Direct Interaction of Mitochondrial Targeting Presequences with Purified Components of the TIM23 Protein Complex
Direct Interaction of Mitochondrial Targeting Presequences with Purified Components of the TIM23 Protein Complex
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DOI：
10.1074/jbc.m111.261040
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发表时间：
2011-12-23
影响因子：
4.8
通讯作者：
Azem, Abdussalam
中科院分区：
文献类型：
--
作者：
Marom, Milit;Dayan, Dana;Azem, Abdussalam
从细胞质溶胶进入线粒体基质的前体蛋白携带带正电荷的两亲性前体序列，并在TIM23复合物的重要组分的帮助下穿过内膜。目前尚不清楚TIM23复合体的哪些亚基识别并直接与序列结合。在这里，我们分析了前序肽与纯化的TIM23复合物组分的结合。研究了三种不同序列与酵母纯化的可溶性结构域Tim50（Tim50(IMS)）、Tim23（Tim23(IMS)）和全长Tim44的相互作用。利用化学交联和表面等离子体共振，我们首次证明了纯化的Tim50IMS和Tim44能够直接与酵母Hsp60前序相互作用。我们还分析了它们与酵母线粒体70-kDa热休克蛋白（mHsp70）和牛细胞色素P450（SCC）前体序列的相互作用。此外，我们表征了相互作用的性质，并确定了它们的K(D)s。基于我们的研究结果，我们提出了一种易位机制，其中通道反侧的前体序列相互作用更强，支持前体蛋白通过TIM23进入基质。
Precursor proteins that are imported from the cytosol into the matrix of mitochondria carry positively charged amphipathic presequences and cross the inner membrane with the help of vital components of the TIM23 complex. It is currently unclear which subunits of the TIM23 complex recognize and directly bind to presequences. Here we analyzed the binding of presequence peptides to purified components of the TIM23 complex. The interaction of three different presequences with purified soluble domains of yeast Tim50 (Tim50(IMS)), Tim23 (Tim23(IMS)), and full-length Tim44 was examined. Using chemical cross-linking and surface plasmon resonance we demonstrate, for the first time, the ability of purified Tim50IMS and Tim44 to interact directly with the yeast Hsp60 presequence. We also analyzed their interaction with presequences derived from precursors of yeast mitochondrial 70-kDa heat shock protein (mHsp70) and of bovine cytochrome P450(SCC). Moreover, we characterized the nature of the interactions and determined their K(D)s. On the basis of our results, we suggest a mechanism of translocation where stronger interactions of the presequences on the trans side of the channel support the import of precursor proteins through TIM23 into the matrix.