A Genetic Screen Targeted on the FO Component of Mitochondrial ATP Synthase in Saccharomyces cerevisiae
A Genetic Screen Targeted on the FO Component of Mitochondrial ATP Synthase in Saccharomyces cerevisiae
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DOI：
10.1074/jbc.m110.214825
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发表时间：
2011-05-20
影响因子：
4.8
通讯作者：
di Rago, Jean-Paul
中科院分区：
文献类型：
--
作者：
Godard, Francois;Tetaud, Emmanuel;di Rago, Jean-Paul
在酵母中，ATP 合酶的两个主要 F-O 质子易位亚基（亚基 6/a 和 9/c）由线粒体 DNA (mtDNA) 编码。不幸的是，使 F-O 失活的突变通常会导致 rho(-)/rho(0) 细胞形式的 mtDNA 丢失。因此，我们设计了一种新的遗传策略来规避这个问题。它利用了之前的发现，即编码 ATP 合酶亚基 delta 的核 ATP16 基因的无效突变会导致大量致命的 F-O 介导的质子穿过线粒体内膜泄漏。因此，在这些条件下，可以积极选择使 F-O 失活的突变作为细胞活力拯救事件。对第一组七个突变体进行了分析，正如预期的那样，所有突变体都显示出非常严重的 F-O 缺陷。两个突变体在 c 亚基表达所需的已知基因（AEP1、AEP2）中携带核突变。其他五个突变位于线粒体 DNA 中。其中，三个影响亚基a转录物的合成或稳定性，最后两个影响亚基c中的单个氨基酸替换。 c 亚基突变之一特别有趣。它包括与亚基 c 的必需谷氨酸（位置 59）相邻的亚基 c 位置 60 处的丙氨酸至缬氨酸的变化，该谷氨酸与亚基 a 的必需精氨酸 186 相互作用。该突变体的特性表明，亚基a和十个亚基c环结构之间的接触区仅涉及仅限于亚基a和c环之间质子交换的区域的关键瞬时相互作用。
In yeast, the two main F-O proton-translocating subunits of the ATP synthase (subunits 6/a and 9/c) are encoded by mitochondrial DNA (mtDNA). Unfortunately, mutations that inactivate the F-O typically result in loss of mtDNA under the form of rho(-)/rho(0) cells. Thus, we have designed a novel genetic strategy to circumvent this problem. It exploits previous findings that a null mutation in the nuclear ATP16 gene encoding ATP synthase subunit delta results in massive and lethal F-O-mediated protons leaks across the inner mitochondrial membrane. Mutations that inactivate the F-O can thus, in these conditions, be selected positively as cell viability rescuing events. A first set of seven mutants was analyzed and all showed, as expected, very severe F-O deficiencies. Two mutants carried nuclear mutations in known genes (AEP1, AEP2) required for subunit c expression. The five other mutations were located in mtDNA. Of these, three affect synthesis or stability of subunit a transcripts and the two last consisted in a single amino acid replacement in subunit c. One of the subunit c mutations is particularly interesting. It consists in an alanine to valine change at position 60 of subunit c adjacent to the essential glutamate of subunit c (at position 59) that interacts with the essential arginine 186 of subunit a. The properties of this mutant suggest that the contact zone between subunit a and the ten subunits c-ring structure only involves critical transient interactions confined to the region where protons are exchanged between the subunit a and the c-ring.