ACTIN NH2-TERMINAL PROCESSING AND ITS SIGNIFICANCE

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Actin, a major contractile and structural protein in both muscle and nonmuscle cells, is processed at its NH2-terminus in a series of novel reactions not used for other proteins: an acetyl-methionine or acetyl-cysteine residue is removed from a completed polypeptide chain following completion of translation, and the new NH2-terminal amino acid is acetylated to make the mature form of actin. The significance of this unique processing event is not understood. In the proposed research, we will determine the requirement for the involvement of acetylation in removing NH2-terminal methionine and cysteine residues from newly synthesized class II actins in reticulocyte lysate preparations. By pulse-labeling mouse L-cells with 35S-methionine, we will determine whether or not the actin must be processed before it can enter the cell's cytoskeletal structures. By pulse-labeling BC3H1 smooth muscle cells with 35S-cysteine, we will determine how class I and class II actins are processed and deposited into cytoskeletal structures in the same cell at the same time. We will establish whether organisms which make only class I actins can faithfully process class II actins and visa versa. We will purify and characterize the class I actin acetylmethionine cleaving enzyme(s) from rat liver, make antibodies to it and use these to determine whether, in mammalian cells, separate enzymes exist for processing class I and class II actins, and how closely these enzymes are related to similar enzymes in lower eukaryotes. Finally, myosin is known to bind to actin at or near the actin NH2-terminus. We will make unprocessed actins in a cell-free translation system and use these to determine the effect of actin amino-terminal processing on the ability of actin to bind to the myosin S1 fragment. These studies will help us to understand the role of actin processing in cytoskeletal assembly and in the ability of actin to work with myosin to generate contractile force, two processes required for proper cell function and organismic viability.