Structural and Functional Requirements for Activity of the Tim9-Tim10 Complex in Mitochondrial Protein Import
Structural and Functional Requirements for Activity of the Tim9-Tim10 Complex in Mitochondrial Protein Import
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DOI：
10.1091/mbc.e08-09-0903
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发表时间：
2009-02-01
影响因子：
3.3
通讯作者：
Ryan, Michael T.
中科院分区：
文献类型：
--
作者：
Baker, Michael J.;Webb, Chaille T.;Ryan, Michael T.
Tim9-Tim10复合体在线粒体蛋白的导入过程中起着至关重要的作用，它通过伴随选择的疏水前体蛋白穿过膜间隙。该复合物如何与前体相互作用尚不清楚，尽管已经提出Tim10在底物识别中起作用，而Tim9在复合物稳定中起作用。在这项研究中，我们报道了酵母Tim9-Tim10六聚体组装的结构，确定为2.5埃，并在酵母中进行了突变分析，以评估Tim9和Tim10的具体作用。与人类的对应体一样，每个Tim9和Tim10亚基都包含一个中心环，其两侧是二硫键，将两个延伸的N端和c端触角状螺旋分开。埋在高度保守的赖氨酸和谷氨酸残基之间的盐桥连接着交替的亚基。这些残基的突变使复合物不稳定，导致前体底物的进口缺陷，并导致酵母生长缺陷。截断分析显示，在缺少Tim9的N端区域的情况下，即使与Tim10仍有接触，六聚体复合物也不再能够有效地捕获进入的底物。我们得出的结论是，Tim9起着重要的功能作用，包括促进前体底物转移到膜间空间的初始步骤。
The Tim9-Tim10 complex plays an essential role in mitochondrial protein import by chaperoning select hydrophobic precursor proteins across the intermembrane space. How the complex interacts with precursors is not clear, although it has been proposed that Tim10 acts in substrate recognition, whereas Tim9 acts in complex stabilization. In this study, we report the structure of the yeast Tim9-Tim10 hexameric assembly determined to 2.5 angstrom and have performed mutational analysis in yeast to evaluate the specific roles of Tim9 and Tim10. Like the human counterparts, each Tim9 and Tim10 subunit contains a central loop flanked by disulfide bonds that separate two extended N- and C-terminal tentacle-like helices. Buried salt-bridges between highly conserved lysine and glutamate residues connect alternating subunits. Mutation of these residues destabilizes the complex, causes defective import of precursor substrates, and results in yeast growth defects. Truncation analysis revealed that in the absence of the N- terminal region of Tim9, the hexameric complex is no longer able to efficiently trap incoming substrates even though contacts with Tim10 are still made. We conclude that Tim9 plays an important functional role that includes facilitating the initial steps in translocating precursor substrates into the intermembrane space.