GTP BINDING PROTEINS IN MITOCHONDRIAL PROTEIN IMPORT

线粒体是产生哺乳动物组织几乎所有能量的细胞器。它们对重要的细胞过程至关重要，如程序性细胞死亡，它们功能的异常直接导致许多疾病，如糖尿病、心脏病和中风。由于线粒体基因组只编码参与其功能的数百种蛋白质中的少数几种，因此它们必须通过双膜输入大部分蛋白质。这一过程很复杂，在哺乳动物中也不为人所知，但输入缺陷会导致甲基丙二酸血症等疾病。我们发现了一种新的途径，通过这种途径，蛋白质被靶向并运输到线粒体膜上。此外，我们在线粒体膜中发现了一组新的gtp结合蛋白，它调节了这一过程，我们是唯一拥有这一数据的实验室。简而言之，线粒体输入的前体蛋白被认为是在细胞质中合成，并由线粒体膜中的受体复合物输入（翻译后输入模型）。然而，我们发现核糖体特异性靶向并结合线粒体膜。g蛋白和前体蛋白靶向序列的性质控制着这种结合。这一提议背后的假设是，蛋白质进入线粒体是一个共翻译事件，即核糖体特异性靶向线粒体表面，前体蛋白在翻译时被输入。本方案有三个具体目的：1)确定核糖体与线粒体结合的机制。转运肽的突变、竞争性结合试验和蛋白酶保护试验将确定核糖体-线粒体结合的成分。2)鉴定和分析线粒体表面的核糖体受体。该受体的蛋白酶敏感性及其位置将被确定。竞争性结合试验将确定受体对核糖体及其新生链的特异性。3)鉴定线粒体膜GTP结合蛋白G54。GTP水解控制核糖体与线粒体膜的结合，我们最近发现的这些膜G蛋白之一，称为G54，与核糖体相互作用。对G54进行纯化和测序，并对其核基因和cDNA进行鉴定。确定核糖体-线粒体相互作用的组成部分，以及这些新型g蛋白的功能，对于理解线粒体如何生长、分裂和自我修复至关重要。

Mitochondria are organelles that generate virtually all of the energy for mammalian tissues. They are crucial to important cellular processes, such as programmed cell death, and abnormalities in their function are directly responsible for many diseases such as diabetes, heart disease, and stroke. Because the mitochondrial genome encodes only a few of the hundreds of proteins involved in their function, they must import most of their proteins across their double membranes. This process is complex and poorly understood in mammals, but defects in import can cause diseases such as methylmalonic acidemia. We have discovered a novel pathway whereby proteins are targeted and transported to the mitochondrial membranes. Furthermore, we have found a novel group of GTP-binding proteins in the mitochondrial membrane that regulates this process, and we are the only laboratory to have this data. In brief, precursor proteins destined for mitochondrial import are believed to be synthesized in the cytosol and imported by a receptor complex in the mitochondrial membrane (the post-translational import model). However, we have found that ribosomes specifically target and bind to the mitochondrial membrane. G-proteins, and the nature of the targeting sequence of the precursor protein, control this binding. The hypothesis behind this proposal is that import of proteins into mitochondria is a co-translational event, that is, ribosomes specifically target the mitochondrial surface and precursor proteins are imported as they are translated. This proposal has three specific aims: 1) Determine the mechanism of ribosome binding to mitochondria. Mutations in the transit peptide, competitive binding assays, and protease protection assays will identify the components of ribosome-mitochondrial binding. 2) Identify and analyze the ribosome receptor on the mitochondrial surface. Protease sensitivity of the receptor and its location will be determined. Competitive binding assays will determine the specificity of the receptor for the ribosome and its nascent chain. 3) Identify the mitochondrial membrane GTP- binding protein, G54. GTP hydrolysis controls ribosome binding to the mitochondrial membrane and one of these membrane G- proteins we recently discovered, termed G54, interacts with ribosomes. G54 will be purified and sequenced, and its nuclear gene and cDNA characterized. Identifying the components of this ribosome-mitochondrial interaction, and the function of these novel G-proteins, is crucial to understanding how mitochondria grow, divide, and repair themselves.