Perlin D S, Harris S L, Monk B C, Seto-Young D, Na S, Anand S, Haber J E
Department of Biochemistry, Public Health Research Institute, New York.
Acta Physiol Scand Suppl. 1992;607:183-92.
The H(+)-ATPase from Saccharomyces cerevisiae has been probed by a random genetic approach that has led to the isolation of primary and secondary site mutations. These H(+)-ATPase (PMA1) mutants help define specific functional, as well as interacting, regions of the H(+)-ATPase. Cellular resistance to hygromycin B has been an important selection tool for the isolation of pmal mutants. One prominent hygromycin B-resistant mutant, pmal-105, was found to have a S368F mutation near the site of phosphorylation (D378) in the catalytic core. This mutation prevents growth in low pH or NH(4+)-containing medium and induces an acid-sensitive Vmax for ATP hydrolysis, as well as a pronounced insensitivity to vanadate. The prominent cellular and biochemical phenotypes of this strain facilitated a detailed revertant analysis to identify protein structure domains that interact directly or indirectly with the localized region defined by the F368 mutation. Partial revertants were isolated which were resistant to low pH or NH4+ but retained hygromycin resistance. Second site mutations were found within the first and second cytoplasmic loop domains, as well as in transmembrane segments 1-3 & 7. All of the revertant enzymes have a stable Vmax but some show changes in the pH optimum for ATP hydrolysis; all display vanadate sensitivities ranging between the insensitive F368 mutant and the fully-sensitive wild type enzyme. Revertant analyses have also been performed on two other pma1 mutants which carry the mutations A135V and G158D in transmembrane segments 1 and 2, respectively. Compensating second site mutations to these mutations were identified in transmembrane segments 1, 2, 4 & 7, as well as within the central catalytic domain. These analyses have helped identify interacting protein structure domains that may participate in coupling ATP hydrolysis to proton transport. Furthermore, they facilitate the construction of structural models to account for these interactions.
通过一种随机遗传方法对酿酒酵母的H(+)-ATP酶进行了研究，该方法已导致分离出一级和二级位点突变。这些H(+)-ATP酶（PMA1）突变体有助于定义H(+)-ATP酶的特定功能区域以及相互作用区域。对潮霉素B的细胞抗性一直是分离pmal突变体的重要选择工具。一个突出的潮霉素B抗性突变体pmal-105，发现在催化核心的磷酸化位点（D378）附近有一个S368F突变。该突变阻止了在低pH或含NH(4+)培养基中的生长，并诱导了对ATP水解的酸敏感Vmax，以及对钒酸盐的明显不敏感性。该菌株突出的细胞和生化表型有助于进行详细的回复突变分析，以鉴定与由F368突变定义的局部区域直接或间接相互作用的蛋白质结构域。分离出了对低pH或NH4+有抗性但保留潮霉素抗性的部分回复突变体。在第一和第二细胞质环结构域以及跨膜片段1-3和7中发现了第二位点突变。所有回复突变酶都有稳定的Vmax，但有些在ATP水解的最适pH值上有变化；所有酶对钒酸盐的敏感性介于不敏感的F368突变体和完全敏感的野生型酶之间。还对另外两个分别在跨膜片段1和2中携带A135V和G158D突变的pma1突变体进行了回复突变分析。在跨膜片段1、2、4和7以及中央催化结构域中鉴定出了对这些突变的补偿性第二位点突变。这些分析有助于鉴定可能参与将ATP水解与质子运输偶联的相互作用蛋白质结构域。此外，它们有助于构建结构模型来解释这些相互作用。
