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.
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