Abstract
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.
Highlights
Our laboratory previously constructed an actin mutant (GG) in which the first two residues in the subdomain 3/4 loop were replaced by glycines [4, 5]
Polymerization can be restored by inclusion of phalloidin or beryllium fluoride (BeFx),1 the effects of these agents on GG-actin polymerization differ in a temperature-dependent fashion
When Sac6p was added at an actin:fimbrin ratio of 10:1, we observed an immediate increase in light scattering suggesting the fimbrininduced formation of actin filaments
Summary
Our laboratory previously constructed an actin mutant (GG) in which the first two residues in the subdomain 3/4 loop were replaced by glycines [4, 5]. The polymerization defect of the GG-mutant, coupled with its compatibility with yeast viability, contradicts the prediction of Holmes and colleagues [2, 3] cited above. This result immediately raises the question of what allows GG actin to function in vivo, since F-actin is required for actin-based processes. Actin function is modulated by a number of actin monomer-binding proteins and filament capping, cross-linking, severing, and bundling proteins [9]. MATa act1::HIS3 sac6::LEU2 leu GG-URA3 his trp rate at 23 °C and cannot grow at 37 °C These null mutant cells do not have normal actin cables and exhibit an abnormal cortical actin distribution. It has been demonstrated that deletion of SAC6 leads to a substantial increase in the G/F actin ratio, presumably because of filament destabilization [20]
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