MAPK related phenotypic decisions in yeast
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Date
2015-03-26
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Abstract
The evolutionarily conserved mitogen-activated protein kinase (MAPK) network is a signalling module, which enables the coordination and processing of various extracellular stimuli. It thereby guarantees a specific biological response to a precise dose of a given stimuli. The haploid yeast Saccharomyces cerevisiae uses this network to select particular mating partners by quantitatively interpreting the pheromone concentration gradient generated by potential mates. Activation of the mating MAPK module occurs only above a certain pheromone concentration threshold and relies on the pheromone-induced recruitment of a protein complex consisting of the scaffold Ste5 and the MAPKs Ste11, Ste7 and Fus3. This module ensures a robust morphological response in form of a mating projection to a defined pheromone concentration and allows cells to gauge the distance to a potential mating partner. Yet it remains unclear which of the module’s features interpret the pheromone concentration to decide when and where to generate a mating projection.
To infer the network structure of the mating MAPK module, we developed a reverse engineering approach, which is based on the detection of pheromone response dependent changes in protein complex abundances. Interactions within the MAPK module were measured by fluorescence correlation spectroscopy (FCS), of which all possible protein-species in the MAPK module were resolved by applying a linear regression analysis (LRA). Using this approach, we were able to identify a cytosolic kinase-substrate interaction between Fus3 and the upstream Ste11, which constitutes a hitherto uncharacterized negative feedback. It affects the readout of the pheromone gradient and provides robustness to changes in the components involved in the loop. This negative feedback occurs by phosphorylation of S243 on Ste11 that hinders its binding to the scaffold Ste5 and thereby uncouples Ste11 from Fus3 activity. Controlling this mechanism provides ultrasensitivity at the first step of the MAPK cascade, as part of the hierarchical cascade arrangement, ensures a switch-like mating response and triggers shmoo formation at the right distance to a partner. This cytoplasmic feedback has a spatial component that confines the cytoplasmic Fus3 phosphorylation gradient. It thereby generates and maintains a localized source of active Fus3 at the mating tip, which in turn spatially restricts shmoo formation.
This work shows how a network motif in the MAPK module enables the interpretation of the pheromone concentration gradient to sense potential mates, and how this extracellular gradient is translated into an intracellular activity gradient by spatial control of signalling, ultimately deciding both when and where to respond.