Synthetic Lethal Analysis Implicates Ste20p, a p21-activated Protein Kinase, in Polarisome Activation

The p21-activated kinases Ste20p and Cla4p carry out undefined functions that are essential for viability during budding in Saccharomyces cerevisiae. To gain insight into the roles of Ste20p, we have used a synthetic lethal mutant screen to identify additional genes that are required in the absence of Cla4p. Altogether, we identified 65 genes, including genes with roles in cell polarity, mitosis, and cell wall maintenance. Herein, we focus on a set that defines a function carried out by Bni1p and several of its interacting proteins. We found that Bni1p and a group of proteins that complex with Bni1p (Bud6p, Spa2p, and Pea2p) are essential in a cla4Δ mutant background. Bni1p, Bud6p, Spa2, and Pea2p are members of a group of polarity determining proteins referred to as the polarisome. Loss of polarisome proteins from a cla4Δ strain causes cells to form elongated buds that have mislocalized septin rings. In contrast, other proteins that interact with or functionally associate with Bni1p and have roles in nuclear migration and cytokinesis, including Num1p and Hof1p, are not essential in the absence of Cla4p. Finally, we have found that Bni1p is phosphorylated in vivo, and a substantial portion of this phosphorylation is dependent on STE20. Together, these results suggest that one function of Ste20p may be to activate the polarisome complex by phosphorylation of Bni1p

Golgi Vesicle Proteins Are Linked to the Assembly of an Actin Complex Defined by mAbp1

Recent studies indicate that regulation of the actin cytoskeleton is important for protein trafficking, but its precise role is unclear. We have characterized the ARF1-dependent assembly of actin on the Golgi apparatus. Actin recruitment involves Cdc42/Rac and requires the activation of the Arp2/3 complex. Although the actin-binding proteins mAbp1 (SH3p7) and drebrin share sequence homology, they are differentially segregated into two distinct ARF-dependent actin complexes. The binding of Cdc42 and mAbp1, which localize to the Golgi apparatus, but not drebrin, is blocked by occupation of the p23 cargo-protein-binding site on coatomer. Exogenously expressed mAbp1 is mislocalized and inhibits Golgi transport in whole cells. The ability of ARF, vesicle-coat proteins, and cargo to direct the assembly of cytoskeletal structures helps explain how only a handful of vesicle types can mediate the numerous trafficking steps in the cell