Saccharomyces cerevisiae Afr1 Protein Is a Protein Phosphatase 1/Glc7-Targeting Subunit That Regulates the Septin Cytoskeleton during Mating

Abstract

Glc7, the type1 serine/threonine phosphatase in the yeast Saccharomyces cerevisiae, is targeted by auxiliary subunits to numerous locations in the cell, where it regulates a range of physiological pathways. We show here that the accumulation of Glc7 at mating projections requires Afr1, a protein required for the formation of normal projections. AFR1-null mutants fail to target Glc7 to projections, and an Afr1 variant specifically defective in binding to Glc7 [Afr1(V546A F548A)] forms aberrant projections. The septin filaments in mating projections of AFR1 mutants initiate normally but then rearrange asymmetrically as the projection develops, suggesting that the Afr1-Glc7 holoenzyme may regulate the maintenance of septin complexes during mating. These results demonstrate a previously unknown role for Afr1 in targeting Glc7 to mating projections and in regulating the septin architecture during mating.How cells make appropriate morphological changes in response to external and internal cues is a central question in biology. The budding yeast Saccharomyces cerevisiae has been used extensively as a model to investigate the regulation of morphological changes. In dividing cells, growth is largely restricted to the bud, the site of which is determined by a complex set of internal cues that depend on cell type and the position of the previous bud (9). Once the site of polarization is established, a complex of septin proteins forms a ring at the polarization site through which new growth and cell wall expansion are directed. In mating competent cells, mating pheromones induce growth into mating projections rather than into buds. The site of polarization and the overall shape of the projection are determined both by internal cues and by a concentration gradient of pheromone (9). This program of directional growth allows cells of opposite mating types to fuse efficiently in order to form a zygote, which initiates the diploid vegetative cycle.Septins, a collection of related structural proteins, play a central role in morphological regulation during both vegetative growth and the mating response. They are evolutionarily conserved and have physiological roles in cytokinesis, vesicle trafficking, and the secretory pathway in many eukaryotes (15,30,57). There are seven septin proteins in Saccharomyces cerevisiae, of which five, Cdc3, Cdc10, Cdc11, Cdc12, and Shs1, are expressed in vegetatively growing cells (42). Four of these, Cdc3, Cdc10, Cdc11, and Cdc12, form stoichiometric complexes in vivo and in vitro (22,59,60). Septins form a ring at the site of polarization prior to bud formation. The ring transforms into an hourglass-shaped collar at bud initiation and separates into two rings prior to cytokinesis (reviewed in reference 42). This collar acts both as a diffusion barrier to separate membrane-associated proteins in the mother cell from those in the bud (3, 16, 56) and as a scaffold for proteins that direct chitin synthesis, actin nucleation, cell cycle control, and other signaling pathways (reviewed in reference 27). T...

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