Nelson H, Mandiyan S, Nelson N
Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110.
J Biol Chem. 1994 Sep 30;269(39):24150-5.
Yeast vacuoles contain an H(+)-ATPase that acidifies the vacuolar lumen and generates an electrochemical gradient of protons across their membranes. Five polypeptides, denoted as subunits A to E in the order of decreasing molecular masses from 69 to 27 kDa, were identified in the catalytic sector of the enzyme. However, other polypeptides may be necessary for the activity of the enzyme. When a 14-kDa polypeptide was suggested to be a subunit of a specialized V-ATPase in Manduca sexta, and a homologous short sequence was detected downstream of the UGA1 gene in yeast, we cloned this counterpart gene from yeast. The gene VMA7 encodes a protein Vma7p of about 14 kDa. The predicted protein is highly homologous to the above mentioned M. sexta protein. The delta vma7::URA3 null mutant exhibits growth characteristics typical of other VMA disruptant mutants in genes encoding subunits of the catalytic sector. The delta vma7::URA3 null mutants are not able to grow on a medium buffered at pH 7.5, they fail to accumulate quinacrine into their vacuoles and the other subunits of the catalytic sector are not assembled onto the vacuolar membrane in its absence. Epitope-tagged Vma7p was constructed and the proton uptake activity of isolated vacuoles from this yeast strain was markedly inhibited by a monoclonal antibody against the epitope-tag. A cold inactivation experiment demonstrated that Vma7p is a genuine subunit of the catalytic sector of V-ATPase and it is denoted as subunit F.
酵母液泡含有一种H(+)-ATP酶，该酶可使液泡内腔酸化，并在其膜上产生质子电化学梯度。在该酶的催化部分鉴定出了五种多肽，按照分子量从69 kDa到27 kDa递减的顺序分别命名为亚基A至E。然而，该酶的活性可能还需要其他多肽。当一种14 kDa的多肽被认为是烟草天蛾中一种特殊V-ATP酶的亚基，并且在酵母的UGA1基因下游检测到同源短序列时，我们从酵母中克隆了这个对应基因。VMA7基因编码一种约14 kDa的蛋白质Vma7p。预测的蛋白质与上述烟草天蛾蛋白质高度同源。缺失vma7::URA3的突变体表现出催化部分亚基编码基因中其他VMA破坏突变体典型的生长特征。缺失vma7::URA3的突变体无法在pH 7.5缓冲的培养基上生长，它们无法将喹吖因积累到液泡中，并且在其缺失时催化部分的其他亚基无法组装到液泡膜上。构建了表位标记的Vma7p，来自该酵母菌株的分离液泡的质子摄取活性被针对表位标记的单克隆抗体显著抑制。冷失活实验表明Vma7p是V-ATP酶催化部分的一个真正亚基，它被命名为亚基F。
