Figure 1

Clpx^−/−MEL cells have a heme synthetic defect distinct from the role of CLPX in the CLPXP protease.A, predicted model for the role of CLPXP in heme regulation. In yeast, CLPX activates the ALAS enzyme that catalyzes the committed step of heme synthesis. In mammalian cells, the CLPXP protease regulates ALAS stability and heme synthesis (16, 17). We hypothesized that CLPX-deficient erythroid cells will accumulate inactive ALAS protein due to defective activation by CLPX. Erythroid cells were predicted to be heme-deficient. B, Western blot analysis of CLPX and CLPP expression in WT, Clpx^−/−, and Clpp^−/− MEL cell lysates (N = 4). HSP60 was used as a loading control. C, qPCR analysis of Clpx and Clpp mRNA expression in WT, Clpx^−/−, and Clpp^−/− MEL cells. Gene expression was normalized to β-actin mRNA levels. D, benzidine staining of hemoglobin in Clpx^−/− and Clpp^−/− differentiated MEL cells. Clpx^−/− cells were hemoglobin-deficient, while Clpp^−/− cells produced more hemoglobin than WT cells. E, Quantitation of heme synthesis by ⁵⁵Fe labeling. Clpx^−/− cells had a heme synthesis defect. In contrast, Clpp^−/− cells had increased heme synthesis. F, Differentiated Clpx^−/− cells had decreased Hbb-b1 and Hbb-b2 expression. Undifferentiated Clpx^−/− cells also had decreased Hbb-b2 expression. ∗p < 0.05. Error bars indicate mean ± standard deviation (SD).