Mutations that enhance the cap2 null mutant phenotype in Saccharomyces cerevisiae affect the actin cytoskeleton, morphogenesis and pattern of growth.

Abstract

Mutations conferring synthetic lethality in combination with null mutations in CAP2, the gene encoding the beta subunit of capping protein of Saccharomyces cerevisiae, were obtained in a colony color assay. Monogenic inheritance was found for four mutations, which were attributed to three genetic loci. One mutation, sac6-69, is in the gene encoding fimbrin, another actin-binding protein, which was expected because null mutations in SAC6 and CAP2 are known to be synthetic-lethal. The other two loci were designated slc for synthetic lethality with cap2. These loci include the mutations slc1-66, slc1-87 and slc2-107. The slc mutations are semi-dominant, as shown by incomplete complementation in slc/SLC cap2/cap2 heterozygotes. The slc mutations and sac6-69 interact with each other, as shown by enhanced phenotypes in diheterozygotes. Moreover, the haploid slc2-107 sac6-69 double mutant is inviable. In a CAP2 background, the slc mutations lead to temperature and osmotic sensitivity. They alter the distribution of the actin cytoskeleton, including deficits in the presence of actin cables and the polarization of cortical actin patches. The slc mutations also lead to a pseudomycelial growth pattern. Together these results suggest that slc1 and slc2 encode components of the actin cytoskeleton in yeast and that the actin cytoskeleton can regulate the patterns of growth.

Actin filaments in yeast are unstable in the absence of capping protein or fimbrin.

“…Previously, CP and fimbrin mutations were found to be synthetic lethal (1,17). This enhanced phenotype is specific for this combination; combinations with many other actin-binding protein mutations did not show enhancement (1), and a screen for mutations synthetic lethal with a CP null mutation did not uncover other actin-binding protein genes (17 and herein).…”

“…A synthetic lethal screen with cap2A yielded two alleles, slcl-66 and slcl-87 (17), of a gene that we now identify as actl. Strain YJC 963 carrying slcl-87 and cap2A and a plasmid with CAP2 was transformed with a cDNA library under control of the GALl promoter (23), kindly provided by Anthony Bretscher (Cornell University, Ithaca, NY) and screened for rescue of synthetic lethality by colony color sectoring.…”

“…Several different genomic libraries produced no clones that rescued, and ACT1 with its own promoter on a CEN plasmid provided only partial rescue for one allele. Both alleles were previously observed as semidominant, with slcl-87 more so than slcl-66 (17). Therefore, the levels of the rescuing wt Actlp presumably needed to be in excess of those of the mutant Actlp.…”

“…Two-dimensional gel electrophoresis and immunoblotting were per-formed as described (33), using goat anti-yeast actin antibodies (17). Actin was purified as described (18), except that a MonoQ column was substituted for the DEAE column.…”

“…To determine total actin, cells were completely disrupted with glass beads for SDS-PAGE. Actin was measured by a quantitative immunoblot using either affinity-purified goat antiyeast actin (17) or the mouse antiactin mAb C4 (20) as the first Ab and 125I-labeled secondary Abs as described (15). Five aliquots of cells were assayed for each strain, with three serial twofold dilutions.…”