MOLECULAR GENETIC ANALYSES OF CYTOCHROMES C BIOGENESIS

细胞色素 C 生物发生的分子遗传学分析

• 批准号: 2185326
• 负责人: Robert G. Kranz
• 金额: $ 12.75万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-09-01 至 1997-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2185326
• 关键词: Rhodospirillales; SDS polyacrylamide gel electrophoresis; bacterial genetics; biological transport; cytochrome c; electron transport; heme; hemoprotein biosynthesis; laboratory rabbit; lipid bilayer membrane; mitochondria; molecular genetics; oxidation reduction reaction; protein purification; site directed mutagenesis; thioredoxin; western blottings

Recent discoveries of genes involved in prevalent genetic-based human diseases have stimulated interest in understanding the molecular and biochemical basis for these disease processes. For example, a number of neurological syndromes and encephalomyopathies have been attributed to mutations in genes encoding mitochondrial electron transfer components. These results have stifled interest in understanding the biogenesis and function of individual mitochondrial components. As with most complex systems, the molecular processes involved in the biogenesis and function of these electron transfer components are likely to be elucidated by studying analogous simplified systems that are more amenable to genetic and biochemical analyses. Such a simplified system is described, a bacterium that is closely related to the ancestral mitochondrial endosymbiont; seven genes involved specifically in the biogenesis of c- type cytochromes have already been isolated (called helABCDX and ccl1 and ccl2). The c-type cytochromes possess heme that is covalently ligated to the apocytochrome and these cytochromes are present in distinct compartments within eucaryotes (i.e., mitochondrial intermembrane space) and procaryotes (i.e., external to the cytoplasmic membrane). Major questions that remain concern what factors are needed for their assembly and how these individual factors are themselves transported to the proper compartments. Specifically, using appropriate mutants and/or wild type strains we will: (1) Develop and use heme reporters to further study a putative heme transporter encoded by helABC genes (and the defects in Ccl- strains). (2) Isolate heme-correcting delta-helAB mutants (to test the heme transporter function for helABC). (3) Produce and use antisera to the Hel and Ccl proteins to investigate localization and macromolecular complex interactions. (4) Overproduction, purification and functional analyses of HelX, a periplasmic thioredoxin-like protein. The long-term objective of this work is to understand at a detailed molecular level the specific proteins and events that are required for the biogenesis of c-type cytochromes. Moreover, since heme is required for many different processes in eucaryotes and procaryotes, the mechanism by which heme is transported across bilayer membranes from its site of biosynthesis, the mitochondrial matrix or the bacterial cytoplasm, is a fundamental question in biology. Completion of the specific aims proposed here will answer questions concerning intracellular heme targeting, specific red-ox reactions within the cell, and cytochrome c biogenesis. A combination of bacterial genetics, immunological analyses, and molecular biology will be used to accomplish these goals.

流行性遗传病相关基因的新发现 疾病激发了人们对了解分子和 这些疾病过程的生化基础。例如多个 神经系统综合征和脑肌病已被归因于 编码线粒体电子传递成分的基因突变。 这些结果扼杀了人们对了解生物起源的兴趣， 单个线粒体成分的功能。与大多数复杂的 系统，参与生物发生和功能的分子过程 这些电子转移组件可能被阐明， 研究类似的简化系统， 和生化分析。 描述了这样的简化系统， 与祖先线粒体密切相关的细菌 内共生体;七个基因参与的生物发生的c- 型细胞色素已经被分离出来（称为helABCDX和ccl 1， CCl 2）。C型细胞色素具有血红素，其共价连接到 脱辅基细胞色素和这些细胞色素以不同形式存在 真核细胞内的隔室（即，线粒体膜间隙） 和原核生物（即，细胞质膜外）。主要 仍然存在的问题是，它们的组装需要哪些因素 以及这些个体因素是如何传递到 隔间 具体地，使用适当的突变体和/或野生型菌株，我们将： (1)开发和使用血红素报告基因进一步研究一个假定的血红素 由helABC基因编码的转运蛋白（以及Ccl-菌株中的缺陷）。（二） 分离血红素校正δ-helAB突变体（以测试血红素转运蛋白 helABC的函数）。(3)生产和使用抗Hel和Ccl的抗血清 研究定位和大分子复合物的蛋白质 交互. (4)超高效生产、纯化及功能分析 HelX，一种周质硫氧还蛋白样蛋白。 这项工作的长期目标是详细了解 分子水平上的特定蛋白质和事件所需的 C型细胞色素的生物发生。此外，由于血红素是必需的 在真核生物和原核生物中有许多不同的过程， 其中血红素从它的位点穿过双层膜被运输， 生物合成，线粒体基质或细菌细胞质，是一种 生物学的基本问题。实现所提出的具体目标 这里将回答有关细胞内血红素靶向的问题， 细胞内的特定red-ox反应和细胞色素c生物合成。一 细菌遗传学、免疫学分析和分子生物学的结合， 生物学将被用来实现这些目标。