The significance of membrane segments M6 and M8 in the biogenesis and functioning of the yeast PMA1 H<sup>+</sup>-ATPase

Abstract. The membrane domain of PMA1 H+-ATPase of the yeast plasma membrane is formed by 10 transmembrane segments (M1–M10), of which segments M6 and M8 are especially important. To study them, alanine-scanning mutagenesis was used, replacing each of the residues forming the segments with alanine. The enzymes were expressed from the plasmid pma1 gene in secretory vesicles under heat shock. In M6, half of the mutant proteins lost activity (0–7%), but were expressed at a level of 15–87% of the wild type. In M8, one third of the mutants showed a block in biogenesis (0–7%) or a significant decrease in expression (up to 16–17%), accompanied by an almost complete loss of enzymatic activity (0–10%). Since expression in secretory vesicles requires the use of elevated temperatures, the effect of mutations causing disturbance of expression and ATPase activity on the biogenesis and functioning of the enzyme in the absence of heat shock was tested by expressing them in plasma membranes from the chromosomal PMA1 gene at a permissive temperature. In the case of M6, only one mutant (F728A) out of the ten inactive enzymes was expressed in the plasma membrane and had activity at the wild type level; the remaining mutants were nonviable. In the case of M8, only mutants Q798A and I799A were unable to express at the plasma membrane level, while I794A, F796A, L797A, and L801A were expressed by 35–89% and had an activity of 14–65% of the wild type level. The effect of mutations F728A and F796A on the structural and functional organization of PMA1 ATPase and its regulation due to glucose-dependent activation of the enzyme was compared. Both mutations reduced ATPase activity by 30–50% and the degree of its activation by 30–40%. The data allow us to conclude that substitutions in the M6 segment primarily affect the functioning of the enzyme and, to a lesser extent, its conformation and biogenesis, suggesting the participation of the studied amino acid residues in the transport process. Residues in M8, on the contrary, play a major role in ATPase biogenesis. Overall, the results confirm the important role of amino acid residues in M6 and M8 for the structural and functional organization of PMA1 H+-ATPase and indicate that M6 contains more residues that affect the functioning of the enzyme.