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某些原核生物呼吸所需的酶复合物。酶复合物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。电子转移与质子泵相结合，随后的质子梯度用于驱动ATP的合成。复合物中亚基的数量是可变的，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，亚基I、II和III在真核生物的线粒体DNA上编码，并且与在一些原核生物中发现的三个亚基同源。因此，这些编码的亚基很可能代表了酶的催化重要亚基。亚基I是电子传输和质子泵送所必需的。亚基II已被证明携带双核铜中心，这是从细胞色素c的电子转移的初始位点。与亚基I和II相反，亚基III的功能还不清楚，并且尚未证明其在电子转移或质子泵送中具有作用。亚基II和III由简单的、未剪接的基因编码，并且所有这三种基因都含有在已研究基因的所有生物体中保守的氨基酸序列区域。 为了更好地了解细胞色素c氧化酶的亚基II和III的功能，已经鉴定了由于这两个线粒体基因突变而导致酶缺陷的酵母菌株。这种缺陷导致丧失进行细胞呼吸的能力。已经鉴定了一些在编码亚基II（COXII）的基因中携带突变和在编码亚基III（COXIII）的基因中携带三个移码突变的菌株，并且已经完成了对其中一些菌株的分析。目前正在对其余菌株进行详细调查。此外，将通过鉴定额外的自发突变体和通过使用定点诱变构建感兴趣的特定突变来扩大这两个基因中携带突变的菌株的收集。一旦鉴定/构建了具有可能阐明亚基功能的突变的菌株，研究将扩展到这些菌株的回复突变体，其中回复突变体已恢复功能。可以获得的回复突变体的类别是恢复野生型序列的真回复突变体、其中不同氨基酸密码子取代最初存在于该位点的氨基酸密码子的假回复突变体、其中多肽链的一个区域中的突变可以补偿另一个区域中的改变的基因内抑制子以及携带基因外抑制子的菌株。在后一组中，可能是核和线粒体的基因产物，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，并可能产生有关氧化酶亚基之间相互作用的信息。 本科生将参与研究和结果分析。 由于亚基的氨基酸序列是如此的高度保守，这些蛋白质在酵母中的功能推断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在产生能量的过程中如何使用氧气是重要的。

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.