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。 CcO中亚基的数量是可变的，从细菌中的三个或四个到牛酶复合物中的多达13个。 三个最大的亚基，Cox1p，Cox2p和Cox3p在真核生物的线粒体DNA上编码，与原核生物中发现的三个亚基密切相关。 因此，很可能这些编码的亚基代表了真核酶的催化重要亚基。 Cox2p已被证明携带双核铜中心，这是电子从细胞色素c进入的初始位点。 为了更好地理解Cox2p的功能，已经鉴定或创建了由于COX2基因突变而具有细胞呼吸缺陷的酵母菌株。 对于在该基因中产生的定点突变，将对突变菌株进行基因型和表型表征。 对于每种突变菌株，将鉴定和表征大量恢复呼吸功能的回复突变菌株，并确定恢复功能的遗传变化的性质。 还将分析携带氨基酸取代的突变菌株，以确定每种突变对Cox2p的定位、稳定性、加工和组装的影响。 一些突变可能会干扰其中一个方面，但不会干扰其他方面。 例如，一些突变可以产生稳定的蛋白质，其与其它考克斯多肽组装成复合物，然而，其是无活性的。 由于Cox2p的氨基酸序列是高度保守的，因此推测该蛋白在酵母中的功能有助于阐明该蛋白在高等生物中的功能。 这样的理解是重要的，因为蛋白质对从细菌到灵长类动物的广泛生物体的基本能量代谢的深刻影响。 除了扩大我们对这种重要蛋白质的了解外，该项目还将涉及所有阶段的本科合作者，包括进行实际实验，起草手稿以及在科学会议上展示其结果。 在研究过程中为学生提供的实践培训将帮助他们为研究生和专业学校做好准备，并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.