PROJECT SUMMARY/ABSTRACT Mitochondria are central hubs of cellular metabolism, and their dysfunction is linked to a host of age-related and metabolic disorders. As a major source of cellular energy production and metabolite biosynthesis, mitochondria continually take up and release a variety of intracellular nutrients. This renders mitochondria vulnerable to changes in nutrient concentrations frequently observed in disorders associated with metabolic overload. Indeed, recent work from our lab showed that mitochondria are uniquely sensitive to elevations in intracellular amino acid load, and that amino acid toxicity is a key driver of age-related mitochondrial decline. Moving forward, a major goal of our lab is to understand mechanisms by which elevated amino acids impair mitochondrial function, and identify pathways that protect mitochondria from amino acid stress. In work during the last project period, we identified a key role for the amino acid cysteine in regulating mitochondrial respiration, and uncovered a new cellular structure, called the Mitochondrial-Derived Compartment (MDC), that protects cells from harmful effects of excess amino acids. Our current data suggests that MDCs are dynamic, organelle-like structures that are generated from mitochondria in response to intracellular amino acid elevation. Upon formation, MDCs selectively sort and remove proteins of the mitochondrial carrier superfamily, key mediators of metabolite transport across the mitochondrial inner membrane, away from the rest of the mitochondrial network. MDCs are conserved from yeast to humans, and loss of the MDC pathway renders cells susceptible to amino acid overload. These results have led to our current working model that MDC formation represents a new mechanism to acutely regulate mitochondrial nutrient transporters in response to changes in cellular metabolic supply. Currently, our understanding of the biogenesis, nutrient regulation, and role of MDCs in cellular metabolism is far from complete. During the next project period, we will work in both yeast and mammalian systems to elucidate mechanisms and machinery involved in MDC biogenesis, identify nutrient cues and sensors that control MDC activation, and determine the role of this pathway in mitochondrial and cellular metabolism. Because mitochondrial metabolite transporters are key regulatory points of cellular metabolism, we anticipate that elucidating the function of this new cellular pathway will have significant impact on our understanding of mitochondrial physiology and its role in human disease.