NH2-terminal processing of Dictyostelium discoideum actin in vitro.

When Dictyostelium discoideum actin is synthesized in a rabbit reticulocyte lysate system, it is made as a 43,000-dalton polypeptide with the NHz-terminal sequence Ac-Met-Asp-Gly . . . even though in vivo the NHz-terminal sequence is Ac-Asp-Gly . . . (Rubenstein, P., Smith, P., Deuchler, J., and Redman, K. (1981) J. Biol. Chem 256,8149-8155). Here we describe the subsequent fate of the NHz-terminal methionine residue of the actin synthesized in vitro. D. discoideum actin was synthesized in a rabbit reticulocyte lysate in the presence of ~-[~~S]methionine. This fully translated and labeled actin was placed in a fresh aliquot of rabbit reticulocyte lysate under conditions where further protein acetylation was inhibited. This treatment resulted in the shift of the isoelectric point of actin to a more basic value characteristic of nonacetylated actin. Experiments using either [3bS]Met-tRNA$e' as a sole source of label or tryptic and thermolytic digests of actin uniformly labeled with [35S]methionine prove that this modification results from the removal of the NHzterminal acetyl and methionyl residues from the polypeptide chain to generate a polypeptide terminating in aspartic acid. Removal of the methionine apparently requires its prior acetylation. Once the aspartic acid residue is exposed at the NHz terminus, the actin can again be acetylated in an acetyl-CoA-dependent reaction to yield a polypeptide probably identical with D. discoideum actin found in vivo. All of these reactions can occur post-translationally following release of the completed polypeptide chain from the ribosome. This series of experiments defines a pathway for actin NH2terminal processing that is different from the path used for other proteins studied so far.