Spa2p Interacts with Cell Polarity Proteins and Signaling Components Involved in Yeast Cell Morphogenesis

Abstract

The yeast protein Spa2p localizes to growth sites and is important for polarized morphogenesis during budding, mating, and pseudohyphal growth. To better understand the role of Spa2p in polarized growth, we analyzed regions of the protein important for its function and proteins that 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⌬, pea2⌬, and bud6⌬ mutants display similar phenotypes, suggesting that these three proteins are involved in the same biological processes. Coimmunoprecipitation experiments demonstrate that Spa2p and Pea2p are tightly associated with each other in vivo. Velocity sedimentation experiments suggest that a significant portion of Spa2p, Pea2p, and Bud6p cosediment, raising the possibility that these proteins form a large, 12S multiprotein complex. Bud6p has been shown previously to interact with actin, suggesting that the 12S complex functions to regulate the actin cytoskeleton. Deletion analysis revealed that multiple regions of Spa2p are involved in its localization to growth sites. One of the regions involved in Spa2p stability and localization interacts with Pea2p; this region 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 defects, suggesting that other regions are required for Spa2p function. By using the two-hybrid system, Spa2p and Bud6p were also found to interact with components of two mitogen-activated protein kinase (MAPK) pathways important for polarized cell growth. Spa2p interacts with Ste11p (MAPK kinase [MEK] kinase) and Ste7p (MEK) of the mating signaling pathway 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 mapped to the N-terminal putative regulatory domain. Bud6p interacts with Ste11p. The MEK-interacting region of Spa2p corresponds to the highly conserved SHD-I domain, which is shown to be important for mating and MAPK signaling. spa2 mutants exhibit reduced levels of pheromone signaling and an elevated level of Slt2p kinase activity. We thus propose that Spa2p, Pea2p, and Bud6p function together, perhaps as a complex, to promote polarized morphogenesis through regulation of the actin cytoskeleton and signaling pathways.

From Function to Shape: A Novel Role of a Formin in Morphogenesis of the FungusAshbya gossypii

“…One possible answer is that homologous polar growth components may exert not only common functions but also (Knechtle et al, 2003). These are properties specific for A. gossypii, because ScSpa2p, the homolog in S. cerevisiae, does not localize permanently but only transiently to the bud tip and because a cellular process, controlling maximal bud tip speed, is unknown in morphogenesis of S. cerevisiae (Sheu et al, 1998). Because ScSpa2p is part of the polarisome, we speculated that A. gossypii homologues of other components of the polarisome complex, in particular the formin homology protein ScBni1p Sagot et al, 2002a), could fulfill, in addition to common functions, also specific functions in sustained hyphal tip growth and tip splitting.…”

Functional characterization of Fusarium verticillioides CPP1, a gene encoding a putative protein phosphatase 2A catalytic subunit

“…We next identified well-characterized Saccharomyces cerevisiae polarity genes BNI1, BUD3, BUD6 and SPA2 (Amberg et al, 1997;Chant et al, 1995;Sagot et al, 2002;Sheu et al, 1998). Homologues of these genes in filamentous fungi are known to be associated with cellpolarity maintenance and cytokinesis (Harris et al, 1997;Virag & Harris, 2006;Wendland, 2003).…”

Stable and Dynamic Axes of Polarity Use Distinct Formin Isoforms in Budding Yeast

“…Bud6p and Spa2p are two components of a Bni1p-binding protein complex called the polarisome (Sheu et al, 1998) that localizes to growth sites with Bni1p (Snyder, 1989;Amberg et al, 1997). Bud6p cooperates with Bni1p in promoting actin assembly in vivo Sagot et al, 2002a) and in vitro (Moseley et al, 2004) and associates with secretory vesicles (Jin and Amberg, 2000), and in the absence of Bud6p, cables are very sparse (Amberg et al, 1997).…”