The mating pathway in Saccharomyces cerevisiae has been the focus of considerable research effort, yet many quantitative aspects of its regulation still remain unknown. Using an integrated approach involving experiments in microfluidic chips and computational modelling, we studied gene expression and phenotypic changes associated with the mating response under well-defined pheromone gradients. Here we report a combination of switch-like and graded pathway responses leading to stochastic phenotype determination in a specific range of pheromone concentrations. Furthermore, we show that these responses are critically dependent on mitogen-activated protein kinase (MAPK)-mediated regulation of the activity of the pheromone-response-specific transcription factor, Ste12, as well as on the autoregulatory feedback of Ste12. In particular, both the switch-like characteristics and sensitivity of gene expression in shmooing cells to pheromone concentration were significantly diminished in cells lacking Kss1, one of the MAP kinases activated in the mating pathway. In addition, the dynamic range of gradient sensing of Kss1-deficient cells was reduced compared with wild type. We thus provide unsuspected functional significance for this kinase in regulation of the mating response.

Mating pheromones promote cellular differentiation and fusion of yeast cells with those of the opposite mating type. In the absence of a suitable partner, high concentrations of mating pheromones induced rapid cell death in ϳ25% of the population of clonal cultures independent of cell age. Rapid cell death required Fig1, a transmembrane protein homologous to PMP-22/EMP/MP20/Claudin proteins, but did not require its Ca 2؉ influx activity. Rapid cell death also required cell wall degradation, which was inhibited in some surviving cells by the activation of a negative feedback loop involving the MAP kinase Slt2/Mpk1. Mutants lacking Slt2/Mpk1 or its upstream regulators also underwent a second slower wave of cell death that was independent of Fig1 and dependent on much lower concentrations of pheromones. A third wave of cell death that was independent of Fig1 and Slt2/Mpk1 was observed in mutants and conditions that eliminate calcineurin signaling. All three waves of cell death appeared independent of the caspase-like protein Mca1 and lacked certain "hallmarks" of apoptosis. Though all three waves of cell death were preceded by accumulation of reactive oxygen species, mitochondrial respiration was only required for the slowest wave in calcineurin-deficient cells. These findings suggest that yeast cells can die by necrosis-like mechanisms during the response to mating pheromones if essential response pathways are lacking or if mating is attempted in the absence of a partner. INTRODUCTIONProgrammed cell death (PCD) occurs in metazoans as a means of eliminating unwanted cells during development and removing damaged, weak, infected, or malignant cells from the organism to avoid potentially harmful consequences (Danial and Korsmeyer, 2004). PCD is highly coordinated and regulated at multiple levels. Inputs from a variety of sources can impact on a core set of enzymes that coordinate destruction of key cellular components necessary for cell survival. Apoptosis, one form of PCD, typically requires activation of cysteine-aspartyl proteases (caspases) by signaling factors derived from mitochondria or the plasma membrane. Conservation of PCD factors among all animals (Koonin and Aravind, 2002) is consistent with a very early origin of the PCD mechanism, potentially even before the divergence of animals and fungi.The occurrence of PCD in fungi has received support from numerous studies using the budding yeast Saccharomyces cerevisiae (reviewed in Madeo et al., 2002(reviewed in Madeo et al., , 2004Longo et al., 2005). Several so-called "hallmarks" of apoptosis can be observed in populations of yeast cells that have been mortally wounded by environmental stresses such as high heat, high salt, hypertonic shock, DNA damaging agents, food preservatives, and hydrogen peroxide. Starvation and aging also seem to induce apoptosis-like cell death in a minority of cells in the dying population (reviewed in Longo et al., 2005). Expression of mammalian Bax in yeast also leads to mitochondrial dysfunction, accumulation of reactive oxygen s...

SUMMARY mRNAs encoding polarity and secretion factors (POLs) target the incipient bud site in yeast for localized translation during division. In pheromone-treated cells, we now find that these mRNAs are also localized to the shmoo tip. However, in contrast to the budding program, neither the She2 nor She3 proteins are involved. Instead, the Scp160 RNA-binding protein binds POL and mating pathway mRNAs and regulates their spatial distribution in a Myo4- and cortical ER-dependent fashion. RNA-binding by Scp160 is stimulated by activation of Gpa1, the G-protein α-subunit regulated by the pheromone receptor, and is required for pheromone gradient sensing, as well as subsequent chemotropic growth and cell-cell mating. These effects are incurred independently of obvious changes in translation, thus, mRNA trafficking is required for chemotropism and completion of the mating program. This is the first demonstration of ligand-activated RNA targeting in the development of a simple eukaryote.

SUMMARY Signal transduction networks can display complex dynamic behavior such as oscillations in the activity of key components [1-6], but it is often unclear if such dynamic complexity is actually important for the network's regulatory functions [7, 8]. Here we found that the mitogen-activated protein kinase (MAPK) Fus3, a key regulator of the yeast mating pheromone response undergoes sustained oscillations in its phosphorylation/activation state during continuous pheromone exposure. These MAPK activity oscillations led to corresponding oscillations in mating gene expression. Oscillations in MAPK activity and gene expression required the negative regulator of G-protein signaling Sst2, and partially required the MAPK phosphatase Msg5. Peaks in Fus3 activation correlated with periodic rounds of cell morphogenesis, with each peak preceding the formation of an additional mating projection. Preventing projection formation did not eliminate MAPK oscillation, but preventing MAPK oscillation blocked the formation of additional projections. A mathematical model was developed that reproduced several features of the observed oscillatory dynamics. These observations demonstrate a role for MAPK activity oscillation in driving a periodic downstream response, and explain how the pheromone signaling pathway, previously thought to desensitize after 1-3 hours, controls morphology changes that continue for a much longer time.

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