Defective H(+)-ATPase of hygromycin B-resistant pma1 mutants fromSaccharomyces cerevisiae.
Defective H(+)-ATPase of hygromycin B-resistant pma1 mutants fromSaccharomyces cerevisiae.
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DOI：
10.1016/s0021-9258(20)88263-5
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发表时间：
1989-12
期刊：
影响因子：
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通讯作者：
D. Perlin;S. Harris;D. Seto-Young;J. Haber
中科院分区：
文献类型：
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作者：
D. Perlin;S. Harris;D. Seto-Young;J. Haber
最近研究表明，酿酒酵母的质膜H+- atp酶基因（PMA1）突变可导致整个细胞膜电位的显著去极化（Perlin， D. S., Brown， C. L., and Haber， J. E., 1988）。医学杂志。化学。263,18118-18122)。在这篇报道中，描述了四个突出的抗湿霉素b突变体pma1-105、pma1-114、pma1-147和pma1-155的H+- atp酶的生化和遗传特性。在pma1-105、pma1-114和pma1-147中发现单碱基对变化，分别导致Ser-368→Phe、Gly-158→Asp、Pro-640→Leu的氨基酸取代。在pma1-155 mRNA的5 ' -非翻译区−39处也发现了A→G过渡突变。这种突变产生了一个帧外的上游AUG起始密码子，明显减少了pma1的正常翻译。与已发表的菌株S288C序列相比，菌株Y55的pma1序列发现了9个碱基对的替换，导致非保守区有6个氨基酸的变化。四个pma1突变体中有三个的质膜含有正常量的H+- atp酶；mpma1-155的膜含酶量为野生型的62%。pma1-105和pma1-147酶的ATP水解动力学发生了最强烈的变化，这表明两者的kmandvmax都发生了变化。这些参数对mpma1-105酶有显著的pH依赖性，导致inkmandvmax在pH 6.5以下急剧下降。pma1-105和pma1-147酶的ATP水解对钒酸盐的抑制不敏感。与野生型和钒酸盐敏感突变型酶相比，这些酶对MgATP和钒酸盐或Pialone诱导的胰蛋白酶失活保护较差。这些结果与钒酸盐诱导的酶抑制机制有关，表明Ser-368和Pro-640影响了磷酸结合位点对Pi的亲和力。所有突变酶催化atp诱导的pH梯度形成纯化和重组脂质体。最后，这些结果进一步证明了潮霉素B作为分离多种质膜atp酶突变体的通用筛选工具的有效性。
Mutations in the plasma membrane H+-ATPase gene (PMA1) ofSaccharomyces cerevisiaethat confer growth resistance to hygromycin B have been shown recently to cause a marked depolarization of whole cell membrane potential (Perlin, D. S., Brown, C. L., and Haber, J. E. (1988)J. Biol. Chem. 263, 18118-18122). In this report, the biochemical and genetic properties of H+-ATPases from four prominent hygromycin B-resistantpma1mutants,pma1-105,pma1-114,pma1-147, andpma1-155, are described. Single base pair changes were identified inpma1-105,pma1-114, andpma1-147 that resulted in amino acid substitutions of Ser-368→Phe, Gly-158→Asp, Pro-640→Leu, respectively. An A→G transition mutation at −39 in the 5′-untranslated region of the mRNA ofpma1-155 was also found. This mutation creates an out-of-Frame upstream AUG initiation codon that apparently reduces normal translation ofPMA1. DNA sequence analysis ofPMA1from strain Y55 identified 9 base pair substitutions that resulted in 6 amino acid changes in nonconserved regions when compared to the published sequence for strain S288C. Plasma membranes of three of the fourpma1mutants contained normal amounts of H+-ATPase; membranes frompma1-155contained enzyme at 62% of the wild-type level. The kinetics of ATP hydrolysis were most strongly altered for enzymes frompma1-105andpma1-147which showed changes in bothKmandVmax. A striking pH dependence for these parameters was found for enzyme frompma1-105which resulted in a precipitous decline inKmandVmaxbelow pH 6.5. ATP hydrolysis by enzymes frompma1-105andpma1-147was insensitive to inhibition by vanadate. These enzymes, in contrast to wild-type and vanadate-sensitive mutant enzymes, were poorly protected from trypsin-induced inactivation by MgATP and vanadate or Pialone. These results are pertinent to the mechanism of vanadate-induced enzyme inhibition and suggest that Ser-368 and Pro-640 influence the affinity of the phosphate-binding site for Pi. All mutant enzymes catalyzed ATP-induced pH gradient formation following purification and reconstitution into liposomes. Finally, these results further demonstrate the usefulness of hygromycin B as a generalized screening tool for isolating diverse plasma membrane ATPase mutants.