Expression of bovine F1-ATPase with functional complementation in yeast Saccharomyces cerevisiae
Expression of bovine F1-ATPase with functional complementation in yeast Saccharomyces cerevisiae
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DOI：
10.1074/jbc.m411113200
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发表时间：
2005-06-10
影响因子：
4.8
通讯作者：
Mueller, DM
中科院分区：
文献类型：
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作者：
Puri, N;Lai-Zhang, J;Mueller, DM
线粒体F1F0-ATP合成酶是由至少16个独特的多肽组成的多聚体酶复合体，总分子质量约为600 kDa。F-1-ATPase由α(3)-β(3)-γ-β-epsilon组成，总分子质量为370 kDa。编码牛F-1-ATPase的基因已在酿酒酵母五重缺失突变体(DeltaαDeltaβDelta Gamma Delta Delta epsilon)中表达。这株表达牛F-1的菌株不能在含有非发酵碳源(YPG)的培养基上生长，表明该酶不起作用。然而，子代菌株很容易被选择用于在YPG培养基上生长，而这些子代菌株是为了在YPG培养基上促进生长而进化的。这些菌株的进化可能是由于突变，但编码牛F-1-ATPase亚单位的基因突变并不是细胞在YPG培养基上生长所必需的。在酵母中表达的牛酶被部分纯化，其比活力约为从牛心脏线粒体中纯化的酶的一半。这些结果表明，组装线粒体ATP合成酶所需的分子机制在牛和酵母中是保守的，表明酵母可能有助于哺乳动物F-1-或F1F0-ATP合成酶的表达、突变和分析。
The mitochondrial F1F0- ATP synthase is a multimeric enzyme complex composed of at least 16 unique peptides with an overall molecular mass of similar to 600 kDa. F-1-ATPase is composed of alpha(3)beta(3)gamma delta epsilon with an overall molecular mass of 370 kDa. The genes encoding bovine F-1-ATPase have been expressed in a quintuple yeast Saccharomyces cerevisiae deletion mutant (Delta alpha Delta beta Delta gamma Delta delta Delta epsilon). This strain expressing bovine F-1 is unable to grow on medium containing a non-fermentable carbon source (YPG), indicating that the enzyme is non-functional. However, daughter strains were easily selected for growth on YPG medium and these were evolved for improved growth on YPG medium. The evolution of the strains was presumably due to mutations, but mutations in the genes encoding the subunits of the bovine F-1-ATPase were not required for the ability of the cell to grow on YPG medium. The bovine enzyme expressed in yeast was partially purified to a specific activity of about half of that of the enzyme purified from bovine heart mitochondria. These results indicate that the molecular machinery required for the assembly of the mitochondrial ATP synthase is conserved from bovine and yeast and suggest that yeast may be useful for the expression, mutagenesis, and analysis of the mammalian F-1- or F1F0-ATP synthase.