Lai-Zhang J, Mueller D M
Department of Biochemistry and Molecular Biology, The Chicago Medical School, Chicago, IL 60064, USA.
Eur J Biochem. 2000 Apr;267(8):2409-18. doi: 10.1046/j.1432-1327.2000.01253.x.
The F1F0 ATP synthase is composed of the F1-ATPase which is bound to F0, in the inner membrane of the mitochondrion. Assembly and function of the enzyme is a complicated task requiring the interactions of many proteins for the folding, import, assembly, and function of the enzyme. The F1-ATPase is a multimeric enzyme composed of five subunits in the stoichiometry of alpha3beta3gammadeltaepsilon. This study demonstrates that four of the five bovine subunits of the F1-ATPase can be imported and function in an otherwise yeast enzyme effectively complementing mutations in the genes encoding the corresponding yeast ATPase subunits. In order to demonstrate this, the coding regions of each of the five genes were separately deleted in yeast providing five null mutant strains. All of the strains displayed negative or a slow growth phenotype on medium containing glycerol as the carbon source and strains with a null mutation in the gene encoding the gamma-, delta- or epsilon-gene became completely, or at a high frequency, cytoplasmically petite. The subunits of bovine F1 were expressed individually in the yeast strains with the corresponding null mutations and targeted to the mitochondrion using a yeast mitochondrial leader peptide. Expression of the bovine alpha-, beta-, gamma-, and epsilon-, but not the delta-, subunit complemented the corresponding null mutations in yeast correcting the corresponding negative phenotypes. These results indicate that yeast is able to import, assemble subunits of bovine F1-ATPase in mitochondria and form a functional chimeric yeast/bovine enzyme complex.
F1F0 ATP合酶由与F0结合的F1 - ATP酶组成，位于线粒体内膜。该酶的组装和功能是一项复杂的任务，需要许多蛋白质相互作用以实现酶的折叠、导入、组装和功能。F1 - ATP酶是一种多聚体酶，由α3β3γδε化学计量比的五个亚基组成。本研究表明，F1 - ATP酶的五个牛亚基中的四个可以被导入，并在另一种酵母酶中发挥作用，有效地补充编码相应酵母ATP酶亚基的基因突变。为了证明这一点，在酵母中分别删除了五个基因的编码区，得到了五个缺失突变株。所有菌株在以甘油作为碳源的培养基上均表现出阴性或生长缓慢的表型，并且在编码γ -、δ - 或ε - 基因的基因中存在缺失突变的菌株完全或高频地变为细胞质小菌落。牛F1的亚基在具有相应缺失突变的酵母菌株中单独表达，并使用酵母线粒体前导肽靶向线粒体。牛α -、β -、γ - 和ε - 亚基（而非δ - 亚基）的表达补充了酵母中相应的缺失突变，纠正了相应的阴性表型。这些结果表明，酵母能够在线粒体中导入、组装牛F1 - ATP酶的亚基，并形成功能性的嵌合酵母/牛酶复合物。
