Studies of Mitochondrial Biogenesis in Yeast

具体而言，将在缺乏细胞色素B（该复合物中发现的唯一线粒体翻译产物）的酵母突变株中研究细胞色素b-c1复合物在线粒体膜中的组装。最近，该实验室报道，缺乏细胞色素B的突变体含有显著减少（50-75%）的两个核心蛋白以及铁硫蛋白，基本上没有亚基VII（Q结合蛋白）。相比之下，细胞色素c1是光谱和免疫检测在所有的突变体研究的数量等于在野生型。随后对这些蛋白质从突变体之一合成和输入线粒体的研究表明，细胞色素B对于核心蛋白I、铁硫蛋白和Q-结合蛋白的正确加工和组装是必需的，但对于细胞色素c1不是必需的。细胞色素B在铁硫蛋白（ISP）的加工和组装中的作用将使用体外转录/翻译系统进行研究，一旦ISP的基因被克隆到合适的质粒中。将比较ISP向野生型和细胞色素b缺陷型线粒体的输入，以及ISP前体形式裂解的线粒体内位点。随后，将修改ISP的前序列以确定ISP的结合和输入所需的氨基酸。所得结果将确立细胞色素B在细胞色素b-c1复合物整体组装中的作用。本项目的长期目标是阐明酵母中控制膜形成的因素。这种生物体是一种方便的实验系统，因为它易于在实验室中操作，并且它的遗传学得到了很好的研究。被研究的膜是线粒体内膜。质子是细胞的能量工厂。内膜的生物合成为我们提供了一个机会，以了解生物功能的基础上产生的结构信息，并了解两个遗传系统，一个在细胞核和其他在细胞核中，如何相互作用，以指导大型生物聚合物的合成。

Specifically, the assembly of the cytochrome b-c1 complex in the mitochondrial membrane will be investigated in mutant strains of yeast lacking cytochrome b, the only mitochondrial translation product found in this complex. Recently, this lab reported that the mutants lacking cytochrome b contain significantly diminished (50-75%) amounts of the two core proteins as well as the iron-sulfur protein and essentially none of subunit VII (the Q binding protein). By contrast, cytochrome c1 was both spectrally and immunologically detectable in all the mutants studied in amounts equal to that in the wild type. Subsequent studies of the synthesis and import of these proteins into mitochondria from one of the mutants suggested that cytochrome b is necessary for the proper processing and assembly of core protein I, the iron- sulfur protein and the Q-binding protein, but not for cytochrome c1, into complex III of the inner mitochondrial membrane. The role of cytochrome b in the processing and assembly of the iron-sulfur protein (ISP) will be investigated using an in vitro transcription/translation system once the gene for the ISP has been cloned in a suitable plasmid. The import of the ISP into both wild-type and cytochrome b-deficient mitochondria will be compared plus the intramitochondrial sites for cleavage of the precursor form of the ISP. Subsequently, the presequence of the ISP will be modified to establish which amino acids are necessary for binding and import of the ISP. The results obtained will establish a role for cytochrome b in the overall assembly of the cytochrome b-c1 complex. %%% The long term goal of this project is elucidate the factors controlling membrane formation in yeast. This organism is a convenient experimental system because of its ease in manipulation in the laboratory and its well studied genetics. The membrane being studied is the inner mitochondrial membrane. The mitochondrion is the energy factory for the cell. The biosynthesis of the inner membrane affords us an opportunity to understand biological function based on the structural information being generated and to understand how two genetic systems, one in the mitochondrion and the other in nucleus, interact to direct the synthesis of large biopolymers.