Profilin plays an important role in actin organization in all eukaryotic cells through mechanisms that are still poorly understood. We had previously shown that Mid2p, a transmembrane protein and a potential cell wall sensor, is an effective multicopy suppressor of the profilin-deficient phenotype in Saccharomyces cerevisiae. To better understand the role of Mid2p in the organization of the actin cytoskeleton, we isolated five additional multicopy suppressors of pfy1Δ cells that are Rom1p, Rom2p, Rho2p, Smy1p, and the previously uncharacterized protein Syp1p. The problems of caffeine and NaCl sensitivity, growth defects at 30° and 37°, the accumulation of intracellular vesicular structures, and a random budding pattern in pfy1Δ cells are corrected by all the suppressors tested. This is accompanied by a partial repolarization of the cortical actin patches without the formation of visible actin cables. The overexpression of Mid2p, Rom2p, and Syp1p, but not the overexpression of Rho2p and Smy1p, results in an abnormally thick cell wall in wild-type and pfy1Δ cells. Since none of the suppressors, except Rho2p, can correct the phenotype of the pfy1-111/rho2Δ strain, we propose a model in which the suppressors act through the Rho2p signaling pathway to repolarize cortical actin patches.
Profilin is an actin monomer-binding protein implicated in the polymerization of actin filaments. In the budding yeast Saccharomyces cerevisiae, the pfy1-111 rho2Δ double mutant has severe growth and actin cytoskeletal defects. The GEA1 and GEA2 genes, which code for paralog guanosine exchange factors for Arf proteins, were identified as multicopy suppressors of the mutant phenotype. These two genes restored the polarized distribution of actin cortical patches and produced visible actin cables in both the pfy1-111 rho2Δ and pfy1Δ cells. Thus, overexpression of GEA1 or GEA2 bypassed the requirement for profilin in actin cable formation. In addition, gea1 gea2 double mutants showed defects in budding and in actin cytoskeleton organization, while overexpression of GEA1 or GEA2 led to the formation of supernumerary actin cable-like structures in a Bni1p/Bnr1p-dependent manner. The ADP-ribosylation factor Arf3p may be a target of Gea1p/Gea2p, since overexpression of ARF3 partially suppressed the profilin-deficient phenotype and a deletion of ARF3 exacerbated the phenotype of a pfy1-111 mutant. Gea1p, Gea2p, Arf1p, and Arf2p but not Arf3p are known to function in vesicular transport between the endoplasmic reticulum and the Golgi. In this work, we demonstrate a role for Gea1p, Gea2p, and Arf3p in the organization of the actin cytoskeleton.
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