GENETIC ANALYSIS OF YEAST MITOCHONDRIAL PROTEIN

大多数线粒体蛋白由核基因编码， 在细胞质中合成并直接靶向细胞器。 两 这些因素有助于蛋白质输入的特异性， 线粒体 首先是标记蛋白质的前序列 第二，细胞器的组成部分， 识别前序列并促进蛋白质转运到 细胞器 几个实验室正在进行研究， 线粒体所必需的前序列的结构要求 本地化 本提案中的研究旨在确定和 表征指导线粒体蛋白质输入的细胞组分 通过分析酵母（酿酒酵母）染色体突变体， 线粒体生物发生有缺陷。 该分析将包括 三个部分。 a）选择和初步表征在以下方面有缺陷的突变体： 线粒体蛋白靶向。 酵母ATP 2基因之间的基因融合 （B亚基F1-ATPase）和E.大肠杆菌lacZ基因编码 当表达于细胞中时引起呼吸缺陷表型的蛋白质， 酵母 该表型的Gly+回复突变体包括具有降低的Gly+突变体。 将杂合蛋白质靶向至靶向子。 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）。 耐温 mtf突变体的回复突变体包括菌株（错线粒体合成） 在线粒体中野生型B亚基的水平非常低 并且不能在不可发酵的碳源上生长。 B）通过一系列的免疫细胞化学方法系统地筛选这些mtf和mis突变体。 线粒体结构和功能的体内和体外测定 将提供深入了解线粒体生物发生中的任何缺陷， 找出最有趣的突变体做进一步分析 c）深入分析一些最有趣的突变体，以确定 个别蛋白质在输入过程中的具体作用。

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.