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.
| Lee, Cho-Yin; Lou, Jizhong; Wen, Kuo-Kuang et al. (2016) Regulation of actin catch-slip bonds with a RhoA-formin module. Sci Rep 6:35058 |
| Wen, Kuo-Kuang; McKane, Melissa; Stokasimov, Ema et al. (2011) Mutant profilin suppresses mutant actin-dependent mitochondrial phenotype in Saccharomyces cerevisiae. J Biol Chem 286:41745-57 |
| Stark, Benjamin C; Wen, Kuo-Kuang; Allingham, John S et al. (2011) Functional adaptation between yeast actin and its cognate myosin motors. J Biol Chem 286:30384-92 |
| Kudryashov, Dmitri S; Grintsevich, Elena E; Rubenstein, Peter A et al. (2010) A nucleotide state-sensing region on actin. J Biol Chem 285:25591-601 |
| Wen, Kuo-Kuang; McKane, Melissa; Stokasimov, Ema et al. (2010) A potential yeast actin allosteric conduit dependent on hydrophobic core residues val-76 and trp-79. J Biol Chem 285:21185-94 |
| Scoville, Damon; Stamm, John D; Altenbach, Christian et al. (2009) Effects of binding factors on structural elements in F-actin. Biochemistry 48:370-8 |
| Stokasimov, Ema; Rubenstein, Peter A (2009) Actin isoform-specific conformational differences observed with hydrogen/deuterium exchange and mass spectrometry. J Biol Chem 284:25421-30 |
| Wen, Kuo-Kuang; Rubenstein, Peter A; DeMali, Kris A (2009) Vinculin nucleates actin polymerization and modifies actin filament structure. J Biol Chem 284:30463-73 |
| Stokasimov, Ema; McKane, Melissa; Rubenstein, Peter A (2008) Role of intermonomer ionic bridges in the stabilization of the actin filament. J Biol Chem 283:34844-54 |
| Wen, Kuo-Kuang; McKane, Melissa; Houtman, Jon C D et al. (2008) Control of the ability of profilin to bind and facilitate nucleotide exchange from G-actin. J Biol Chem 283:9444-53 |
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