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.

大多数线粒体蛋白在细胞质中合成为前体蛋白，并借助蛋白质易位机在外部和内部机制中进口到线粒体中，分别称为TOM复合物和TIM复合物。在本研究中，我们分析了蛋白质通量对线粒体的控制和调节的机制。基于易位中间体的位点特异性光叠链链接的结果，我们已经确定了TIM50，这是酵母TIM23进口机械的新成分，它介导了跨层次跨元素的presequence probrakeR跨膜片的旋转。 TIM50固定在线粒体内膜上，将C末端结构域暴露于膜间空间。 TIM50与TIM23的N末端膜间空间结构域相互作用。 TIM50的功能缺陷可以通过蛋白质的耗竭或添加抗TIM50抗体阻止蛋白质易位跨内膜。在TOM复合物处积累的一个易位中间体被交联至TIM50。我们建议TIM50与TIM23合作，有助于将易位蛋白从TOM复合物转移到Tim23复合物中。我们采用了整个蛋白质组蛋白在体外进口的蛋白质组方法，以找到一组底物蛋白，以识别TOM70。将总酵母RNA的翻译产物进口到孤立的野生型线粒体和没有TOM70的野生型线粒体中。在野生型和ΔTOM70线粒体之间的2D电泳凝胶上进口蛋白的比较使我们能够系统地识别受TOM70缺失影响的蛋白质。