Analysis of Bni4: Revealing new roles for Glc7 at the bud neck in Saccharomyces cerevisiae

Many proteins bind asymmetrically to the septin scaffold at the bud neck of Saccharomyces cerevisiae. Yeast chitin synthase III (CSIII) is targeted to the mother side of the bud neck, where it is thought to be tethered by the septin-associated protein Bni4. Bni4 also associates with the yeast protein phosphatase 1 (PP1) catalytic subunit, Glc7. To identify regions of Bni4 necessary for its 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 bni4Delta 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 towards substrates necessary for CSIII recruitment and synthesis of chitin at the bud neck. The asymmetric localization of Bni4 is a robust feature and is retained even in mutants that disrupt septin architecture. However, Bni4 asymmetry is lost under different types of cellular stress, including glucose starvation or activation of cell cycle checkpoints. Our data indicate that loss of Bni4 asymmetry can occur via at least two distinct mechanisms, and may regulate cell cycle checkpoints.