COTRANSLATIONAL PROCESSING AND PROTEIN TURNOVER

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The co-translational processing of eukaryotic proteins generally produces four main classes of proteins: those with and without initiator Met and those with and without N-alpha-acetylation. Methionine aminopeptidase (MAP) and N-alpha-acetyltransferase (NAT), enzymes associated with the ribosomes, apparently affect these modifications. Importantly, the structures generated apparently dictate the long-term stability of many intracellular proteins in eukaryotes and can direct the turnover of these proteins via the ubiquitin-based degradation system. Two main lines of experimentation are proposed to further clarify these relationships. In the first, porcine liver NAT and MAP, which act co-translationally, and protein N- terminal, asparagine deamidase, which acts post-translationally, will be examined with respect to structure, properties and specificity. Full-length cDNA sequences will be obtained as will precipitating antibodies directed against each of the 3 enzymes. The combined effect of these agents, either in sub-groups or en bloc, is to provide (presumably well regulated) access of selected intracellular proteins to the degradation machinery. The second part of the proposal deals with the function of these enzymes in protein degradation using rabbit reticulocyte lysate. Lysate will be used to express transcripts (both native and altered) to generate appropriate proteins and to induce degradation by the ubiquitin-dependent pathway. Participants in the modification and turnover pathways will be manipulated (or neutralized) with antisera and/or inhibitors, to determine their role in the process. In the whole cell experiments, transcripts will be generated in situ from appropriately tailored plasmids and the degradation of the resulting proteins monitored. Two proteins (asparagine synthetase and hypoxanthine phosphoribosyl transferase) will be used as the principal substrates in these studies. Appropriate nucleic acid and immunological reagents are either in-hand or will be generated.