PROJECT SUMMARY Metabolic pathways function in response to cellular needs, but they also can direct signaling and even influence how our genes are read. If we can understand the underlying mechanisms of metabolic-dependent signaling, it could broaden the range of interventions when combatting diseases to include modulation of cell metabolism. It is currently challenging to accurately measure concentrations and fluctuations of the metabolic intermediates that participate in these signaling pathways. By understanding the intracellular concentrations and regulation of these signaling metabolites, we can determine how the availability of a specific metabolite is poised to impact enzymatic activity, as well as the extent and timing that their levels may fluctuate over the course of physiology and disease. These metabolites are highly compartmentalized even within individual cells, and an accurate measurement for signaling needs to distinguish its free concentration from bound pools. Moreover, signaling metabolites often have distinct roles in different parts of the cells and their concentrations can be differentially regulated. We are deconvoluting the signaling roles of metabolites by developing small single-fluorescent protein biosensors that are genetically encoded and selective for specific intracellular metabolites. These sensors can be localized subcellularly and measured changes in their fluorescence reflect changes in concentration for specific intermediary metabolites. Together the data will determine how that metabolite regulates signaling. In this proposal, we use mitochondrial NAD+ sensors to elucidate the mechanisms of transport for human mitochondrial carrier family member, SLC25A51, and determine its roles in disease. We also develop new sensors to expand our investigations to additional signaling metabolites, including NAD-derived metabolites.