Molecular mechanism of recognition of mitochondrial protein precursor by mitochondria

大多数线粒体蛋白质在胞质溶胶中的游离多核糖体上合成，作为前体，在其N-末端部分具有前序列，输入线粒体并位于各种线粒体隔室（外膜、膜间隙、内膜和基质）中的其自身位置。在这个输入过程中，许多细胞蛋白质，如细胞溶质因子和线粒体蛋白前体的受体，需要作为输入机制，我们从兔网织红细胞裂解物中，纯化均匀的28 kDa的蛋白质，这是需要输入的前体蛋白质的亲和柱使用合成肽含有鸟氨酸氨基转移酶前体作为配体。该28 kDa蛋白质（28 kDa靶向因子）是构建携带线粒体前体的因子的组分。抗28 kDa靶向因子IgG特异性抑制前体的输入以及其与线粒体的结合。 ...更多信息 通过亲和柱色谱法，使用与用于纯化28 kDa靶向因子相同的合成肽，从线粒体膜级分中广泛纯化机械。纯化的级分含有两个主要的蛋白质，分子量为29和52 kDa。虽然这两种蛋白质对鸟氨酸氨基转移酶前序列有亲和力，但29 kDa蛋白质比52 kDa蛋白质能更紧密地结合前序列。此外，42 kDa蛋白质也被纯化为与29和/或52 kDa蛋白质的复合物。还显示，抗29、42和52 kDa蛋白Fab片段强烈抑制鸟氨酸氨基转移酶前体向线粒体的输入。对29和52 kDa蛋白质在线粒体中的定位进行了研究，发现这些蛋白质大多位于线粒体外膜上的“接触位点”。进一步分析证实这三个蛋白质是线粒体前体蛋白的转运机制。少

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