Protein Import into Mitochondria

本提案的目的是调查发生在 将酵母酿酒酵母的胞质溶胶在 在蛋白质进入线粒体的过程中。 重点 线粒体前体蛋白之间的相互作用， 胞质组分。 将确定这些组成部分， 在评估进口过程中的作用。 的影响 将分析胞质因子对前体构象的影响。 将进行实验以确定前体蛋白质 在细胞质中紧密折叠，随后在细胞质中合成。 细胞或防止折叠或主动展开 在进口反应之前。 还将进行实验 评估共翻译导入的整体重要性 机制，并确定前体合成和进口是否 体内偶联。 为了实现这些目标，S。酿酒厂将是 用于进行体外和体内实验。 一 完全同源的酵母体外导入系统， 最近开发的将是该方法的基本组成部分。 由于体外前体细胞所需的胞质提取物 合成和线粒体分离自相同类型的细胞， 可以研究这两种细胞成分之间的相互作用 并且结果更直接地应用于体内情况。 线粒体是真核生物的重要细胞器成分， 细胞，由来自两个基因组来源的蛋白质组成： 线粒体蛋白质由线粒体自身的DNA编码 并通过自身的转录和翻译 机器，而另一些则由核基因组编码， 由细胞质翻译机器合成。 的 这些蛋白质是如何从它们的合成位点 到他们的功能位置在南卡罗来纳州是一个重要的 也是被广泛研究的一个 解决问题的两个方面 在这个建议中，即（1）是否胞质因子， 参与输入核编码的线粒体蛋白 进入线粒体，以及（2）是否有些蛋白质 共同易位到线粒体中，也就是说， 虽然它们仍在合成，而不是后- 从理论上讲，也就是说，在蛋白质合成之后， 完成，是重要的，有点争议，及时。 它 预计这些研究将导致新的基础 深入了解线粒体生物发生的机制。

The goal of this proposal is to investigate events that occur in the cytosol of the yeast Saccharomyces cerevisiae prior to or during the import of proteins into mitochondria. Emphasis is placed on interactions between mitochondrial precursor proteins and cytosolic components. Such components will be identified and their function in the import process assessed. The influence of cytosolic factors on precursor conformation will be analyzed. Experiments will be performed to determine if precursor proteins fold tightly in the cytosol subsequent to their synthesis in the cell or are either prevented from folding or actively unfolded prior to the import reaction. Experiments will also be conducted to evaluate the overall importance of a cotranslational import mechanism and to determine if precursor synthesis and import are coupled in vivo. To achieve these goals, S. cerevisiae will be used to perform both in vitro and in vivo experiments. A completely homologous yeast in vitro import system that has been recently developed will be a fundamental component of the approach. Since both the cytosolic extract required for in vitro precursor synthesis and mitochondria are isolated from the same type of cell, interactions between these two cellular components can be studied and the results more directly applied to the situation in vivo. The mitochondrion, a vital organellar component of the eukaryotic cell, is made up of proteins from two genomic sources: some of mitochondrial proteins are encoded by the mitochondrion's own DNA and synthesized by its own transcriptional and translational machinery, while others are encoded by the nuclear genome and synthesized by the cytoplasmic translational machinery. The question of how these proteins get from their sites of synthesis to their functional locations in the mitochondrion is an important and widely-studied one. The two aspects of the problem addressed in this proposal, namely (1) whether or not cytosolic factors are involved in the import of nuclear-encoded mitochondrial proteins into the mitochondria, and (2) whether or not some proteins are translocated into the mitochondria co-translationally, that is, while they are still being synthesized, as opposed to post- translationally, that is, after the synthesis of the protein is completed, are important, somewhat controversial, and timely. It is anticipated that these studies will lead to fundamental new insights into the mechanism of mitochondrial biogenesis.