SPA2P INTERACTS WITH CELL POLARITY PROTEINS AND SIGNALING COMPONENTS INVOLVED IN YEAST-CELL MORPHOGENESIS

The yeast protein Spa2p localizes to growth sites and is important for polarized morphogenesis during budding, mating, and pseudohyphal grow th. To better understand the role of Spa2p in polarized growth, we ana lyzed regions of the protein important for its function and proteins t hat interact with Spa2p. Spa2p interacts with Pea2p and Bud6p (Aip3p) as determined by the two-hybrid system; all of these proteins exhibit similar localization patterns, and spa2 Delta, pea2 Delta, and bud6 De lta mutants display similar phenotypes, suggesting that these three pr oteins are involved in the same biological processes. Coimmunoprecipit ation experiments demonstrate that Spa2p and Pea2p are tightly associa ted with each other in vivo. Velocity sedimentation experiments sugges t that a significant portion of Spa2p, Pea2p, and Bud6p cosediment, ra ising the possibility that these proteins form a large, 12S multiprote in complex. Bud6p has been shown previously to interact with actin, su ggesting that the 12S complex functions to regulate the actin cytoskel eton. Deletion analysis revealed that multiple regions of Spa2p are in volved in its localization to growth sites. One of the regions involve d in Spa2p stability and localization interacts with Pea2p; this regio n contains a conserved domain, SHD-II. Although a portion of Spa2p is sufficient for localization of itself and Pea2p to growth sites, only the full-length protein is capable of complementing spa2 mutant defect s, suggesting that other regions are required for Spa2p function. By u sing the two-hybrid system, Spa2p and Bud6p were also found to interac t with components of two mitogen-activated protein kinase (MAPK) pathw ays important for polarized cell growth. Spa2p interacts with Ste11p ( MAPK kinase [MEK] kinase) and Ste7p (MEK) of the mating signaling path way as well as with the MEKs Mkk1p and Mkk2p of the Slt2p (Mpk1p) MAPK pathway; for both Mkk1p and Ste7p, the Spa2p-interacting region was m apped to the N-terminal putative regulatory domain. Bud6p interacts wi th Ste11p. The MEK-interacting region of Spa2p corresponds to the high ly conserved SHD-I domain, which is shown to be important for mating a nd MAPK signaling. spa2 mutants exhibit reduced levels of pheromone si gnaling and an elevated level of Slt2p kinase activity. We thus propos e that Spa2p, Pea2p, and Bud6p function together, perhaps as a complex , to promote polarized morphogenesis through regulation of the actin c ytoskeleton and signaling pathways.