In vitro analysis of the interaction between yeast fimbrin and actin: A model for a mechanism of allele-specific suppression

Abstract

The actin cytoskeleton in yeast plays a role in many morphological events such as cell growth, secretion, polarity, bud emergence, and endocytosis. Some proteins have been identified that regulate the elongation, nucleation, and stability of the actin cytoskeleton. One of the many proteins controlling the state of the actin cytoskeleton is an actin bundling protein encoded by the gene SAC6 (suppressor of actin). In this study, I have examined some of the phenotypic consequences of overexpression of Sac6p and analyzed interactions between Sac6p and actin in vitro. I have investigated the molecular basis of allele-specific suppression observed between combination of act1 mutations and sac6 mutations by analyzing in vitro the interaction between wild-type and mutant actin and wild-type and mutant Sac6p. The biochemical assays show in vitro suppression of binding and crosslinking activity between Sac6 suppressor protein and mutant actin protein. In addition, the Sac6 suppressor proteins tested have an increased affinity to wild-type actin. This analysis, as well as previous genetic data, is consistent with the idea that suppression results from an overall increase in affinity to actin rather than a strict "lock and key" mechanism previously hypothesized. Overexpression of Sac6p under the control of a galactose inducible promoter results in growth inhibition, accumulation of multinucleated cells, and altered actin cytoskeletal organization. To better understand why overexpression of Sac6p has detrimental effects, I devised a screen to isolate genomic mutations that can suppress the growth defects resulting from elevated levels of Sac6p. I have found that an act1-3 strain is able to suppress Sac6p overexpression and one of the mutants isolated in the screen is also an act1 mutant allele. In addition, the temperature sensitive and osmotic sensitive phenotypes are not complemented by an act1-3 strain. These results suggest that the lethality is mediated through the interaction of Sac6p with actin. In addition, this analysis shows that the proper stoichiometry of Sac6p is critical for proper function of actin in vivo.