Sla1p Is a Functionally Modular Component of the Yeast Cortical Actin Cytoskeleton Required for Correct Localization of Both Rho1p-GTPase and Sla2p, a Protein with Talin Homology

Abstract

SLA1 was identified previously in budding yeast in a genetic screen for mutations that caused a requirement for the actin-binding protein Abp1p and was shown to be required for normal cortical actin patch structure and organization. Here, we show that Sla1p, like Abp1p, localizes to cortical actin patches. Furthermore, Sla1p is required for the correct localization of Sla2p, an actin-binding protein with homology to talin implicated in endocytosis, and the Rho1p-GTPase, which is associated with the cell wall biosynthesis enzyme ␤-1,3-glucan synthase. Mislocalization of Rho1p in sla1 null cells is consistent with our observation that these cells possess aberrantly thick cell walls.Expression of mutant forms of Sla1p in which specific domains were deleted showed that the phenotypes associated with the full deletion are functionally separable. In particular, a region of Sla1p encompassing the third SH3 domain is important for growth at high temperatures, for the organization of cortical actin patches, and for nucleated actin assembly in a permeabilized yeast cell assay. The apparent redundancy between Sla1p and Abp1p resides in the C-terminal repeat region of Sla1p. A homologue of SLA1 was identified in Schizosaccharomyces pombe. Despite relatively low overall sequence homology, this gene was able to rescue the temperature sensitivity associated with a deletion of SLA1 in Saccharomyces cerevisiae.

The Deubiquitinating Enzyme Doa4p Protects Cells from DNA Topoisomerase I Poisons

“…6A), enlarged and fragmented vacuoles are evident in ϳ30% of the doa4-10 cells grown at 36°C. As reported for sla1⌬ cells (44,46,59), thick cell walls were also evident in sla1-10 mother cells but not in the bud. Significant cell lysis was apparent, with discontinuities in cell wall structure visible at the mother-bud juncture (Fig.…”

“…However, the ts synthetic lethality of doa4 mutants in combination with sla1 or sla2 mutants in the absence of DNA damage was surprising. Morphological inspection of the single and double mutants indicated that the defects in cell wall structure, reported for sla1 mutants (44,46,59), was exacerbated by the loss of Doa4p DUB activity. The result was a pronounced increase in cell lysis, particularly at the largebudded stage of the cell cycle.…”

“…Doa4p also functions in the endocytic pathway to remove ubiquitin from plasma membrane proteins targeted for vacuolar degradation (43). Sla1p and Sla2p function in the assembly of cortical actin patches and the actin cytoskeleton (44,45) and in endocytosis (46).…”

Characterization of the Yeast Amphiphysins Rvs161p and Rvs167p Reveals Roles for the Rvs Heterodimer In Vivo

“…In mutants with defects in endocytosis, cell wall synthesis may go unchecked and recycling of plasma membrane will not occur. Many mutants with defects in endocytosis (e.g., sla2⌬ [Mulholland et al, 1997], sla1⌬ [Ayscough et al, 1999], pan1-4 and end3⌬ [Tang et al, 2000], and myo3⌬ myo5⌬ [Goodson et al, 1996]) have defects in cell wall assembly, often having thickened cell walls.…”

“…In mutants with defects in endocytosis, cell wall synthesis may go unchecked and recycling of plasma membrane will not occur. Many mutants with defects in endocytosis (e.g., sla2⌬ [Mulholland et al, 1997], sla1⌬ [Ayscough et al, 1999], pan1-4 and end3⌬ [Tang et al, 2000], and myo3⌬ myo5⌬ [Goodson et al, 1996]) have defects in cell wall assembly, often having thickened cell walls.A related category of genes that have synthetic genetic interactions with RVS167 and RVS161 contains genes encoding components of the Sin3p/Rpd3p histone deacetylase complex. PHO23 (Loewith et al, 2001, Gavin et al, 2002, SDS3 (Lechner et al, 2000;Gavin et al, 2002), SAP30 (Zhang et al, 1998;Gavin et al, 2002), DEP1 (Lamping et al, 1994;Gavin et al, 2002), and RXT2 (Gavin et al, 2002) encode proteins associated with the Sin3p-Rpd3p histone deacetylase complex (Kasten et al, 1997).…”