Identification of subunit g of yeast mitochondrial F1F0-ATP synthase, a protein required for maximal activity of cytochrome c oxidase
Identification of subunit g of yeast mitochondrial F1F0-ATP synthase, a protein required for maximal activity of cytochrome c oxidase
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DOI：
10.1046/j.1432-1327.1999.00345.x
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发表时间：
1999-06-01
期刊：
影响因子：
--
通讯作者：
Prescott, M
中科院分区：
文献类型：
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作者：
Boyle, GM;Roucou, X;Prescott, M
通过对酵母基因组数据库的检索，我们在酿酒酵母第16号染色体上发现了一个开放阅读框，其编码的蛋白质与牛线粒体F1F0-ATP合成酶的11.3 kDa亚基g有53%的氨基酸相似性。我们将其命名为ORF ATP20及其产物亚基g。通过将HIS3基因插入到ATP20的编码区构建的零突变株保留了氧化磷酸化功能。在没有g亚基的情况下，atp20缺失菌株的F1F0-ATP合成酶的组装没有受到影响，线粒体中对寡霉素敏感的ATP水解酶活性水平是正常的。从atp20缺失细胞制备的线粒体裂解物中免疫沉淀F1F0-ATP合成酶，表明该多肽与酵母复合体中其他特征良好的亚基有关。虽然从atp20缺失菌株分离的线粒体具有与对照菌株相同的氧化磷酸化效率(ATP：O)，但atp20缺失菌株的呼吸量和ATP合成速率都显示出大约30%的下降。G亚基的缺失也降低了细胞色素C氧化酶的活性，并改变了该复合体的动力学控制，氰化物滴定ATP合成活性的实验证明了这一点。这些结果表明，g亚基与F1F0-ATP合成酶有关，是酵母最高水平的呼吸、ATP合成和细胞色素c氧化酶活性所必需的。
By means of a yeast genome database search, we have identified an open reading frame located on chromosome XVI of Saccharomyces cerevisiae that encodes a protein with 53% amino acid similarity to the 11.3-kDa subunit g of bovine mitochondrial F1F0-ATP synthase. We have designated this ORF ATP20, and its product subunit g. A null mutant strain, constructed by insertion of the HIS3 gene into the coding region of ATP20, retained oxidative phosphorylation function. Assembly of F1F0-ATP synthase in the atp20-null strain was not affected in the absence of subunit g and levels of oligomycin-sensitive ATP hydrolase activity in mitochondria were normal. Immunoprecipitation of F1F0-ATP synthase from mitochondrial lysates prepared from atp20-null cells expressing a variant of subunit g with a hexahistidine motif indicated that this polypeptide was associated with other well-characterized subunits of the yeast complex. Whilst mitochondria isolated from the atp20-null strain had the same oxidative phosphorylation efficiency (ATP : O) as that of the control strain, the atp20-null strain displayed approximately a 30% reduction in both respiratory capacity and ATP synthetic rate. The absence of subunit g also reduced the activity of cytochrome c oxidase, and altered the kinetic control of this complex as demonstrated by experiments titrating ATP synthetic activity with cyanide. These results indicate that subunit g is associated with F1F0-ATP synthase and is required for maximal levels of respiration, ATP synthesis and cytochrome c oxidase activity in yeast.