The general objective of this program is to understand protein methylation from three perspectives: (i) The role of methylation in the dynamics of protein biosynthesis and intracellular processing and trafficking, (ii) the physico-chemical and functional modulation of protein by enzyme-catalyzed methylation, and (iii) a study of the protein methyltransferases themselves, looking specifically at substrate-binding and/or active sites. In order to achieve the above, the following specific research objectives must be met. (1) Elucidation of possible roles for methylation in the dynamics of cytochrome c biosynthesis (elongation, termination, proteolytic resistance of the nascent protein), utilizing in vitro translation systems containing exogenously added mRNA which is transcribed in vitro from plasmid containing yeast iso-l-cytochrome c gene and purified protein-lysine N-methyltransferase. (2) Utilizing in vitro systems containing isolated mitochondria, examination of possible roles for methylation in the posttranslational uptake of cytochrome c apoprotein into the mitochondria and the subsequent attachment of the heme prosthetic group. (3) Utilizing physical methods, characterization of the precise physico-chemical effects exerted by methylation on cytochrome c, and characterization of the effects of methylation on the molecular interaction of cytochrome c with cytochrome c oxidase, reductase, and peroxidase. (4) Identification and subsequent purification of two enzymes implicated to exist from observations during the past grant period. (a) An enzyme which dealkylates methylated cytochrome c in vivo. (b) An enzyme which methylates the Res-86 lysine of cytochrome c. (5) Identification and characterization of the protein structure contributing to the active sites of highly purifiedprotein methyltransferase: Cytochrome c-specific protein-lysine N- methyltransferase from Neurospora crassa and protein-carboxyl O- methyltransferase from calf brain by photoaffinity labeling with analogues of S-adenosyl-L-methionine, and subsequent analysis of labeled peptides generated by enzymatic degradation of the modified methyltransferases.
