and/or blockage in biogenesis. Given that heat shock may affect PMA1 biogenesis, the inactive mutants were integrated into the chromosomal copy of the PMA1 gene. All but one mutant (F728A) were unable to support growth. F728A was expressed and exhibited activity close to the wild-type level. However, the F728A ATPase ability for glucose-dependent activation dropped almost twice.

Conclusion: M6 is a very important segment in maintaining enzyme structure-function relationships. Further studies of the substitutions' effect will help to reveal details of the mechanism of PMA1 H⁺-ATPase regulation/functioning.