Mason A B, Kardos T B, Monk B C
Molecular Microbiology Laboratory, Department of Oral Sciences, School of Dentistry and the Centre for Gene Research, University of Otago, P.O. Box 647, Dunedin, New Zealand.
Biochim Biophys Acta. 1998 Jul 17;1372(2):261-71. doi: 10.1016/s0005-2736(98)00065-0.
Constitutive, chromosomal expression of yeast pma1 deletion alleles in Saccharomyces cerevisiae yielded functional, truncated forms of the plasma membrane H+-ATPase which were independently capable of supporting wild type yeast growth rates. Deletion of 27 amino-terminal residues affected neither the enzyme's activity nor its responsiveness to changes in glucose metabolism. By contrast, removal of 18 carboxy-terminal amino acids produced an enzyme with a Vmax that was relatively insensitive to glucose-dependent metabolic status and with a Km that was significantly lower than that of the wild type enzyme. These effects were exaggerated when the amino- and carboxy-terminal deletions were combined in a bilaterally truncated H+-ATPase, suggesting that the amino terminus may have a subtle role in modulating ATPase activity. In pma1DeltaDelta cells cultured at pH 6, plasma membrane H+-ATPase levels were much lower than those in cells expressing a wild type ATPase. Increased expression levels could be achieved by growing the pma1DeltaDelta mutant at pH 3, a result that was at least partially due to a sustained, elevated transcription of pma1DeltaDelta mRNA. Our observations suggest that intracellular proton balance can be maintained by regulation of the activity and/or quantity of H+-ATPase in the plasma membrane.
酿酒酵母中酵母pma1缺失等位基因的组成型染色体表达产生了功能性的截短形式的质膜H⁺-ATP酶，这些截短形式能够独立支持野生型酵母的生长速率。缺失27个氨基末端残基既不影响该酶的活性，也不影响其对葡萄糖代谢变化的反应性。相比之下，去除18个羧基末端氨基酸产生了一种酶，其Vmax对葡萄糖依赖性代谢状态相对不敏感，Km显著低于野生型酶。当氨基末端和羧基末端缺失组合在双向截短的H⁺-ATP酶中时，这些效应会被放大，这表明氨基末端可能在调节ATP酶活性中起微妙作用。在pH 6培养的pma1ΔΔ细胞中，质膜H⁺-ATP酶水平远低于表达野生型ATP酶的细胞。通过在pH 3培养pma1ΔΔ突变体可以实现表达水平升高，这一结果至少部分归因于pma1ΔΔ mRNA的持续、升高转录。我们的观察结果表明，细胞内质子平衡可以通过调节质膜中H⁺-ATP酶的活性和/或数量来维持。
