RUI: Effect of Amino Acid Substitutions on the Production, Localization, Processing, Stability and Function of Subunit II of Cytochrome c Oxidase in Yeast

细胞色素c氧化酶(CcO)是所有真核生物和某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。CcO中的亚基数量是可变的，从细菌中的三到四个到牛酶复合体中的多达13个。Cox1p、Cox2p和Cox3p这三个最大的亚基在真核生物线粒体DNA上编码，与原核生物中发现的三个亚基密切相关。因此，这些线粒体编码的亚基很可能代表真核生物酶的催化重要亚基。Cox2p已被证明携带一个双核铜中心，这是细胞色素c电子进入的初始位置。为了更好地了解Cox2p的功能，已经鉴定或创造了由于COX2基因突变而导致细胞呼吸障碍的酵母菌株。对于在该基因中产生的定点突变，突变菌株将同时具有基因和表型特征。对于每个突变菌株，将鉴定和鉴定大量已恢复呼吸功能的突变菌株，并将确定恢复功能的遗传变化的性质。还将分析带有氨基酸替换的突变菌株，以确定每个突变对Cox2p的定位、稳定性、加工和组装的影响。其中一些突变可能会干扰这些方面中的一个，但不会干扰其他方面。例如，一些突变可能产生一种稳定的蛋白质，该蛋白质与其他COX多肽组装成一个复合体，但该复合体是不活跃的。其他突变可能会影响稳定性或定位到正确的细胞隔间。由于Cox2p氨基酸序列是如此高度保守，推断出的有关该蛋白在酵母中功能的信息应该有助于阐明该蛋白在高等生物中的功能。这样的理解很重要，因为蛋白质对从细菌到灵长类动物等各种生物的基本能量代谢有着深远的影响。除了扩大我们对这一重要蛋白质的了解外，该项目还将包括所有阶段的本科生合作者，包括进行实际实验、起草手稿以及在科学会议上展示他们的结果。在研究过程中向学生提供的实践培训将有助于他们为研究生和专业学校做好准备，并将为他们提供技术和批判性思维技能。

Cytochrome c oxidase (CcO) is an enzyme complex required for respiration in all eukaryotes and some prokaryotes. The enzyme complex is the terminal member of the electron transport chain and is responsible for transferring electrons from cytochrome c to molecular oxygen. The number of subunits in CcO is variable, ranging from three or four in bacteria to as many as 13 in the bovine enzyme complex. The three largest subunits, Cox1p, Cox2p, and Cox3p are encoded on mitochondrial DNA in eukaryotes and are closely related to the three subunits found in prokaryotes. Therefore, it is likely that these mitochondrially encoded subunits represent the catalytically significant subunits of the eukaryotic enzyme. Cox2p has been shown to carry a binuclear copper center, which is the initial site of electron entry from cytochrome c. To better understand the function of Cox2p, yeast strains with defects in cellular respiration due to mutations in the COX2 gene have been identified or created. For the site-directed mutations which have been created in this gene, the mutant strains will be characterized both genotypically and phenotypically. For each mutant strain, a large collection of revertant strains which have recovered respiratory function will be identified and characterized, and the nature of the genetic change which restores function will be determined. The mutant strains which bear amino acid substitutions will also be analyzed to determine the effect of each of the mutations on the localization, stability, processing, and assembly of Cox2p. Some of the mutations may interfere with one of these aspects but not others. For example, some mutations may produce a stable protein, which is assembled with the other Cox polypeptides into a complex, which is however, inactive. Other mutations may affect stability or localization to the correct cellular compartment.Because the Cox2p amino acid sequence is so highly conserved, information deduced about the function of this protein in yeast should help to elucidate the function of the protein in higher organisms. Such an understanding is of importance because of the profound effect of the protein on the basic energy metabolism of a broad range of organisms, from bacteria to primates. In addition to expanding our knowledge of this important protein, the project will involve undergraduate collaborators at all stages, including those of carrying out the actual experiments, drafting manuscripts, and presenting their results at scientific conferences. The hands-on training provided to students in the course of the research will help prepare them for graduate and professional school and will provide them with both technical and critical thinking skills.