RUI: Genetic Analysis of the Function of Cytochrome Oxidase Subunits Encoded on Mitochondrial DNA in Yeast

卡梅隆9727738细胞色素c氧化酶是所有真核生物和一些原核生物呼吸所必需的酶复合体。酶复合体是电子传递链的末端成员，负责将电子从细胞色素c转移到分子氧。电子转移耦合到质子泵浦，随后的质子梯度被用来驱动ATP的合成。该复合体中的亚基数量是可变的，从细菌中的三到四个到牛酶中的多达13个。三个最大的亚基I、II和III编码在真核生物的线粒体DNA上，并与一些原核生物中的三个亚基同源。因此，这些线粒体编码的亚基很可能代表酶的催化重要亚基。亚基I对于电子传输和质子泵浦都是必需的。亚基II被证明携带一个双核铜中心，这是细胞色素c电子转移的初始位置。与亚基I和亚基II相比，亚基III的功能还不是很清楚，也没有证明它在电子转移或质子泵中起作用。亚基II和III由简单的非剪接基因编码，这三个基因都含有氨基酸序列的区域，这些区域在所有研究过这些基因的生物体中都是保守的。为了更好地了解细胞色素C氧化酶II和III亚基的功能，已鉴定出由于这两个线粒体基因突变而导致该酶缺陷的酵母菌株。这些缺陷会导致丧失进行细胞呼吸的能力。已鉴定了一些编码亚基II(COXII)基因突变和编码亚基III(COXIII)基因移码突变的菌株，并对其中一些菌株进行了分析。其余的菌株正在进行详细的调查。此外，通过鉴定更多的自发突变和利用定点突变构建感兴趣的特定突变，将扩大携带这两个基因突变的菌株的集合。一旦鉴定/构建了具有可能揭示亚单位功能的突变的菌株，研究将扩展到这些菌株的回复菌株，在这些回复菌株中，回复菌株已经恢复了功能。可获得的逆转株包括恢复野生型序列的真逆转株、用不同的氨基酸密码子取代原来位于该位点的氨基酸密码子的假逆转株、多肽链一个区域的突变可以补偿另一个区域的改变的基因内抑制株，以及携带基因外抑制物的菌株。在后一类中，可能是基因产物，包括核和线粒体，它们直接与这两个亚基相互作用或参与它们的表达。这种分析将产生有关蛋白质中功能关键氨基酸的信息，可能还会产生关于氧化物酶亚基之间相互作用的信息。本科生将参与结果的研究和分析。由于亚基氨基酸序列高度保守，推测酵母中这些蛋白质功能的信息应该有助于阐明这些蛋白质在高等生物中的功能。这样的理解对于理解生物体如何在产生能量的过程中利用氧气具有重要意义。

Cameron 9727738 Cytochrome c oxidase is an enzyme complex required for respiration in all eucaryotes and some procaryotes. The enzyme complex is the terminal member of the electron transport chain, responsible for transferring electrons from cytochrome c to molecular oxygen. Electron transfer is coupled to proton pumping, and the ensuing proton gradient is used to drive the synthesis of ATP. The number of subunits in the complex is variable, ranging from three or four in bacteria to as many as 13 in the bovine enzyme. The three largest subunits, subunit I, II and III are encoded on mitochondrial DNA in eucaryotes and are homologous to the three subunits found in some procaryotes. Therefore, it is likely that these mitochondrially encoded subunits represent the catalytically significant subunits of the enzyme. Subunit I is required for both electron transport and for proton pumping. Subunit II has been shown to carry a binuclear copper center, which is the initial site of electron transfer from cytochrome c. In contrast to subunits I and II, the function of subunit III is not well understood, and it has not been demonstrated to have a role in either electron transfer or proton pumping. Subunits II and III are encoded by simple, unspliced genes and all three of these genes contain regions of amino acid sequence which are conserved in all organisms in which the genes have been studied. To better understand the function of subunits II and III of cytochrome c oxidase, yeast strains with defects in the enzyme due to mutations in these two mitochondrial genes have been identified. Such defects lead to loss of the ability to carry out cellular respiration. A number of strains bearing mutations in the gene encoding subunit II ( COXII ) and three frameshift mutations in the gene encoding subunit III( COXIII ) have been identified and analysis of some of these strains has been completed. The remaining strains are being investigated in detail. In addition, the collection of strains bearing mutations in t hese two genes will be enlarged by identification of additional spontaneous mutants and by construction of specific mutations of interest using site-directed mutagenesis. Once strains with mutations that have the potential to shed light on the function of the subunits have been identified/constructed, the investigation will be extended to revertants of these strains, where the revertants have recovered function. Classes of revertants that may be obtained are true revertants that restore the wild type sequence, pseudo-revertants where a different amino acid codon is substituted for the one which originally resided at that site, intragenic suppressors where a mutation in one region of a polypeptide chain can compensate for an alteration in another region, and strains carrying extragenic suppressors. Among the latter group may be gene products, both nuclear and mitochondrial, that interact directly with these two subunits or are involved in their expression. This analysis will yield information concerning functionally critical amino acids in the proteins and possibly about interactions between oxidase subunits. Undergraduate students will be involved in both the research and analysis of the results. Because the subunit amino acid sequences are so highly conserved, information deduced about the function of these proteins in yeast should help to elucidate the function of these proteins in higher organisms. Such an understanding is of importance to understanding how organisms use oxygen in the process of generating energy.