Functioning of yeast Pma1 H⁺-ATPase under changing charge: Role of Asp739 and Arg811 residues

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• 作者：V. V. Petrov
• 关键词：yeast ; plasma membrane ; secretory vesicles ; Pma1 H+ ; ATPase ; transmembrane segments ; H+ transport ; sitedirected mutagenesis
• 刊名：Biochemistry (Moscow)
• 出版年：2017
• 出版时间：January 2017
• 年：2017
• 卷：82
• 期：1
• 页码：46-59
• 全文大小：
• 刊物类别：Biomedical and Life Sciences
• 刊物主题：Biochemistry, general; Bioorganic Chemistry; Microbiology; Biomedicine, general;
• 出版者：Pleiades Publishing
• ISSN：1608-3040
• 卷排序：82

文摘

The plasma membrane Pma1 H+-ATPase of the yeast Saccharomyces cerevisiae contains conserved residue Asp739 located at the interface of transmembrane segment M6 and the cytosol. Its replacement by Asn or Val (Petrov et al. (2000) J. Biol. Chem., 275, 15709-15716) or by Ala (Miranda et al. (2011) Biochim. Biophys. Acta, 1808, 1781-1789) caused complete blockage of biogenesis of the enzyme, which did not reach secretory vesicles. It was proposed that a strong ionic bond (salt bridge) could be formed between this residue and positively charged residue(s) in close proximity, and the replacement D739A disrupted this bond. Based on a 3D homology model of the enzyme, it was suggested that the conserved Arg811 located in close proximity to Asp739 could be such stabilizing residue. To test this suggestion, single mutants with substituted Asp739 (D739V, D739N, D739A, and D739R) and Arg811 (R811L, R811M, R811A, and R811D) as well as double mutants carrying charge-neutralizing (D739A/R811A) or charge-swapping (D739R/R811D) substitutions were used. Expression of ATPases with single substitutions R811A and R811D were 38-63%, and their activities were 29-30% of the wild type level; ATP hydrolysis and H+ transport in these enzymes were essentially uncoupled. For the other substitutions including the double mutations, the biogenesis of the enzyme was practically blocked. These data confirm the important role of Asp739 and Arg811 residues for the biogenesis and function of the enzyme, suggesting their importance for defining H+ transport determinants but ruling out, however, the existence of a strong ionic bond (salt bridge) between these two residues and/or importance of such bridge for structure–function relationships in Pma1 H+-ATPase.