PROJECT SUMMARY Organelle deterioration is a common feature of aging, and contributes to a number of age-related diseases. A major interest of my lab is to understand how organelles are functionally interconnected within cells, and uncover mechanisms by which organelle dysfunction perturbs cellular homeostasis during aging. Our work in this area during the previous project period focused on the lysosome (or vacuole in yeast), an acidic organelle that functions in protein degradation as well as metabolite storage and signaling. Impaired lysosomal function is a hallmark of aging across species and is a major contributor to age-related cellular dysfunction. Because many pathways converge at the lysosome that rely on the acidification of the organelle, how perturbations in lysosome acidity impact cell function has remained unclear. Answering this important question is a long-term goal of our lab. In the previous project period, we employed budding yeast as a model system to interrogate how vacuole dysfunction impacts cell health during aging, with a specific focus on dissecting the nature of its functional link to another metabolic organelle, the mitochondrion. We found that the essential function of vacuoles in supporting mitochondrial respiration is not linked to its well-known function in autophagy, but rather, it was tied to the role of vacuoles in amino acid compartmentalization and storage. We found that cysteine, which is stored in vacuoles in healthy cells, disrupts iron homeostasis when its vacuole compartmentation is perturbed during aging. Loss of iron homeostasis in turn alters the function of mitochondria, as well as numerous other iron-dependent cellular processes. Importantly, the connection between lysosome function, mitochondria, and iron homeostasis was validated by two other groups in human cells. Altogether, these results raise a new hypothesis to explain how loss of lysosome function causes age-related deterioration, and identify amino acids as potential drivers of cellular decline during aging. In the next project period, we plan to build on this discovery and new preliminary data by identifying pathways that coordinate with vacuoles to maintain amino acid homeostasis (Aim 1), and dissecting cellular mechanisms of amino acid toxicity and their impact on cellular decline during aging (Aim 2). The results of these studies will have a significant impact on our understanding of cellular pathways that coordinate with vacuoles to maintain amino acid organization, and provide key insight into the degree to which the 20 proteogenic amino acids act as toxic agents during aging when their organization is compromised.