Project Summary/Abstract Mitochondria house the metabolism of all building blocks of life and are central for cellular metabolism, however, how metabolites translocate across the mitochondrial membrane is poorly understood; and the impact on neuronal cellular metabolism is yet to be explored. The mitochondrial carrier SLC25 family represents the largest protein family critical for mitochondrial metabolite transport, in which approximately 20 out of the 53 human SLC25 genes remain of unknown function. Our overarching research program is to characterize mitochondrial metabolite transport by identifying the endogenous metabolite ligands for SLC25 transporters. Recently, my laboratory applied mitochondrial metabolomics to discover the role of a poorly characterized mitochondrial transporter SLC25A39 in glutathione uptake (supported by R00). Here, building upon this recent progress, we propose to expand the strategy to other SLC25 transporters using loss-of-function and gain-of-function study in cells. As a distinct and complementary approach, we will apply sequence and structure analysis towards SLC25 transporter to guide in vitro mitochondrial metabolite uptake screen with a goal to characterize ligand recognition mechanism. To study mitochondrial metabolism in neurons, we will adopt iPSC-derived neurons as an in vitro model to profile the impact of SLC25 transporters loss on mitochondrial and synaptic metabolism. Together, our study of SLC25 transporters would reveal fundamental biology in mitochondrial metabolite transport, an emerging new frontier in cellular metabolism. Our expertise in cellular metabolism, uniquely in applying state-of-art, high- resolution mass spec metabolomics to fundamental biochemistry and cell biology, is perfectly suited for the research direction. The ESI-MIRA program enables launching my research career in cellular metabolism as well as moving into a new research area in neuronal cell biology. The flexibility in the funding mechanism allows this ambitious project to systematically decode mitochondrial metabolite transport.