FUNCTIONAL SIGNIFICANCE OF THE ACTIN N-TERMINAL REGION

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The overall goal of our work is to understand the nature of the interactions controlling the association of actin with myosin and with various proteins that modulate actin's behavior within the cell. We are particularly interested in actin's N-terminal region, the site of an unusual processing reaction involving the posttranslational removal of acetyl amino acids by a specific processing enzyme. This region of the actin also forms close contacts with proteins such as myosin, gelsolin, fragmin, depactin, and troponin I and therefore may be important in controlling cellular actin function. This grant addresses the importance of three conserved elements of the 18 amino acid actin N-terminal region: the acidic amino acids at the N-terminus of mature processed actin; the conserved Ala-Leu-Val beginning at residue 6; and the conserved Asp-Asn- Gly-Ser-Gly beginning at position 10 in what otherwise structurally is a hypervariable part of the actin polypeptide. We will use a yeast transformation system in which a centromeric plasmid carrying a mutated intronless yeast actin gene will be introduced into a diploid strain of S. cerevisiae containing one wild type actin gene and one inactive actin gene. Using site-directed mutagenesis, mutations will be made in one of the three structural elements just described or will be inserted between Met-1 and Asp1 to prevent N-terminal processing. Haploid cells will be tested for the ability to remain viable with the mutated actin alone. The transformed diploid will be tested for dominant effects exerted by the mutant actin on the cell. We will examine affected cells for changes in actin deposition patterns by immunofluorescent staining techniques. We will also examine the effects of the mutations on chitin deposition and invertase secretion, two processes that are dependent on proper actin function. Peptide mapping of the isolated mutant actin will indicate whether alterations affect removal of the Met-i by the processing enzyme. We will isolate the mutant actins in active form and determine the effects of the mutations on actin polymerization, cation and nucleotide binding, and the interaction of actin with myosin and gelsolin. These experiments will provide answers for the role of N-terminal processing and the three conserved structural elements of the N-terminal region both in vivo and in vitro in actin function and will allow us to prepare large quantities of mutant actins for biochemical analysis.