Analysis of scaffold-protein membrane recruitment dynamics during pheromone response in Saccharomyces cerevisiae

REPETTO M. V.; BUSH A.; COLMAN LERNER A.

Congreso; XLVII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2011

The yeast mating pheromone response pathway is a prototypical example of a signaling pathway that uses a scaffold protein. The scaffold, Ste5, has several interaction domains, some of which mediate essential steps necessary for the activation of its associated three-tiered MAPK cascade, while others function in higher-order regulatory behaviors, such as feedback phosphorylation by the MAPK Fus31 and inhibitory phosphorylation by the cell-cycle dependent kinase2. This works is aimed at determining the role of Ste5 interaction domains in the dynamics of the pheromone response. To do this, we followed in real-time recruitment of Ste5 to the membrane in single cells upon pheromone treatment, analyzing the redistribution of a Ste5 YFP fusion with a confocal microscope. We obtained quantitative measurements from single-cells using Cell-ID coupled data analysis with R, following a method previously developed in our lab3. We measured several Ste5 mutants to assess the role of Ste5 modular domains in pathway dynamics. Mutations at Ste5 regulatory sites, mimicking constitutive (de)phosphorylation, produced changes in the dynamic of membrane binding, suggesting the importance of such modifications for the fine-tuning of the response. 1Yu et al. Nature 456:755(2008) 2Strickfaden et al. Cell 128:519(2007) 3Chernomoretz et al. Curr Protoc Mol Biol 84:14.18.1(2008)