Scaffold number in yeast signaling system sets tradeoff between system output and dynamic range

Abstract

Although the proteins comprising many signaling systems are known, less is known about their numbers per cell. Existing measurements often vary by more than 10-fold. Here, we devised improved quantification methods to measure protein abundances in the Saccharomyces cerevisiae pheromone response pathway, an archetypical signaling system. These methods limited variation between independent measurements of protein abundance to a factor of two. We used these measurements together with quantitative models to identify and investigate behaviors of the pheromone response system sensitive to precise abundances. The difference between the maximum and basal signaling output (dynamic range) of the pheromone response MAPK cascade was strongly sensitive to the abundance of Ste5, the MAPK scaffold protein, and absolute system output depended on the amount of Fus3, the MAPK. Additional analysis and experiment suggest that scaffold abundance sets a tradeoff between maximum system output and system dynamic range, a prediction supported by recent experiments.quantitative immunoblotting | single-cell fluorescence quantification | genetic algorithmic model optimization T he Saccharomyces cerevisiae pheromone response system has been useful for understanding eukaryotic cell signaling (1-3). In haploid cells, the pheromone response system detects mating pheromone in the extracellular environment secreted by yeast of the opposite mating type. The system triggers a number of responses, including induction of gene expression, arrest in cell cycle progression, changes in morphology, and eventually, mating. The molecules and interactions by which the system operates are relatively well-understood (Fig. 1).Changes in the number of molecules per cell (hereafter called abundances) of signaling proteins can alter quantitative signaling behaviors. For example, in the pheromone response system, overexpression of Fus3 increases pheromone-induced sensitivity to cell cycle arrest (4), and overexpression of Gpa1 decreases pheromone-induced transcription (5). Similarly, changes in the abundances of MAPK cascade scaffold proteins (Ste5 in the yeast pheromone response system) ( Fig. 1) can alter system behavior. In models, when scaffold is limiting, increasing scaffold abundance first increases system output and then, eventually sequesters the associated protein kinases onto separate scaffolds, diminishing the number of fully assembled complexes and system output (6). This peak and decline behavior has been experimentally shown for Ste5 in this yeast system (7) and the Kinase Suppressor of Ras (KSR) scaffold protein in Ras signaling in Xenopus oocytes (8).Previous investigators reported focused and genome-wide inventories of pheromone system protein abundances (9-13). These measurements differed by up to 12-fold (Fig. 2) (14, 15). We thought some discrepancies might be because of differences and systematic biases in quantification methods (Discussion). We developed improved measurement methods and used these methods to quantify system proteins. We used...

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