Pheromone-induced anisotropy in yeast plasma membrane phosphatidylinositol-4,5- bis phosphate distribution is required for MAPK signaling

Abstract

During response of budding yeast to peptide mating pheromone, the cell becomes markedly polarized and MAPK scaffold protein Ste5 localizes to the resulting projection (shmoo tip). We demonstrated before that this recruitment is essential for sustained MAPK signaling and requires interaction of a pleckstrin homology (PH) domain in Ste5 with phosphatidylinositol 4,5-bisphosphate [PtdIns (4,5)P 2 ] in the plasma membrane. Using fluorescently tagged highaffinity probes specific for PtdIns(4,5)P 2 , we have now found that this phosphoinositide is highly concentrated at the shmoo tip in cells responding to pheromone. Maintenance of this strikingly anisotropic distribution of PtdIns(4,5)P 2 , stable tethering of Ste5 at the shmoo tip, downstream MAPK activation, and expression of a mating pathway-specific reporter gene all require continuous function of the plasma membrane-associated PtdIns 4-kinase Stt4 and the plasma membrane-associated PtdIns4P 5-kinase Mss4 (but not the Golgi-associated PtdIns 4-kinase Pik1). Our observations demonstrate that PtdIns(4,5)P 2 is the primary determinant for restricting localization of Ste5 within the plasma membrane and provide direct evidence that an extracellular stimulus-evoked selfreinforcing mechanism generates a spatially enriched pool of PtdIns (4,5)P 2 necessary for the membrane anchoring and function of a signaling complex.α-factor | phosphoinositides | scaffold protein | yeast mating response E ukaryotic cells respond to extracellular stimuli and spatial cues via signaling responses that can dramatically alter cell polarity. Ample evidence from diverse cell types indicates that membrane phosphoinositides, especially phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ] (1), are key regulators of processes, like cytoskeletal remodeling (2) and vesicle-mediated trafficking (3, 4), which are required for polarized growth, cell morphogenesis, and cell division (5). In both mammalian leukocytes and migratory Dictyostelium discoideum amoebae, exposure to chemoattractant results in spatial restriction of PtdIns(3,4,5)P 3 , as well as of the enzymes that catalyze its synthesis (PtdIns 3-kinase) and breakdown [phosphatidylinositol 3-phosphatase and tensin homolog (PTEN)] (6). Likewise, in mammalian kidney cells, localized action of the PtdIns(3,4,5)P 3 phosphatase PTEN plays a key role in determining epithelial cell polarity by defining the content of this lipid in the apical and basolateral membranes (7), thereby affecting the distribution of small GTPases that dictate cell shape (8). Membrane phosphoinositides were also shown to play an important role in driving anchor cell invasion during Caenorhabditis elegans development (9). These examples all indicate that membrane subdomains enriched for specific phosphoinositides are crucial for attracting signaling components specific for establishment and/or maintenance of cell polarity.Likewise, in Saccharomyces cerevisiae, phosphoinositides in the plasma membrane have been implicated in the recruitment of proteins involved in polarized...

Dynamic Ubiquitination of the Mitogen-activated Protein Kinase Kinase (MAPKK) Ste7 Determines Mitogen-activated Protein Kinase (MAPK) Specificity

“…Finally, the phosphatidylinositol 3-kinase Vps34 promotes activation of Fus3 but not Kss1 (88). With the exception of Ste5, however, there is little evidence that these regulators are subject to dynamic regulation by pheromone stimulation (86). In contrast, ubiquitination of Ste7 is strictly dependent on pheromone stimulation and subsequent phosphorylation by Ste11 (33).…”

The Putative Lipid Transporter, Arv1, Is Required for Activating Pheromone-Induced MAP Kinase Signaling in Saccharomyces cerevisiae

“…It has been shown that polarized PI(4,5)P 2 in the plasma membrane interacts with the pleckstrin homology (PH) domain in Ste5 to cluster Ste5 (Winters et al 2005;Garrenton et al 2006Garrenton et al , 2010 and that ergosterol promotes PI(4,5)P 2 polarity ( Jin et al 2008). To determine whether the sterol trafficking defects in arv1 cells contribute to defects in PI(4,5)P 2 polarization in response to pheromone, we examined the localization of GST-GFP-PH PLCd after treating cells with a-factor.…”

“…The Ste5 scaffolding protein recruits the MAP kinases Ste11, Ste7, and Fus3 in response to pheromone to initiate signaling. In turn, Ste5 is recruited to the plasma membrane by pheromone/receptor binding and subsequent interaction with the Gbg dimer Ste4-Ste18, but also interacts with phosphatidyinositol 4,5-bisphosphate [PI(4,5)P 2 ] in the plasma membrane through its pleckstrin homology (PH) domain (Winters et al 2005;Garrenton et al 2006Garrenton et al , 2010. Activation of the pheromone response pathway results in G1 cell cycle arrest, mating-specific gene transcriptional induction, and changes in cytoskeletal structure, which allows for polarized cell growth and alterations in nuclear architecture, ultimately leading to cell fusion and formation of an a/a diploid (Wang and Dohlman 2004).…”

“…The following plasmids were a gift from Peter Pryciak: 2m HIS3 pGAL1-STE12 (Strickfaden and Pryciak 2008), CEN TRP1 pGAL1-STE11-Cpr (Strickfaden et al 2007), CEN TRP1 pGAL1-GST-Ste11DN (Moskow et al 2000), CEN URA3 STE5-GFPx3 (Strickfaden et al 2007), CEN URA3 pGAL1-STE5 Q59L (Winters et al 2005), and CEN TRP1 pGAL1-STE5DN-CTM (Pryciak and Huntress 1998). The following plasmid was a gift from Jeremy Thorner: CEN LEU2 pGAL1-GST-GFP-PH PLCd (Winters et al 2005;Garrenton et al 2010).…”

“…Protein extraction and immunoblotting: Protein was extracted using the NaOH/TCA cell lysis/protein extraction protocol as previously described (Garrenton et al 2010). 3HA-tagged Arv1 constructs were detected using anti-HA (clone 16B12) from Covance (Princeton, NJ).…”