METHYLATION OF ATYPICAL PROTEIN ASPARTYL RESIDUES

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A protein methyltransferase with a specificity for structurally altered aspartyl residues is widely distributed in bacteria and eucaryotic cells. The intracellular concentration of the enzyme is similar in extracts from bacteria, amphibian oocytes, human erythrocytes and transformed mammalian cell lines, suggesting that the enzyme is of fundamental importance to cell function. The protein aspartyl residues which serve as the substrates for the enzyme have arisen by spontaneous racemization and deamidation-linked isomerization events, and it has been proposed that the methyltransferase may function in the correction or metabolism of these damaged sequences. We will be investigating specific functional roles for the methyltransferase using Xenopus laevis oocytes. In these experiments radiolabeled protein or peptides containing altered aspartyl residues will be injected into oocytes and the methylation-dependent processing of these substrates will be monitored using HPLC. Individual metabolites will be analyzed to determine the nature of the structural changes initiated by the methyl transfer reactions. We will also analyze the impact of protein methylation reactions on the rates of aspartyl residue racemization and asparaginyl residue deamidation in oocyte proteins. The presence of high concentrations of the methyltransferase in all cell types studied would seem to imply that racemization and deamidation events occur more frequently than previously anticipated. In these experiments Xenopus proteins will be pulse-labeled with either L-[C14] aspartic acid or L[14C] asparagine, and the production of derivatized aspartyl residues will be measured in the absence and presence of methyltransferase inhibitors. These rates will be compared to the rate of protein carboxyl methylation measured in parallel groups of oocytes microinjected with S-adenosyl-L-[methyl-H-3] methionine. This comparison will provide a measure of the efficiency of methylation-dependent processing reactions. Finally, we will undertake a thorough characterization of the endogenous substrates for the oocyte methyltransferase. Particular attention will be paid to the prevalent proteins which are stored for extended periods of time during oogenesis. These proteins include the ribosomal, cytoskeletal, karyoplasmic and mitochondrial proteins. In the course of these studies, we may also uncover other kinds of regulatory methyltransferases or methyltransferases which are restricted to intracellular organelles.