Bachhawat A K, Manolson M F, Murdock D G, Garman J D, Jones E W
Department of Biological Science, Carnegie Mellon University, Pittsburgh, PA 15213.
Yeast. 1993 Feb;9(2):175-84. doi: 10.1002/yea.320090208.
Strains bearing the vph2 mutation are defective in vacuolar acidification. The VPH2 gene was isolated from a genomic DNA library by complementation of the zinc-sensitive phenotype of the mutant. Deletion analysis localized the complementing activity to a 1.2 kb DNA fragment. Sequence analysis of this fragment revealed the presence of a single open reading frame that encoded a protein of 215 amino acids. Computer analysis indicated that the protein, which has a predicted molecular mass of 25,286 Daltons, has two distinct membrane-spanning domains. Biochemical studies indicated that strains bearing the vph2 mutation have greatly reduced levels of vacuolar proton pumping and ATPase activity and that the nucleotide binding subunits of the multimeric vacuolar H(+)-ATPase failed to be correctly targeted to the vacuolar membrane. The vph2 mutant fails to grow on YEP glycerol medium and on media containing 100 mM-CaCl2 or 4 mM-ZnCl2 or buffered to pH 7.5, a phenotype observed in strains carrying deletions in the genes encoding several vacuolar H(+)-ATPase subunits. The VPH2 gene is identical to the VMA12 gene (T. Stevens and Y. Anraku, personal communication).
携带vph2突变的菌株在液泡酸化方面存在缺陷。通过对突变体锌敏感表型的互补作用，从基因组DNA文库中分离出VPH2基因。缺失分析将互补活性定位到一个1.2 kb的DNA片段。对该片段的序列分析揭示了一个单一的开放阅读框，其编码一个215个氨基酸的蛋白质。计算机分析表明，该蛋白质预测分子量为25286道尔顿，具有两个不同的跨膜结构域。生化研究表明，携带vph2突变的菌株液泡质子泵浦和ATP酶活性水平大大降低，并且多聚体液泡H(+)-ATP酶的核苷酸结合亚基未能正确靶向液泡膜。vph2突变体在YEP甘油培养基以及含有100 mM - CaCl2或4 mM - ZnCl2或缓冲至pH 7.5的培养基上无法生长，这种表型在编码几种液泡H(+)-ATP酶亚基的基因缺失的菌株中也有观察到。VPH2基因与VMA12基因相同（T. Stevens和Y. Anraku，个人交流）。
