Measuring compartmentalized metabolism of cofactors

PROJECT SUMMARY Metabolism is the chemical necessity of all living things, allowing the use and interconversion of nutrients for biomass and energy. A fundamental characteristic of eukaryotic metabolism is the compartmentalization of metabolic pathways within and between subcellular compartments (especially mitochondria, cytoplasm, and potentially within the nuclear compartment). This allows the simultaneous existence of competing reactions, underpins regulatory control of major metabolic pathways, and allows metabolites to function as signaling molecules. Acyl-coenzyme A thioesters (acyl-CoAs) are evolutionarily conserved critical cofactors that are made and used in distinct parts of the eukaryotic cell. In different sub-cellular compartments, acyl-CoAs can be used to meet energy needs, act as signaling molecules, and serve as acyl-donors for post-translational modifications (PTMs) of proteins. Our current work has demonstrated that we can measure the compartmentalized pools of major acyl-CoAs and identified specific fates of localized pools. In this proposal we will 1) examine the sources, fates, and effects of pool specific modulation of cofactor metabolism. Simultaneously, we will 2) push the limits of sensitivity to single cell metabolomics and test the generalizability of our approaches to other metabolites. Under the aegis of a MIRA we will have the freedom to innovate and pursue these major research directions in their most important technical and biological directions.