Functional reconstitution of the import of the yeast ADP/ATP carrier mediated by the TIM10 complex

被引:47
作者
Luciano, P [1 ]
Vial, S [1 ]
Vergnolle, MAS [1 ]
Dyall, SD [1 ]
Robinson, DR [1 ]
Tokatlidis, K [1 ]
机构
[1] Univ Manchester, Sch Biol Sci, Manchester M13 9PT, Lancs, England
基金
英国医学研究理事会;
关键词
ADP; ATP carrier; mitochondria biogenesis; protein translocation; reconstitution; TIM10; complex;
D O I
10.1093/emboj/20.15.4099
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Import of the ADP/ATP carrier (AAC) into mitochondria requires the soluble TIM10 complex to cross the intermembrane space. We report here that Tim9 and Tim10 purified from Eschetichia coli can form a complex of the same size as the endogenous complex from yeast mitochondria. This shows that no other mitochondrial protein is required for the formation of the TIM10 complex. Co-expression of both proteins rendered Tim9 more soluble and allowed purification of the reconstituted complex in a single step. Urea/EDTA treatment of recombinant Tim10 allowed its import into tim10-ts mitochondria that lack endogenous Tim10 and cannot import AAC. In this way, we were able to (i) reconstitute the TIM10 complex in the intermembrane space and (ii) restore import of AAC to almost wild-type levels. The reconstituted TIM10 complex not only facilitated passage of AAC across the outer membrane but also ensured its accurate membrane insertion. We conclude that the TIM10 complex can be formed exclusively from Tim9 and Tim10 and that the reconstituted complex efficiently restores AAC import in a strain lacking the TIM10 complex.
引用
收藏
页码:4099 / 4106
页数:8
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