Interaction in vivo and in vitro between the yeast fimbrin, SAC6P, and a polymerization-defective yeast actin (V266G and L267G).
A mutant yeast actin (GG) has decreased hydrophobicity in a subdomain 3/4 hydrophobic plug believed to be involved in a hydrophobic cross-strand "plug-pocket" interaction necessary for actin filament stability. This actin will not polymerize in vitro but is compatible with cell viability. We have assessed the ability of Sac6p, the yeast homologue of the actin filament stabilizing and bundling protein fimbrin, to restore polymerization in vitro and to facilitate GG-actin function in vivo. Sac6p rescues GG-actin polymerization at 25 degrees C but not at 4 degrees C. The actin polymerizes into bundles at room temperature with a fimbrin:actin molar ratio of 1:4. At this ratio, every actin monomer contacts a Sac6p actin binding domain. Following cold-induced depolymerization, actin/Sac6p mixtures repolymerize beginning at 15 degrees C instead of the 25 degrees C required for de novo assembly, because of the presence of residual actin-Sac6p nuclei. Generation of haploid Deltasac6/GG-actin cells from either diploid or haploid cells was unsuccessful. The facile isolation of cells with either mutation alone indicates a synthetic lethal relationship between this actin allele and the SAC6 gene. Sac6p may allow GG-actin function in vivo by stabilizing the actin in bundles thereby helping maintain sufficient levels of an otherwise destabilized actin monomer within the cell.
Cheng
D
D
Department of Biochemistry, University of Iowa College of Medicine, Iowa City, Iowa 52242-1104, USA.
Marner
J
J
Rubenstein
P A
PA
D000199:Actins / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D019943:Amino Acid Substitution; D004171:Diploidy; D005838:Genotype; D006238:Haploidy; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D008840:Microfilament Proteins / Q000737:chemistry / Q000378:metabolism; D008854:Microscopy, Electron; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics
D000199:Actins; D008562:Membrane Glycoproteins; D008840:Microfilament Proteins; C026742:plastin