Scholarlink

Abstract <p>The membrane domain of the yeast plasma membrane PMA1 H<sup>+</sup>-ATPase is formed by 10 transmembrane segments (M1−M10), of which the segments M6 and M8 are especially important. They were investigated using alanine-scanning mutagenesis, with each of the segments’ residues replaced by alanine. The enzymes were expressed from the plasmid <i>pma1</i> gene in secretory vesicles under heat shock. In M6, half of the mutant proteins lost activity (0–7% of the wild type), but were expressed at 15–87% of the wild type level. In M8, one-third of the mutants showed blocks in biogenesis (0−7%) or a significant decrease in expression (16–17% of the wild type), accompanied by an almost complete loss of enzymatic activity (0–10%). Since the enzyme expression in secretory vesicles requires 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 <i>PMA1</i> 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 the mutants Q798A and I799A were incapable of expresssion at the plasma membrane level, while I794A, F796A, L797A, and L801A were expressed at 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 via glucose-dependent activation of the enzyme was compared. Both mutations decreased the ATPase activity by 30–50% and the degree of its activation by 30–40%. These data make it possible 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. On the contrary, residues in M8 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<sup>+</sup>-ATPase and indicate that M6 contains more residues that affect the functioning of the enzyme.</p>

错误：搜索内容不能为空，请输入英文关键词
错误：关键词超出字数限制，请精简
高级检索

The Significance of the M6 and M8 Membrane Segments in the Biogenesis and Functioning of the Yeast PMA1 H+-ATPase

  • 期刊论文

  • 发表日期：

    2025年4月1日

查看全文
  • V. V. Petrov

摘要

Abstract

The membrane domain of the yeast plasma membrane PMA1 H+-ATPase is formed by 10 transmembrane segments (M1−M10), of which the segments M6 and M8 are especially important. They were investigated using alanine-scanning mutagenesis, with each of the segments’ residues replaced by 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% of the wild type), but were expressed at 15–87% of the wild type level. In M8, one-third of the mutants showed blocks in biogenesis (0−7%) or a significant decrease in expression (16–17% of the wild type), accompanied by an almost complete loss of enzymatic activity (0–10%). Since the enzyme expression in secretory vesicles requires 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 the mutants Q798A and I799A were incapable of expresssion at the plasma membrane level, while I794A, F796A, L797A, and L801A were expressed at 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 via glucose-dependent activation of the enzyme was compared. Both mutations decreased the ATPase activity by 30–50% and the degree of its activation by 30–40%. These data make it possible 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. On the contrary, residues in M8 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.