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.

最近发现的基因涉及流行的基于基因的人类 疾病激发了人们对了解分子和疾病的兴趣 这些疾病过程的生化基础。例如，一些 神经系统综合征和脑肌病被归因于 编码线粒体电子传递成分的基因发生突变。 这些结果抑制了理解生物发生和 单个线粒体成分的功能。与最复杂的情​​况一样 系统，涉及生物发生和功能的分子过程 这些电子转移成分可能通过以下方式来阐明 研究更适合遗传的类似简化系统 和生化分析。 描述了这样一个简化的系统， 与祖先线粒体密切相关的细菌 内共生体；七个基因专门参与 c-的生物发生 类型细胞色素已被分离出来（称为 helABCDX 和 ccl1 和 ccl2）。 c 型细胞色素具有共价连接的血红素 脱辅基细胞色素和这些细胞色素以不同的方式存在 真核生物内的区室（即线粒体膜间空间） 和原核生物（即细胞质膜外部）。主要的 仍然存在的问题涉及其组装需要哪些因素 以及这些个体因素本身如何传输到适当的位置 隔间。 具体来说，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来进一步研究假定的血红素 由 helABC 基因编码的转运蛋白（以及 Ccl- 菌株中的缺陷）。 (2) 分离血红素校正 delta-helAB 突变体（以测试血红素转运蛋白 helABC 的函数）。 (3) Hel、Ccl抗血清的制备和使用 研究定位和大分子复合物的蛋白质 互动。 (4) 超量生产、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 分子水平所需的特定蛋白质和事件 c型细胞色素的生物发生。此外，由于血红素是必需的 真核生物和原核生物中有许多不同的过程，其机制是 哪种血红素从其位点跨过双层膜运输 生物合成，线粒体基质或细菌细胞质，是 生物学中的基本问题。完成提出的具体目标 这里将回答有关细胞内血红素靶向的问题， 细胞内特定的氧化还原反应和细胞色素 c 生物发生。一个 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。