PROTEIN IMPORT INTO YEAST MITOCHONDRIA

这项提议的主要目标是了解蛋白质是如何 输入到线粒体中。大多数线粒体蛋白都编码在 细胞核，在细胞质中合成，翻译后 导入细胞器。新合成的线粒体蛋白 必须找到线粒体，穿过一个或两个线粒体 膜，并被分选到正确的亚线粒体室。 这项建议的主要目标是确定新的膜组件 进口途径的一部分。尽管需要许多蛋白质来表达 线粒体蛋白输入最近被确定，许多关键的 蛋白质，如蛋白质转运孔的成分或 海峡，尚未被发现。此外，大多数机械设备 需要将进口蛋白质分选到特定的亚线粒体 车厢也同样无法辨认。将两种方法与 酵母，酿酒酵母，我们已经确定了新的进口 成分，我们正在分析它们在线粒体蛋白中的作用 导入和排序。 确定线粒体的重要组成部分MAS6的功能 内膜Mas6，一个以前未鉴定的进口突变体，是 在进口途径的早期阶段就有缺陷。最近，我们有 结果表明，MAS6编码一种必需的23kD蛋白，定位于 线粒体内膜，并在接触部位丰富。因此，MAS6 表示在内膜中标识的第一个导入组件。 为了确定MAS6蛋白在进口中的确切作用 途径，我们建议扩大我们对MAS6的研究。例如, 我们将分析从中国分离的线粒体的进口或前体 不同的mas6突变体。我们还会问MAS6是否直接与 进口的前体，并确定MAS6是否为复合体或 位于内膜的进口蛋白。我们还建议使用 MAS6鉴定新的内膜进口成分。 需要分选的膜蛋白的鉴定与分析 蛋白质到线粒体膜间空间。许多蛋白质 去往线粒体膜间间隙(IMS)的建议是 首先完全导入到矩阵中，然后再导回 通过一个类似于细菌分泌物的过程穿过内膜。 为了确定IMS分类所需的机器，我们选择了 错位IMS结合融合蛋白的突变体。这些变种人有 用于精确映射在整个 内膜。我们还分离出了一些突变体，它们是 出口途径的部件有缺陷。我们建议使用这些 突变体以确定介导IMS分选的膜蛋白，以及 确定它们在进口中的作用。

The primary objective of this proposal is to understand how proteins are imported into mitochondria. Most mitochondrial proteins are encoded in the nucleus, synthesized in the cytoplasm, and post-translationally imported into the organelle. Newly synthesized mitochondrial proteins must find the mitochondrion, cross one or both of the mitochondrial membranes, and be sorted to the correct submitochondrial compartment. The primary goal of this proposal is to identify new membrane components of the import pathway. Although a number o proteins required for mitochondrial protein import have been recently identified, many critical proteins, such as components of the protein-translocating pore or channel, have yet to be discovered. Furthermore, most of the machinery required to sort imported proteins to specific submitochondrial compartments is likewise unidentified. Using two approaches with the yeast, Saccharomyces cerevisiae, we have identified new import components, and we are analyzing their role in the mitochondrial protein import and sorting. Determine the Function of MAS6, an Import Component of the Mitochondrial Inner Membrane mas6, a previously uncharacterized import mutant, is defective at an early step in the import pathway. Recently, we have shown that MAS6 encodes an essential 23kD protein localized in the mitochondrial inner membrane, and enriched in contact sites. Hence MAS6 represents the first import component identified in the inner membrane. To determine the precise role the MAS6 protein plays in the import pathway, we propose to extend our investigations of MAS6. For example, we will analyze the import or precursors into mitochondria isolated from different mas6 mutants. We will also ask if MAS6 directly interacts with an imported precursor, and determine if MAS6 is member of a complex or import proteins located in the inner membrane. We also propose to use MAS6 to identify new import components of the inner membrane. Identification and Analysis of Membrane Proteins Required to Sort Proteins to the Mitochondrial Intermembrane Space. Many proteins destined for the mitochondrial intermembrane space (IMS) are proposed to be imported first completely into the matrix, and then exported back across the inner membrane by a process analogous to bacterial secretion. To identify the machinery required for IMS sorting, we have selected mutants that mislocalize an IMS-bound fusion protein. These mutants have been used to precisely map the signal required for export across the inner membrane. We have also isolated a number of mutants that are defective in components of the export pathway. We propose to use these mutants to identify membrane proteins that mediate IMS sorting, and determine their function in import.