PROJECT SUMMARY The long-term objectives of this proposal are to elucidate the molecules and mechanisms that mediate heme trafficking and signaling in health and disease. Heme is an essential but potentially cytotoxic metallocofactor, signaling molecule, and nutritional source of iron. All heme-dependent processes require that heme is safely transported and trafficked from sites of uptake or synthesis to heme dependent or regulated proteins located throughout the cell. However, the molecules and mechanisms that mediate the mobilization of heme for trafficking and signaling are poorly understood. Further, the spatio-temporal dynamics of heme trafficking are unknown. This is in large part due to the lack of tools available to image labile heme pools relevant to it biodistribution. The Reddi lab has recently overcome these technological barriers by generating genetically encoded ratiometric fluorescent heme sensors and hemoproteomics approaches to characterize heme trafficking and signaling dynamics. This proposal will focus on integrating molecular genetic, biochemical, biophysical, cell biological, and chemical biology approaches towards elucidating: a. heme transport and trafficking; b. heme-based signal transduction; and c. heme acquisition across a variety of model eukaryotic cell lines, e.g. Saccharomyces cerevisiae and mammalian cell lines, and pathogens, e.g. Mycobacterium tuberculosis and Candida albicans. To probe intracellular heme trafficking, genetically encoded heme sensors will be used in gene deletion and over-expression screens to identify novel pathways for heme distribution and trafficking. To probe heme signaling, hemoproteomics approaches and heme chelators will be deployed to identify how heme signals are initiated, transmitted, and integrated to control metabolism and physiology. To probe heme acquisition by human pathogens, heme sensors and hemoproteomics approaches will be integrated to identify the mechanisms of heme uptake and utilization by Mycobacterium tuberculosis and Candida albicans. Altogether, the proposed research will comprehensively address the mechanisms of heme trafficking and signaling in various contexts relevant to health and disease.