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

大多数线粒体蛋白是在胞浆中作为前体蛋白合成的，并通过外膜和内膜的蛋白质转运机制分别称为TOM复合体和TIM复合体输入到线粒体中。在本研究中，我们分析了蛋白质进入线粒体的调控机制，根据转位中间体的定点光交联结果，我们鉴定了酵母TIM23输入机制中的一个新成分Tim50，它介导了含有前序列的蛋白质跨线粒体内膜的转位。Tim50被锚定在线粒体膜内，将C-末端结构域暴露在膜间隙。TIM50与TIM23的N端膜间空间域相互作用。Tim50的功能缺陷，无论是由于蛋白质的耗尽或添加抗Tim50抗体的加入，都会阻止蛋白质跨内膜的转运。积累在TOM复合体的易位中间体与Tim50交联。我们认为，Tim50与TIM23协同作用，促进了易位蛋白从TOM复合体到TIM23复合体的转移。我们采用了体外蛋白质组输入的方法来寻找一组底物蛋白质，供Tom70识别。将酵母总RNA的翻译产物导入分离的野生型线粒体和不含Tom70的线粒体。通过比较野生型和ΔTom70线粒体2D-电泳胶上的进口蛋白质，我们可以系统地鉴定Tom70缺失影响的蛋白质。

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.