We propose to carryout studies of N-terminal acetylation, mRNA degradation, and protein degradation in Saccharomyces cerevisiae. Although conceptually unrelated, these proposed studies stem from investigations with the iso-cytochromes c system, which constitutes one of the most thoroughly studied gene-protein systems of eukaryotes. Yeast contains three N-terminal acetyltransferases (NATs), designated NatA, NatB and NatC, with each having a different catalytic subunit, Ardip, Nat3p and Mak3p, respectively, and each acetylating different sets of proteins with different N-terminal regions. We propose to identify and characterize all of the subunits of NatB and NatA that co-purify with the catalytic subunits, similar to our work on NatC. The N-terminal acetylation patterns will be determined with mutants deleted in other putative NATs. Biological functions in vivo of acetylation of actin, a NatB substrate, will be deduced from the phenotypes of certain act] mutants. Attempt will be made to characterize mammalian orthologues of the yeast NatA. We will determine if NAT components associate with polysomes and which subunits are responsible for the association. We will test the hypothesis that mRNAs retained in the nucleus are degraded (the DRN pathway), that nuclear mRNA is delivered to the site of degradation by the nuclear cap binding complex containing Cbclp, and that Rrp6p carries out this degradation. DNA microarray technology will be used to identify wild-type mRNAs that are particularly susceptible DRN. Components of the DRN pathway will be identified by suppression of cyc1l-512, which produces abnormally long mRNAs that are highly susceptible to degradation by this pathway. Further studies will carried out with novel protein degradation systems uncovered with the isocytochromes c system. Included are those in which mutationally altered holo-iso-1 having T78I and other replacements are degraded (the RDD pathway). Mutationally altered apo-iso-1 having N-terminal amphipathic structures are degraded independent of ubiquitin system (the MDD pathway); and apo-iso-1 is degraded in the absence of heme and independent of ubiquitin system (the HDD pathway). The latter study would provide the first evidence that apo-cytochrome c and heme interact in vivo in the absence of heme lyase.
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