Mating-Pheromone Response Pathway in Budding Yeast
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Mating-Pheromone Response Pathway in Budding Yeast
The MAPK (Mitogen-Activated Protein Kinase) cascades play a pivotal role in many aspects of cellular functions, and are evolutionarily conserved from yeast to mammals. In Saccharomyces. cerevisiae, there are five MAP kinase signal transduction pathways that regulate mating, filamentous growth, high osmolarity response, maintenance of cellular integrity, and ascospore formation. The best-defined yeast MAPK pathway in S. cerevisae is involved in the mating of haploid cells. Yeast may exist either as haploid or diploid cells. The haploid cells have two sexual phenotypes characterized by the expression of a set of genes involved in mating that are not expressed in diploids. The mating response to generate diploids is stimulated by the release of small peptide mating pheromones, Alpha-factor from Mat-Alpha cells and A-factor from MatA cells, that act on cells of the opposite mating type to prepare that cell for mating. Cellular responses to mating pheromone include polarized growth toward a mating partner, cell cycle arrest in G1, and increased expression of proteins needed for cell adhesion, cell fusion, and nuclear fusion. A pheromone-activated signaling pathway that includes a MAPK cascade helps mediate many of these responses (Ref.1).

The pheromone response pathway is initiated by the binding of a peptide mating pheromone to a cell surface receptor Ste2 or Ste3, which are coupled to a heterotrimeric G-protein. Pheromone binding to its receptor leads to G-protein activation and induces the dissociation of the heterotrimeric G-protein subunits designated Gpa1 (Alpha-subunit), Ste4 (Beta-subunit), and Ste18 (Gamma-subunit). The released Beta-Gamma subunit complex (Ste4/Ste18) activates PAK kinase (Ste20) and interacts with the scaffolding protein Ste5, resulting in the stimulation of the MAPKKK (Ste11)–MAPKK (Ste7)– MAP kinase (Fus3/Kss1) cascade (Ref.2). Fus3 and Kss1 are two partially redundant MAP kinases that regulate the yeast mating processes, including the expression of mating-specific genes, G1 arrest, mating projection (schmoo formation) and cell fusion. The activated form of Fus3 is thought to translocate to the nucleus, where it mediates pheromone induction of transcription of PRE (Pheromone Response Element)-containing genes through phosphorylation and activation of at least three nuclear proteins: Dig1 (also called Rst1)/Dig2 (also called Rst2), Ste12 and Far1. Far1 phosphorylated by MAPK Fus3 binds to and inactivates the cell division control CDC28/Cln kinase complex and thus inhibits cell growth. Phosphorylation of Far1 is critical. Ste12 is a transcription factor containing separate domains for binding to the PRE, activation of transcription, and repression of transcription. One of the functions of Ste12 is to induce Far1 transcription. Thus Fus3 regulates Far1 in two complementary ways: a direct phosphorylation-dependent activation and an increase in transcription through activation of Ste12. Dig1 and Dig2 are related proteins with overlapping function that act as negative regulators of Ste12 function. Dig1 and Dig2 together repress the transcription of pheromone responsive genes. In an unstimulated cell, Dig1 and Dig2 appear to form a complex containing Fus3 (or Kss1) and Ste12. Pheromone stimulation increases Fus3-dependent phosphorylation of Dig1, Dig2, and Ste12 and induces the release of Ste12 from the complex. Putative MAPK phosphorylation sites in the Dig1- and Dig2-interacting domain of Ste12 are not required for Ste12 regulation. Several phosphatases act on the MAPK Fus3: the dual-specificity phosphatase Msg5 and the tyrosine phosphatases Ptp2 and Ptp3. The basal level of Fus3 phosphorylation is controlled mainly by the Ptp3 phosphatase. Pheromone induces the expression of Msg5, which then acts together with Ptp3 to inactivate Fus3. (Ref.3).

Another physiological effect of pheromone, reoriented cellular polarity, requires a different biochemical module, CDC42 (Cell Division Cycle-42), a p21 GTPase of the Ras superfamily. The membrane-tethered, activated G-protein is thought to lead to localize activation of the GEF (Guanine nucleotide Exchange Factor) for CDC42 and, thereby, to localized activation of CDC42. CDC42 in cells exists in a dynamic equilibrium between the GDP-bound and GTP-bound forms. Exchange of GDP for GTP on CDC42 is activated by CDC24 (Cell Division cycle-24), and the hydrolysis of the CDC42-bound GTP to GDP is predicted to be regulated by the GAPs (GTPase-Activating Proteins) Bem3 and Rga1. Once activated, CDC42 can organize the actin cytoskeleton as it does in vegetative cells. CDC42 may also activate Ste20 and influence signaling through the MAP kinase cascade. Bem1, like CDC42, interacts with several proteins important for the function of the actin cytoskeleton in polarized growth (Ref.4). Bem1 associates with actin and with the pheromone response pathway-signaling proteins Ste5 and Ste20. The Bem1-bound Ste5 is complexed to the Ste11-Ste7-Fus3 MAPK cascade. Interaction of Ste20 with Bem1 is required for association of Ste20 with actin. The fraction of these signaling proteins associated with macromolecular complexes in the cell is considerable. Bem1 interacts in cells with other signaling proteins: the CDC42 guanine nucleotide exchange factor CDC24; Far1, a protein needed for pheromone-induced cell cycle arrest; and Boi1 and Boi2, proteins involved in the regulation of the Rho-type GTPase Rho3 and Rho3-dependent growth-related processes. Although there is a fairly sophisticated understanding of signal transmission in the pheromone pathway, how signaling is regulated—for example, how the signal is attenuated so that the cell cycle can reinitiate once mating occurs—is poorly understood. Likewise, how the pheromone pathway may interface with other aspects of cell physiology is also poorly understood. To learn more about regulation of the pheromone pathway and its possible interface with other pathways has become a major challenge for the future (Ref.5).