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Molecular mechanisms of the control and regulation of the mitochondrial protein flux.

线粒体蛋白质通量控制和调节的分子机制。

基本信息

批准号：
13480207
负责人：
ENDO Toshiya
金额：
$ 9.47万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

项目摘要

Most mitochondrial proteins are synthesized as precursor proteins in the cytosol and imported into mitochondria with the aid of protein translocation machineries in the outer and the inner membranes called the TOM complex and the TIM complex, respectively. In the present study, we analyzed the mechanisms of the control and regulation of the protein flux into mitochondria.Based on the results of the site-specific photocrosslinking of the translocation intermediate, we have identified Tim50, a new component of the yeast TIM23 import machinery, which mediates translocation of presequence-containing proteins across the mitochondrial inner membrane. Tim50 is anchored to the inner mitochondrial membrane, exposing the C-terminal domain to the intermembrane space. Tim50 interacts with the N-terminal intermembrane space domain of Tim23. Functional defects of Tim50 either by depletion of the protein or addition of anti-Tim50 antibodies block the protein translocation across the inner membrane. A translocation intermediate accumulated at the TOM complex is crosslinked to Tim50. We suggest that Tim50, in cooperation with Tim23, facilitates transfer of the translocating protein from the TOM complex to the TIM23 complex.We took a proteome-wide approach of mitochondrial protein import in vitro to find a set of substrate proteins for recognition by Tom70. Translation products of total yeast RNA were imported into isolated wild-type mitochondria and those without Tom70. Comparison of the imported proteins on 2D-electrophoresis gels between wild-type and Δtom70 mitochondria allowed us to identify proteins affected by deletion of Tom70 systematically.

大多数线粒体蛋白质在胞质溶胶中作为前体蛋白合成，并借助于分别称为TOM复合物和TIM复合物的外膜和内膜中的蛋白质易位机制输入线粒体。在本研究中，我们分析了蛋白质流入线粒体的控制和调节机制，基于转运中间体的位点特异性光交联的结果，我们鉴定了一个新的酵母TIM 23输入机制的组分Tim 50，它介导含前序列的蛋白质穿过线粒体内膜的转运。Tim 50锚定在线粒体内膜上，使C-末端结构域暴露于膜间空间。Tim 50与Tim 23的N-末端膜间空间结构域相互作用。通过蛋白质的耗尽或抗Tim 50抗体的添加导致的Tim 50的功能缺陷阻断了蛋白质跨内膜的易位。在TOM复合物处积累的易位中间体与Tim 50交联。我们认为Tim 50与Tim 23协同作用，促进了转运蛋白从TOM复合体向TIM 23复合体的转移。我们采用全蛋白质组的方法在体外进行线粒体蛋白质的输入，寻找一组可被Tom 70识别的底物蛋白。将总酵母RNA的翻译产物导入分离的野生型线粒体和没有Tom 70的线粒体中。在2D电泳凝胶上比较野生型和Δ tom 70线粒体之间的输入蛋白，使我们能够系统地鉴定Tom 70缺失影响的蛋白。