Project Summary Metabolism is a tightly controlled and complex process in which different nutrients are taken up and processed to meet variable needs. The regulation of uptake and processing requires that many nutrients and metabolites are sensed. This poses a challenge since some of the molecules that are being sensed are simultaneously subject to processing. This has led to the suspicion that metabolic pathways may directly sense the flux through the pathway, instead of sensing only the concentration of nutrients or metabolites. Recent work has confirmed this by identifying a flux-sensing system in bacteria, although the mechanism used is complex and may not translate to other systems. In preliminary work on the galactose utilization (GAL) pathway of Saccharomyces cerevisiae, we have identified a novel putative mechanism for connecting enzymatic activity to signaling, providing a simple way for metabolic flux to be measured. Because this mechanism is simple, we suspect that it may occur in many pathways. We now propose to investigate this hypothetical mechanism in detail. The GAL pathway is an ideal system in which to identify and mechanistically characterize flux sensing. It is a classic model system for signaling in eukaryotes, and we have extensive methods and genetic tools available. Systematic high- throughput quantitative measurements will be used to develop and refine computational models, which in turn will be used to both guide and interpret experiments and to give insight into the physiological role of both flux and concentration sensors. The insights we gain from the GAL pathway will then simplify the discovery of flux sensors in other pathways. Next, we will bring the tools and models we develop to characterize GAL signaling to bear on identifying and characterizing the sensor modalities in the pathway responsible for metabolizing nitrogen in S. cerevisiae. Based on literature findings, we suspect that this pathway also contains a flux sensor. We anticipate that identifying flux sensors in these pathways will immediately provide insight into the potential for flux sensing in human metabolic pathways, and lead to the identification of promising new therapeutic targets.