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Molecular mechanism of recognition of mitochondrial protein precursor by mitochondria

线粒体识别线粒体蛋白前体的分子机制

基本信息

批准号：
02454542
负责人：
ONO Hideyu
金额：
$ 4.93万
依托单位：
Yamagata University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

项目摘要

Most of mitochondrial proteins are synthesized on free polysomes in cytosol as precursors with a presequence at their N-terminal portions, imported into mitochondria and located in their own locations in various mitochondrial compartments (outer membrane, intermembrane space, inner membrane, and matrix). In this import process, many cellular proteins such as a cytosolic factor and receptor for the mitochondrial protein precursors are required as import machinery.From a rabbit reticulocyte lysate, we purified homogeneously 28 kDa protein which were required for import of the precursor proteins with an affinity column using synthetic peptide containing the presequence of ornithine aminotransferase precursor as a ligand. This 28 kDa protein (28 kDa targeting factor) was a component constructing a factor carrying the precursor to mitochondria. Anti-28 kDa targeting factor IgG specifically inhibited the import of the precursor as well as its binding to mitochondria.The components of import … More machinery were extensively purified from the mitochondrial membrane fraction by affinity column chromatography using the same synthetic peptide as that used for purification of 28 kDa targeting factor. The purified fraction contained two major proteins with molecular masses of 29 and 52 kDa. Although these two proteins had an affinity to the presequence of omithine aminotransferase, the 29 kDa protein could more tightly bind the presequence than the 52 kDa protein. Furthermore, 42 kDa protein was also purified as complex with 29 and/or 52 kDa protein. It was also shown that anti-29, 42, and 52 kDa protein Fab fragments strongly inhibited the import of precursor of ornithine aminotransferase into mitochondria. The localization of 29 and 52 kDa proteins in the mitochondriawas studied, then it was found that most of these proteins were located in the distinct area, so called "the contact site" in the outer mitochondrial membrane. And it was comfirmed by further analysis that these three proteins function as translocation machinery for the mitochondrial protein precursors. Less

大多数线粒体蛋白是在胞质溶胶中的自由多聚体上作为前体合成的，其n端部分有一个前体序列，进入线粒体并位于各种线粒体室（外膜、膜间空间、内膜和基质）中各自的位置。在这个进口过程中，许多细胞蛋白，如细胞质因子和线粒体蛋白前体的受体，需要作为进口机器。我们利用含有鸟氨酸转氨酶前体序列的合成肽作为配体，用亲和柱从兔网织细胞裂解液中纯化了进口前体蛋白所需的28kda蛋白。这个28 kDa蛋白（28 kDa靶向因子）是构建一个携带线粒体前体的因子的成分。抗- 28kda靶向因子IgG特异性抑制前体的输入及其与线粒体的结合。采用与纯化28kda靶向因子相同的合成肽，通过亲和柱层析从线粒体膜部分广泛纯化了进口机械的成分。纯化后的部分含有两种主要蛋白，分子量分别为29和52 kDa。虽然这两种蛋白与氨基转移酶的前序列有亲和力，但29 kDa蛋白比52 kDa蛋白能更紧密地结合前序列。此外，42 kDa蛋白也被纯化为与29和/或52 kDa蛋白的复合物。抗29、42和52 kDa蛋白Fab片段强烈抑制鸟氨酸转氨酶前体进入线粒体。对29和52 kDa蛋白在线粒体中的定位进行了研究，发现这些蛋白大部分位于线粒体外膜的不同区域，称为“接触点”。进一步的分析证实了这三种蛋白作为线粒体蛋白前体的易位机制。少