Protein Phosphatase Type 1 Directs Chitin Synthesis at the Bud Neck in Saccharomyces cerevisiae

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 bni4Δ 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

Ypi1, a Positive Regulator of Nuclear Protein Phosphatase Type 1 Activity in Saccharomyces cerevisiae

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