Yeast actin with a subdomain 4 mutation (A204C) exhibits increased pointed-end critical concentration.
Characterizing mutants of actin that do not polymerize will advance our understanding of the mechanism of actin polymerization and will be invaluable for the production of short F-actin structures for structural studies. To circumvent the problem of expressing dominant lethal nonpolymerizing actin in yeast, we adopted a cysteine engineering strategy. Here we report the characterization of a mutant of yeast actin, AC-actin, possessing a single pointed-end mutation, A204C. Expression of this mutant in yeast results in actin-polymerization-deficient phenotypes. When copolymerized with wild-type actin, ATP-AC-actin is incorporated into filaments. ADP-AC-actin participates in the nucleation and elongation of wild-type filaments only at very high concentrations. At low concentrations, ADP-AC-actin appears to participate only in the nucleation of wild-type filaments, suggesting that Ala-204 is involved in modulating the critical concentration of the pointed end of actin.
Teal
David J
DJ
Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON N1G 2W1, Canada.
Dawson
John F
JF
D000199:Actins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000378:metabolism; D001704:Biopolymers / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D000199:Actins; D001704:Biopolymers; D029701:Saccharomyces cerevisiae Proteins; D000244:Adenosine Diphosphate; D003545:Cysteine