MEASUREMENT OF EFFECTIVE SCAFFOLD PROTEIN AFFINITY TO MEMBRANE BINDING SITES BY IN VIVO SCATCHARD

ALAN BUSH; CHERNOMORETZ A; ALEJANDRO COLMAN LERNER

Puerto Madryn - Chubut

Congreso; SAIB 46th Annual Meeting Argentine Society for Biochemistry and Molecular Biology XLVI Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular.; 2010

Society for Biochemistry and Molecular Biology XLVI Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular.

The yeast, Saccharomyces cerevisiae, pheromone response pathway is one of the best understood signal transduction systems. All key components of the system and their interactions have been identified by more than 30 years of genetic and biochemical investigation. In recent years modeling efforts have been undertaken to try to describe the behavior of the system in a quantitative manner. These models usually contain a large number of parameters to be determined by fitting to a dataset of experimental results. Direct measurement of these parameters can be used to constrain these models and obtain more accurate and predictive descriptions of the pathway. In this work we describe a microscopy imaging technique developed to measure protein relocalization in yeast in a quantitative manner. Using this technique we measured membrane recruitment of the scaffold protein of the mating pathway Ste5, an early event that occurs upon pheromone stimulation. Varying the amount of Ste5 by genetic manipulation allowed us to perform anSaccharomyces cerevisiae, pheromone response pathway is one of the best understood signal transduction systems. All key components of the system and their interactions have been identified by more than 30 years of genetic and biochemical investigation. In recent years modeling efforts have been undertaken to try to describe the behavior of the system in a quantitative manner. These models usually contain a large number of parameters to be determined by fitting to a dataset of experimental results. Direct measurement of these parameters can be used to constrain these models and obtain more accurate and predictive descriptions of the pathway. In this work we describe a microscopy imaging technique developed to measure protein relocalization in yeast in a quantitative manner. Using this technique we measured membrane recruitment of the scaffold protein of the mating pathway Ste5, an early event that occurs upon pheromone stimulation. Varying the amount of Ste5 by genetic manipulation allowed us to perform an in vivo Scatchard to measure the effective binding affinity of the scaffold protein with its membrane associated binding sites. Using this parameter we will refine our mathematical model of the onset of the pheromone response pathway.Scatchard to measure the effective binding affinity of the scaffold protein with its membrane associated binding sites. Using this parameter we will refine our mathematical model of the onset of the pheromone response pathway.