GCS1, an Arf guanosine triphosphatase-activating protein in Saccharomyces cerevisiae, is required for normal actin cytoskeletal organization in vivo and stimulates actin polymerization in vitro

Recent cloning of a rat brain phosphatidylinositol 3,4,5-trisphosphate bind ing protein, centaurin alpha, identified a novel gene family based on homol ogy to an amino-terminal zinc-binding domain, in Saccharomyces cerevisiae, the protein with the highest homology to centaurin alpha is Gcs1p, the prod uct of the GCS1 gene. GCS1 was originally identified as a gene conditionall y required for the reentry of cells into the cell cycle after stationary ph ase growth. Gcs1p was previously characterized as a guanosine triphosphatas e-activating protein for the small guanosine triphosphatase Arf1, and gcs1 mutants displayed vesicle-trafficking defects. Here, we have shown that sim ilar to centaurin alpha, recombinant Gcs1p bound phosphoinositide-based aff inity resins with high affinity and specificity. A novel GCS1 disruption st rain (gcs1 Delta) exhibited morphological defects, as well as mislocalizati on of cortical actin patches. gcs1 Delta was hypersensitive to the actin mo nomer-sequestering drug, latrunculin-B. Synthetic lethality was observed be tween null alleles of GCS1 and SLA2, the gene encoding a protein involved i n stabilization of the actin cytoskeleton. In addition, synthetic growth de fects were observed between null alleles of GCS1 and SAC6, the gene encodin g the yeast fimbrin homologue. Recombinant Gcs1p bound to actin filaments, stimulated actin polymerization, and inhibited actin depolymerization in vi tro. These data provide in vivo and in vitro evidence that Gcs1p interacts directly with the actin cytoskeleton in S. cerevisiae.