ABSTRACT During terminal erythropoiesis, erythroid cells produce significant quantities of heme and heme intermediates which must be coupled to hemoglobin production and iron uptake. Dysregulation of heme synthesis can cause toxic accumulation of heme intermediates and heme deficiency, leading to diseases such as iron overload, anemia and porphyria. We have demonstrated that mitochondrial CLPX, a member of the ubiquitious AAA+ (ATPases associated with various cellular activities) protein unfoldases family, plays a key role in erythroid differentiation by direct regulation of heme synthesis. CLPX functions as a ring-shaped homo-hexamer and is best understood for its function in a proteasome-like enzyme complex with the peptidase CLPP (the CLPXP ATP-dependent protease). In erythroid cells, CLPX is essential for heme synthesis by regulating the terminal steps of porphyrin synthesis and mitochondrial iron metabolism. This finding is conceptually significant as ALA synthesis has until now been understood to be the rate limiting step of porphyrin synthesis. In addition, although the heme synthesis and iron metabolism pathways are coregulated, the mechanisms by which this occurs are poorly understood. This proposal tests the conceptually innovative hypothesis that CLPX coordinates the terminal steps of the heme synthesis pathway with mitochondrial iron metabolism and is a key regulatory node for coupling heme synthesis and iron metabolism to the needs of the erythroid cell. The goal of this proposal is to identify the mechanisms by which CLPX regulates erythoid heme synthesis and erythropoiesis. This will be accomplished by Specific Aim 1, which examines the novel mechanisms by which CLPX regulates the terminal enzymes of the heme synthesis pathway, PPOX and FECH. Specific Aim 2 will identify the mechanisms by which CLPX regulates mitochondrial iron metabolism and the role of iron status in modulating diseases caused by mutations in the CLPX gene. Completion of these specific aims will fundamentally inform our understanding of “housekeeping” proteins like CLPX can have tissue specific functions in erythroid cells. These findings are also of translational significance as they will enable us to determine how to manipulate iron status to treat specific types of iron/heme disorders.