GENETIC ANALYSIS OF YEAST MITOCHONDRIAL PROTEIN

大多数线粒体蛋白是由核基因编码的， 在细胞质中合成，并直接定位于细胞器。二 影响蛋白质进入细胞的特异性的因素 线粒体。首先是标记蛋白质以供传递的前序 到细胞器，第二，细胞机械的组件， 识别前序列并促进蛋白质的运输到 细胞器。几个实验室正在进行研究，以阐明 线粒体所需前序列的结构要求 本地化。这项提案中的研究旨在确定和 描述直接导入线粒体蛋白质的细胞成分 酵母(Saccharomyes Cerevisiae)染色体突变分析 线粒体生物发生有缺陷。本分析将由以下几部分组成 三个部分。 A)有缺陷的突变体的选择和初步鉴定 线粒体蛋白靶向。酵母ATP2基因的基因融合 (B亚基F1-ATPase)和E.ColiLacZ基因编码 表达时导致呼吸缺陷表型的蛋白质 酵母。这一表型的Gly反转变种包括 将杂合蛋白定位于线粒体。其中一些 突变体也是温度敏感的，它们被分配到三个 互补组(MTF-线粒体功能)。耐高温 Mtf突变体的返回体包括菌株(错配线粒体合成) 线粒体中野生型B亚单位的水平非常低 并且不能在不可发酵的碳源上生长。 B)系统地筛选这些MTF和MIS突变体 体内和体外线粒体结构和功能的检测 将提供对线粒体生物发生和 找出最有趣的突变体以供进一步分析。 C)对几个最有趣的突变进行深入分析，以确定 个别蛋白质在进口过程中的具体作用。

The majority of mitochondrial proteins are encoded by nuclear genes, synthesized in the cytoplasm and targeted directly to the organelle. Two factors contribute to the specificity of protein import into the mitochondria. First the presequence which labels the protein for delivery to the organelle, and second, the components of the cellular machinery that recognize the presequence and facilitate transport of the protein into the organelle. Research is in progress in several laboratories to elucidate the structural requirements of the presequences necessary for mitochondrial localization. The research in this proposal is designed to identify and characterize cellular components which direct mitochondrial protein import by the analysis of yeast (Saccharomyes cerevisiae) chromosomal mutants defective in mitochondrial biogenesis. This analysis will be composed of three parts. a) Selection and preliminary characterization of mutants defective in mitochondrial protein targeting. Gene fusions between the yeast ATP2 gene (B-subunit F1-ATPase) and the E. coli lacZ gene have been shown to encode proteins which cause a respiration-defective phenotype when expressed in yeast. Gly+ revertants of this phenotype include mutants with decreased targeting of the hybrid protein to the mitochondrion. Some of these mutants are also temperature-sensitive and they have been assigned to three complementation groups (mtf-mitochondrial function). Temperature-resistant revertants of mtf mutants include strains (mis-mitochondrial synthesis) that have very low levels of wild-type B-subunit within the mitochondria and are unable to grow on a non-fermentable carbon source. b) Systematic screening of these mtf and mis mutants through a series of assays of mitochondrial structure and function both in vivo and in vitro will provide insight into any defects in mitochondrial biogenesis and identify the most interesting mutants for further analysis. c) In depth analysis of a few of the most interesting mutants to determine the specific role of individual proteins in the import process.