Kucharczyk Roza, Ezkurdia Nahia, Couplan Elodie, Procaccio Vincent, Ackerman Sharon H, Blondel Marc, di Rago Jean-Paul
Institut de Biochimie et Génétique Cellulaires CNRS, Université Victor Segalen Bordeaux2, 1 Rue Camille Saint-Saëns, Bordeaux 33077 cedex, France.
Biochim Biophys Acta. 2010 Jun-Jul;1797(6-7):1105-12. doi: 10.1016/j.bbabio.2009.12.022. Epub 2010 Jan 4.
Several human neurological disorders have been associated with various mutations affecting mitochondrial enzymes involved in cellular ATP production. One of these mutations, T9176C in the mitochondrial DNA (mtDNA), changes a highly conserved leucine residue into proline at position 217 of the mitochondrially encoded Atp6p (or a) subunit of the F1FO-ATP synthase. The consequences of this mutation on the mitochondrial ATP synthase are still poorly defined. To gain insight into the primary pathogenic mechanisms induced by T9176C, we have investigated the consequences of this mutation on the ATP synthase of yeast where Atp6p is also encoded by the mtDNA. In vitro, yeast atp6-T9176C mitochondria showed a 30% decrease in the rate of ATP synthesis. When forcing the F1FO complex to work in the reverse mode, i.e. F1-catalyzed hydrolysis of ATP coupled to proton transport out of the mitochondrial matrix, the mutant showed a normal proton-pumping activity and this activity was 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 less efficiently inhibited by oligomycin (60% inhibition versus 85% for the wild type control). Blue Native Polyacrylamide Gel Electrophoresis analyses revealed that atp6-T9176C yeast accumulated rather good levels of fully assembled ATP synthase complexes. However, a number of sub-complexes (F1, Atp9p-ring, unassembled alpha-F1 subunits) could be detected as well, presumably because of a decreased stability of Atp6p within the ATP synthase. Although the oxidative phosphorylation capacity was reduced in atp6-T9176C yeast, the number of ATP molecules synthesized per electron transferred to oxygen was similar compared with wild type yeast. It can therefore be inferred that the coupling efficiency within the ATP synthase was mostly unaffected and that the T9176C mutation did not increase the proton permeability of the mitochondrial inner membrane.
几种人类神经疾病与影响细胞ATP生成过程中线粒体酶的各种突变有关。这些突变之一，线粒体DNA（mtDNA）中的T9176C，将线粒体编码的F1FO - ATP合酶的Atp6p（或a）亚基第217位的一个高度保守的亮氨酸残基变为脯氨酸。这种突变对线粒体ATP合酶的影响仍不清楚。为了深入了解T9176C引发的主要致病机制，我们研究了该突变对酵母ATP合酶的影响，酵母中的Atp6p同样由mtDNA编码。在体外，酵母atp6 - T9176C线粒体的ATP合成速率降低了30%。当迫使F1FO复合体以反向模式工作时，即F1催化的ATP水解与质子从线粒体基质输出相偶联，突变体表现出正常的质子泵活性，并且该活性对ATP合酶质子通道抑制剂寡霉素完全敏感。然而，在最大ATP水解活性条件下，使用非渗透保护的线粒体，突变体的ATP酶活性受寡霉素抑制的效率较低（抑制率为60%，而野生型对照为85%）。蓝色天然聚丙烯酰胺凝胶电泳分析表明，atp6 - T9176C酵母积累了相当高水平的完全组装的ATP合酶复合体。然而，也能检测到一些亚复合体（F1、Atp9p环、未组装的α - F1亚基），推测这是由于Atp6p在ATP合酶内的稳定性降低所致。尽管atp6 - T9176C酵母的氧化磷酸化能力降低，但与野生型酵母相比，每转移到氧的一个电子合成的ATP分子数量相似。因此可以推断，ATP合酶内的偶联效率基本未受影响，并且T9176C突变并未增加线粒体内膜的质子通透性。
