Molecular mechanisms for a switch-like mating
decision in Saccharomyces cerevisiae

Evolution has resulted in numerous innovations that allow organisms to maximize their fitness by choosing particular mating partners, including secondary sexual characteristics, behavioural patterns, chemical attractants and corresponding sensory mechanisms. The haploid yeast Saccharomyces cerevisiae selects mating partners by interpreting the concentration gradient of pheromone secreted by potential mates through a network of mitogen-activated protein kinase (MAPK) signaling proteins. The mating decision in yeast is an all-or-none, or switch-like, response that allows cells to make accurate decisions about which among potential partners to mate with and to filter weak pheromone signals, thus avoiding inappropriate commitment to mating by responding only at or above critical concentrations when a mate is sufficiently close. The molecular mechanisms that govern the switch-like mating decision are poorly understood. In this thesis I demonstrate that the switching mechanism arises from competition between the MAPK Fus3 and a phosphatase Ptc1 for control of the phosphorylation state of four sites on the scaffold protein Ste5. This competition results in a switch-like dissociation of Fus3 from Ste5 that is necessary to generate the switch-like mating response. Thus, the decision to mate is made at an early stage in the pheromone pathway and occurs rapidly, perhaps to prevent the loss of the potential mate to competitors. We argue that the architecture of the Fus3–Ste5–Ptc1 circuit generates a novel ultrasensitivity mechanism that resembles “zero-order ultrasensitivity”, which is robust to variations in the concentrations of these proteins. This robustness helps assure that mating can occur despite stochastic or genetic variation between individuals. I then demonstrate that during the mating response, an early event of Ste5 recruitment to plasma membrane is ultrasensitive and that it is generated by dephosphorylation of eight N-terminal phosphosites on Ste5 by the phosphatase Ptc1 when associated with Ste5 via the polarization protein Bem1. Interference with this mechanism results in loss of ultrasensitivity and reduced amplitude and therefore fidelity of the pheromone signaling response. These changes are reflected in reduced fidelity and accuracy of the morphogenic mating response. Polarized assembly of signaling protein complexes at the plasma membrane surface is a general theme recapitulated in all organisms from bacteria to humans. Such complexes can increase the efficiency, fidelity and specificity of signal transduction. Together with our previous findings, our results demonstrate that ultrasensitivity, accuracy and robustness of the pheromone response occurs through regulation of the stoichiometry of phosphorylation of two clusters of phosphosites on Ste5, by Ptc1, one cluster mediating ultrasensitive recruitment of Ste5 to the membrane and the other, ultrasensitive dissociation and activation of the terminal MAP kinase Fus3. The role of Ste5 as a direct modulator of a cell-fate decision expands the functional repertoire of scaffold proteins beyond providing specificity and efficiency of information processing. Regulation of dynamic signal-response characteristics through such modular regulation of clusters of phosphosites may be a general means by which cell fate decisions are achieved. Similar mechanisms may govern cellular decisions in higher organisms and be disrupted in cancer. Finally, in a related theme, I present the discovery of a novel mechanisms by which the threshold of pheromone response is controlled by a nutrient-sensing pathway, thus adjusting the set-point at which the quantity and quality of nutrients available in the environment set the threshold of pheromone at which yeast will mate. The regulatory subunit of protein kinase A (PKA), Bcy1, a key component of a nutrient sensing signaling network, directly interacts with the α subunit of G-protein, Gpa1, the primary effector of the pheromone signaling network. The Bcy1-Gpa1 interaction is enhanced when cells are grown in their ideal carbon source glucose, lowering the threshold concentration at which the mating response is activated. Disruption of Bcy1-Gpa1 interaction or Bcy1 deletion increased the threshold concentration for the mating response. We argue that by adjusting their sensitivity, yeast can integrate pheromone stimulus with glucose levels and prioritize decisions to survive in a nutrient-starved environment or to continue their sexual cycle by mating.

Keyphrases: Evolution, Signaling dynamics, Ultrasensitivity, Protein-protein interaction, Cellular decision, Robustness, Cross-talk, Accuracy, Amplification du signal, Signal amplification