REGULATION AND GENE EXPRESSION OF CYTOCHROME C

编码线粒体的两个核基因，Cyc1和CyC7 酵母中的蛋白质iso-1-和iso-2-细胞色素c 酿酒酵母是研究最深入的一种 真核生物的基因-蛋白质系统。Cyc1基因表达的所有步骤， 已经被系统地检查过了，并且已经开发出方法来 突变体的检测和选择，用于确定 细胞色素c在体内，并通过改变Cyc1和CyC7基因 用人工合成的寡核苷酸直接转化酵母。我们计划 开展以下方面的研究：(1)Sut1p RNA降解 (2)细胞色素c的降解，包括apo和holo两种形式； 和(3)蛋白质的氨基末端加工，包括 蛋氨酸氨基肽酶和氨基末端乙酰转移酶。(1) 我们将测试最近发现的Sut1p的工作假设 降解系统负责降解细胞核中的RNA，包括 异常Cyc1-512 mRNAs，正常mRNAs滞留在细胞核内，内含子 从rRNA加工而来的mRNA、tRNA内含子和间隔区序列。其他 表型类似于sut1突变体的突变体将被分离并 特色化的。酶活性和相关的其他成分 与Sut1p将进行研究，使用GST-SUT1融合基因，这两个 杂交系统和通过合成致死性获得的突变体 程序。(2)似乎至少有四条降解途径 两种异细胞色素c的全息形式或载脂蛋白形式，包括 通过依赖泛素的途径降解。我们将调查 降解机制：apo-1，而不是apo-2；holo 具有氨基末端的两亲性结构；不含血红素的载脂蛋白 在hem1菌株中；以及依赖于或 不依赖于细胞色素c1和a.a3。(3)我们会识别和 探讨不同蛋氨酸的作用模式 氨基肽酶和不同的氨基末端乙酰转移酶，一些 这些序列都是根据序列相似性发现的。一个 功能性GST-NAT2融合基因将用于研究可能的 氨基末端乙酰转移酶作用所需的其他成分 在具有蛋氨酸末端的蛋白质子集上。我们会调查的 来自突变的蛋白质，这些突变的基因通常编码 不同的氨基末端乙酰基酶或蛋氨酸氨基肽酶。这个 在二维凝胶上表现出迁移性改变的蛋白质将被识别出来 并用质谱仪进行测序。

The two nuclear genes, CYC1 and CYC7, encoding the mitochondrial proteins iso-1- and iso-2-cytochrome c, respectively, in the yeast Saccharomyces cerevisiae, comprise one of the most thoroughly studied gene-protein systems of eukaryotes. All steps of CYC1 gene expression, have been systematically examined, and methods have been developed for the detection and selection of mutants, for determining the levels of cytochrome c in vivo, and for altering the CYC1 and CYC7 genes by transforming yeast directly with synthetic oligonucleotides. We plan to carryout studies on the following: (1) the Sut1p RNA degradation system; (2) the degradation of cytochrome c, both apo and holo forms; and (3) amino-terminal processing of proteins, including the action of methionine aminopeptidases and amino-terminal acetyltransferases. (1) We will test the working hypothesis that the recently identified Sut1p degradation system is responsible for degrading RNA in nuclei, including abnormal cyc1-512 mRNAs, normal mRNAs retained in nuclei, introns of mRNA, introns of tRNA, and spacer sequences processed from rRNA. Other mutants phenotypically similar to sut1 mutants will be isolated and characterized. The enzymatic activity and other components associated with Sut1p will be investigated, using a GST-SUT1 fused gene, the two hybrid system, and mutants obtained by the synthetic lethality procedure. (2) There appears to be at least four pathways for degrading the holo or apo forms of the two iso-cytochromes c, including degradation by the ubiquitin-dependent pathway. We will investigate the mechanisms by which degradation occurs for: apo-1, but not apo-2; holo having amino-terminal amphipathic structures; apo in the absence of heme in hem1 strains; and altered forms of holo, which are dependent or independent on the cytochromes c1 and a.a3. (3) We will identify and investigate the pattern of action of different methionine aminopeptidases and different amino-terminal acetyltransferases, some of which were uncovered on the basis of sequence similarities. A functional GST-NAT2 fused gene will be used to investigate possible other components required for an amino-terminal acetyltransferase acting on a subset of proteins having methionine termini. We will investigate the proteins from mutants with disrupted genes that normally encode different amino-terminal acetylases or methionine aminopeptidases. The proteins that exhibit altered mobilities on 2-D gels will be identified and sequenced by mass spectrometry.