Afr1p has a role in regulating the localization of Mpk1p at the shmoo tip in Saccharomyces cerevisiae

Abstract

Afr1p functions to promote adaptation to pheromoneinduced growth arrest and morphogenesis. We show here that Afr1p regulates polarized localization of the Mpk1p MAP kinase in shmooing cells. Deletion of AFR1 results in mislocalization of Mpk1p although the scaffold protein Spa2p localizes normally at shmoo tip, and overexpression of Spa2 cannot rescue this defect, indicating Afr1p in required for Spa2p to recruit Mpk1 to the site of polarized growth during mating. Overexpression of SPA2 partially suppresses the morphogenetic defect of afr1D cells upon a-factor induction, suggesting the two proteins function in the same genetic pathway with Spa2p acts downstream of Afr1p.

Systematic Definition of Protein Constituents along the Major Polarization Axis Reveals an Adaptive Reuse of the Polarization Machinery in Pheromone-Treated Budding Yeast

“…We therefore undertook a systematic, high-throughput analysis of alpha factor-induced changes in protein localization of GFP-tagged yeast strains. It should be noted that a comprehensive survey of proteins localized to the shmoo tip has not previously been carried out despite many studies − that have implicated key roles for individual proteins in this dynamic cellular compartment. Building on our previous success in constructing and screening yeast cell microarrays, we screened the collection of GFP-tagged yeast strains for pheromone-induced changes in protein localization.…”

Afr1p mediates activation of the Slt2p MAP kinase induced by pheromone, heat shock and hypo-osmotic shock inSaccharomyces cerevisiae

“…However, the protein or proteins that mediate pheromone activation of the cell integrity pathway have not been identified. A recent study demonstrated that Afr1p is required for concentrating Slt2p in the mating projection in response to mating pheromone, although it failed to detect a noticeable change in Slt2p activity in cells lacking Afr1p (Changwei et al ., 2007). Because of the strong correlations between Afr1p and Slt2p with respect to their role in promoting α‐factor resistance, we reinvestigated Slt2p activation in AFR1 deletion and AFR1 overexpression cells by immunoblot analysis using an anti‐p44/42 MAPK antibody that specifically recognizes the doubly phosphorylated form of Slt2p (Giot & Konopka, 1997; Davis et al ., 1998).…”

“…It has been proposed that both septins and Afr1p act together as a barrier at the neck of mating projection to prevent diffusion of pheromone pathway components from the projection tip and restrict the active zone of pheromone signaling to the tip of mating projection (Douglas et al ., 2005). Because Afr1p is also required for localization of Slt2p to the tip of mating projection (Changwei et al ., 2007), where its two upstream functionally redundant MEKs, Mkk1p and Mkk2p, also localize (van Drogen & Peter, 2002), it is applaudable to propose that Afr1p acts as a barrier to restrict, in addition to components of the pheromone signaling pathway, Slt2p to the tip of mating projection for activation by Mkk1p and Mkk2p. Alternatively, Afr1p may function as a scaffold‐like protein or a platform at the base of mating projections to recruit Slt2p to the projection tip and place Slt2p in close proximity to Mkk1p and Mkk2p for activation.…”

“…The methods for construction of bar1 Δ∷ kanMX4 and afr1 Δ∷ LEU2 strains were described previously (Changwei et al ., 2007). The bck1 Δ∷ HIS3 and slt2 Δ∷ HIS3 deletion cassettes were obtained by an EcoRV digestion of plasmid pBCK1D or an EcoRI–SalI digestion of plasmid pSLT2D, respectively.…”

“…Afr1p functions to promote adaptation to pheromone‐induced growth arrest and its constitutive expression in vegetatively growing cells leads to an elongated bud morphology (Konopka, 1993; Davis et al ., 1998; DeMattei et al ., 2000). Afr1p has also been suggested to regulate polarized localization of Slt2p during the mating process (Changwei et al ., 2007). In addition, Afr1p was recently discovered to be required for the accumulation of Glc7p at the mating projection and to regulate the septin cytoskeleton during mating (Bharucha et al ., 2008).…”