Helfenbein Kevin G, Ellis Timothy P, Dieckmann Carol L, Tzagoloff Alexander
Department of Biological Sciences, Columbia University, New York, New York 10027, USA.
J Biol Chem. 2003 May 30;278(22):19751-6. doi: 10.1074/jbc.M301679200. Epub 2003 Mar 18.
Expression of the mitochondrial proton-translocating ATPase of Saccharomyces cerevisiae has been shown to depend on chaperones that target the F1 and F0 sectors of this inner membrane complex. Here we report a new gene, designated ATP22 (reading frame YDR350C on chromosome IV), that provides an essential function in the assembly of F0. ATP22 was cloned by transformation of C208/L2, a strain previously assigned to complementation group G99 of a collection of respiration-defective nuclear pet mutants. C208/L2 and the other atp22 mutants have oligomycin-insensitive F1-ATPase, suggesting that the lesion is confined to F0. This is supported by the sedimentation properties of the mutant ATPase and results of immunochemical analysis of F0 subunit polypeptides. Northern analysis of ATPase transcripts and in vivo pulse labeling of the mitochondrial translation products in the mutant indicate normal expression of subunits 6, 8, and 9, the three mitochondrial gene products of F0. Atp22p therefore functions at a post-translational stage in assembly of F0. Localization studies indicate Atp22p to be a component of the mitochondrial inner membrane. Protease protection experiments further indicate that Atp22p faces the matrix side of the membrane where most of the ATPase proteins are located and assembled.
酿酒酵母线粒体质子转运ATP酶的表达已被证明依赖于伴侣蛋白,这些伴侣蛋白靶向这种内膜复合物的F1和F0区段。在此,我们报道了一个新基因,命名为ATP22(位于第四条染色体上的读码框YDR350C),它在F0的组装中发挥重要作用。通过转化C208/L2克隆了ATP22,C208/L2是一个先前被归为呼吸缺陷型核pet突变体集合中互补组G99的菌株。C208/L2和其他atp22突变体具有对寡霉素不敏感的F1-ATP酶,这表明损伤局限于F0。突变型ATP酶的沉降特性以及F0亚基多肽的免疫化学分析结果支持了这一点。对ATP酶转录本的Northern分析以及突变体中线粒体翻译产物的体内脉冲标记表明,F0的三个线粒体基因产物亚基6、8和9表达正常。因此,Atp22p在F0组装的翻译后阶段发挥作用。定位研究表明Atp22p是线粒体内膜的一个组成部分。蛋白酶保护实验进一步表明,Atp22p面向膜的基质侧,大多数ATP酶蛋白就在此处定位和组装。