Biochemical consequences in yeast of the human mitochondrial DNA 8993T>C mutation in the ATPase6 gene found in NARP/MILS patients
Biochemical consequences in yeast of the human mitochondrial DNA 8993T>C mutation in the ATPase6 gene found in NARP/MILS patients
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DOI：
10.1016/j.bbamcr.2009.02.011
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发表时间：
2009-05-01
影响因子：
5.1
通讯作者：
di Rago, Jean-Paul
中科院分区：
文献类型：
--
作者：
Kucharczyk, Roza;Rak, Malgorzata;di Rago, Jean-Paul
我们已经创建并分析了人类线粒体DNA T8993 C突变的酵母模型的特性，该突变与母系遗传的Leigh综合征和/或神经源性肌无力、共济失调和色素性视网膜炎相关。该突变将ATP合酶Atp 6p亚基中高度保守的亮氨酸变为脯氨酸，该亚基位于人类蛋白质的156位，酵母的183位。在体外，酵母T8993 C线粒体显示ATP合成速率降低40-50%。ATP驱动的质子跨线粒体内膜的易位在突变体中是正常的，并且对ATP合酶质子通道抑制剂寡霉素完全敏感。然而，在最大ATP水解活性的条件下，使用非免疫保护的线粒体，寡霉素对突变体ATP酶活性的抑制很差（40%对野生型细胞中的85%）。这些异常归因于BN-PAGE和线粒体蛋白质合成分析的ATP合酶内的ATP 6p的效率较低的掺入。有趣的是，T8993 C酵母中细胞色素c氧化酶的含量选择性地降低了40-50%，这显然是由于其内源编码的Cox 1 p亚基的合成减少。这一观察结果进一步支持了酵母线粒体中ATP合酶对细胞色素c氧化酶表达的控制。尽管ATP酶的缺陷，atp 6-L183 P突变体的呼吸底物和氧化磷酸化的效率的增长是类似的野生型，表明突变不影响线粒体内膜的质子渗透性。(C)2009爱思唯尔有限公司版权所有。
We have created and analyzed the properties of a yeast model of the human mitochondrial DNA T8993C mutation that has been associated with maternally-inherited Leigh syndrome and/or with neurogenic muscle weakness, ataxia and retinitis pigmentosa. This mutation changes a highly conserved leucine to proline in the Atp6p subunit of the ATP synthase, at position 156 in the human protein, position 183 in yeast. In vitro the yeast T8993C mitochondria showed a 40-50% decrease in the rate of ATP synthesis. The ATP-driven translocation of protons across the inner mitochondrial membrane was normal in the mutant and fully sensitive to oligomycin, an inhibitor of the ATP synthase proton channel. However under conditions of maximal ATP hydrolytic activity, using non-osmotically protected mitochondria, the mutant ATPase activity was poorly inhibited by oligomycin (by 40% versus 85% in wild type cells). These anomalies were attributed by BN-PAGE and mitochondrial protein synthesis analyses to a less efficient incorporation of Atp6p within the ATP synthase. Interestingly,the cytochrome c oxidase content was selectively decreased by 40-50% in T8993C yeast, apparently due to a reduced synthesis of its mitochondrially encoded Cox1p subunit. This observation further supports the existence of a control of cytochrome c oxidase expression by the ATP synthase in yeast mitochondria. Despite the ATPase deficiency, growth of the atp6-L183P mutant on respiratory substrates and the efficiency of oxidative phosphorylation were similar to that of wild type, indicating that the mutation did not affect the proton permeability of the mitochondrial inner membrane. (C) 2009 Elsevier B.V. All rights reserved.