The catalytic subunit of protein phosphatase type 1 (PP1) has an essential role in mitosis, acting in opposition to the Ipl1/Aurora B protein kinase to ensure proper kinetochore-microtubule interactions. However, the regulatory subunit(s) that completes the PP1 holoenzyme that functions in this capacity is not known. We show here that the budding yeast Ypi1 protein is a nuclear protein that functions with PP1 (Glc7) in this mitotic role. Depletion of cellular Ypi1 induces mitotic arrest due to activation of the spindle checkpoint. Ypi1 depletion is accompanied by a reduction of nuclear PP1 and by loss of nuclear Sds22, a Glc7 binding partner that is found in a ternary complex with Ypi1 and Glc7. Expression of a Ypi1 variant that binds weakly to PP1 also activates the spindle checkpoint and suppresses the temperature sensitivity of an ipl1-2 mutant. These results, together with genetic interactions among YPI1, GLC7, and SDS22 mutants, indicate that Ypi1 and Sds22 are positive regulators of the nuclear Glc7 activity that is required for mitosis.

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Yeast chitin synthase III (CSIII) is targeted to the bud neck, where it is thought to be tethered by the septin-associated protein Bni4. Bni4 also associates with the yeast protein phosphatase (PP1) catalytic subunit, Glc7. To identify regions of Bni4 necessary for its targeting functions, we created a collection of 23 deletion mutants throughout the length of Bni4. Among the deletion variants that retain the ability to associate with the bud neck, only those deficient in Glc7 binding fail to target CSIII to the neck. A chimeric protein composed of the septin Cdc10 and the C-terminal Glc7-binding domain of Bni4 complements the defects of a bni4⌬ mutant, indicating that the C-terminus of Bni4 is necessary and sufficient to target Glc7 and CSIII to the bud neck. A Cdc10-Glc7 chimera fails to target CSIII to the bud neck but is functional in the presence of the C-terminal Glc7-binding domain of Bni4. The conserved C-terminal PP1-binding domain of mammalian Phactr-1 can functionally substitute for the C-terminus of Bni4. These results suggest that the essential role of Bni4 is to target Glc7 to the neck and activate it toward substrates necessary for CSIII recruitment and synthesis of chitin at the bud neck.

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...