PROJECT SUMMARY/ ABSTRACT Mitochondria are multi-functional organelles that play central roles in metabolism, protein homeostasis, calcium signaling, immunity, and cell death. Perturbation of these mitochondrial functions is an underlying feature of several common diseases and physiological conditions such as cancer, neurodegenerative diseases and aging. Interaction of mitochondria with other organelles plays a pivotal role in regulation of mitochondrial functions. However these mitochondria-organelle interactions remain poorly understood, partly due to lack of methods that enable isolation and characterization of mitochondria that associate with different organelles. This also hinders investigation of mitochondria-organelle interactions in diseases and their contribution to disease pathogenesis. The goal of this proposal is to overcome this technical challenge in order to investigate the mechanisms of mitochondria regulation through organelle interactions in cell lines and cellular models of mitochondrial diseases. Here, I propose the development of an innovative biochemical workflow that will allow differential tagging and rapid isolation of mitochondria based on their cellular location and organellar interactions. Using this method, we will first characterize the proteome and metabolite composition of mitochondria that are in close proximity to four organelles and the plasma membrane. Next, we will investigate whether specific metabolites and protein modifications regulate mitochondrial functions at organelle contact sites. We will also determine how mitochondria- organelle interactions are altered in cellular models of mitochondrial disease and whether these interactions can be targeted for therapeutic gain. These experiments will be the first comprehensive biochemical analysis of mitochondria-organelle interactions in healthy cells and cells with mitochondrial dysfunction. Our results will lead to identification of signals that originate from different cellular structures to regulate mitochondrial functions and will elucidate their mechanisms. In addition, investigation of mitochondria-organelle interactions in cells with mitochondrial dysfunction has the potential to transform our understanding of mechanisms of mitochondrial diseases, which will lead to novel treatment approaches. The methods developed can easily be applied to investigate other inter-organellar interactions to reveal their mediators and mechanisms at unprecedented biochemical detail.