Vergnolle Mailys A S, Alcock Felicity H, Petrakis Nikos, Tokatlidis Kostas
Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, UK.
J Mol Biol. 2007 Aug 31;371(5):1315-24. doi: 10.1016/j.jmb.2007.06.025. Epub 2007 Jun 15.
The Saccharomyces cerevisiae TIM10 complex (TIM10c) is an ATP-independent chaperone of the mitochondrial intermembrane space, involved in transport of polytopic membrane proteins. The complex is an alpha(3)beta(3) hexamer of Tim9 and Tim10 subunits. We have generated specific mutations in charged residues in the central core domain of each subunit delineated by the characteristic twin CX(3)C motif, and investigated the effect of these mutations on subunit folding, complex assembly and TIM10 function in vitro and in vivo. Any combination of mutations that included a specific glutamate residue, conserved in all known Tim9 and Tim10 sequences, abolished assembly of the TIM10 complex. In vivo complementation analyses using a MET3-TIM10 strain that is selectively inactivated for the expression of wild-type Tim10 showed that (i) an N-terminal deleted version of Tim10 that was previously shown to be defective in substrate binding is lethal under all conditions, but (ii) the charged residues mutant of Tim10 that is defective in assembly with Tim9 can restore growth in glucose, but not in non-fermentable carbon sources. These data suggest that formation of the hexamer is beneficial but not vital for TIM10 function, whilst the N-terminal substrate-binding region of Tim10 is essential in vivo.
酿酒酵母TIM10复合物（TIM10c）是线粒体外膜间隙中一种不依赖ATP的伴侣蛋白，参与多跨膜蛋白的转运。该复合物是由Tim9和Tim10亚基组成的α(3)β(3)六聚体。我们在由特征性双CX(3)C基序界定的每个亚基的中央核心结构域的带电残基中产生了特异性突变，并研究了这些突变对亚基折叠、复合物组装以及TIM10在体外和体内功能的影响。任何包含一个在所有已知Tim9和Tim10序列中都保守的特定谷氨酸残基的突变组合，都会消除TIM10复合物的组装。使用对野生型Tim10表达进行选择性失活的MET3-TIM10菌株进行的体内互补分析表明：（i）先前已证明在底物结合方面存在缺陷的Tim10的N端缺失版本在所有条件下都是致死的，但是（ii）在与Tim9组装方面存在缺陷的Tim10带电残基突变体能够在葡萄糖中恢复生长，但在非发酵碳源中则不能恢复生长。这些数据表明，六聚体的形成对TIM10功能有益但并非至关重要，而Tim10的N端底物结合区域在体内是必不可少的。
