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 h ydrophobic plug believed to be involved in a hydrophobic cross-strand "plug -pocket" interaction necessary for actin filament stability. This actin wil l not polymerize in vitro but is compatible with cell viability. We have as sessed the ability of Sac6p, the yeast homologue of the actin filament stab ilizing and bundling protein fimbrin, to restore polymerization in vitro an d to facilitate GG-actin function in vivo. Sac6p rescues GG-actin polymeriz ation at 25 degrees C but not at 4 degrees C. The actin polymerizes into bu ndles 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 hapl oid Delta sac6/GG-actin cells from either diploid or haploid cells was unsu ccessful. The facile isolation of cells with either mutation alone indicate s a synthetic lethal relationship between this actin allele and the SAC6 ge ne. Sac6p may allow GG-actin function in vivo by stabilizing the actin in b undles thereby helping maintain sufficient levels of an otherwise destabili zed actin monomer within the cell.