CLPX regulates erythroid heme synthesis by control of mitochondrial heme synthesis enzymes and iron utilization

Heme is a prosthetic group that plays a critical role in catalyzing life-essential redox reactions in all cells, including critical metabolic processes. Heme synthesis must be tightly co-regulated with cellular requirements in order to maximize utilization and minimize toxicity. Terminally differentiating erythroid cells have an extremely high demand for heme for hemoglobin synthesis. While the enzymatic reactions of heme synthesis are extremely well studied, the mechanisms by which the mitochondrial homeostatic machinery interacts with and regulates heme synthesis are poorly understood. Knowledge of these regulatory mechanisms are key to understanding how red cells couple heme production with heme demand. Heme synthesis is tightly regulated by the mitochondrial AAA+ unfoldase CLPX, which has been reported to promote heme synthesis by activation of yeast {delta}-aminolevulinate synthase (ALAS/Hem1). CLPX was also reported to mediate heme-induced turnover of ALAS1 in human cells. However, a mutation in the ATP binding domain of CLPX that abrogated ATP binding caused an increase in ALAS activity, contrary to previous predictions that CLPX activated ALAS. Using loss-of-function assays in murine cells and zebrafish, we interrogated the mechanisms by which CLPX regulates erythroid heme synthesis. We found that consistent with previous studies, CLPX is required for erythroid heme synthesis. We show that ALAS2 stability and activity were both increased in the absence of CLPX, suggesting that CLPX primarily regulates ALAS2 by control of its turnover. However, we also showed that CLPX is required for PPOX activity and maintenance of FECH levels, likely accounting for the heme deficiency in the absence of CLPX. Lastly, CLPX is required for iron metabolism during erythroid terminal differentiation. Our results show that the role of CLPX in heme synthesis is not conserved across eukaryotes. Our studies reveal a potential mechanism for the role of CLPX in anemia and porphyria, and reveal multiple nodes at which heme synthesis is regulated by the mitochondrial housekeeping machinery.