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).

clpxb mutant zebrafish embryos develop normal numbers of red cells, but are heme-deficient and have dysregulated red cell morphology. A, qPCR analysis of clpxb mRNA in WT and clpxb mutant embryos. clpxb mRNA is decreased by approximately 50% in mutant embryos (p-value < 0.0001). B, clpxb is required for erythroid hemoglobinization. clpxb mutant embryos are anemic at 48 hpf. C, clpxb is not required for erythroid specification. 72 hpf clpxb/clpxb; Tg(lcr:GFP) zebrafish embryos had similar percentages of GFP⁺ erythroid cells to control Tg(lcr:GFP) embryos as assessed by FACS. D, Compared with control erythroid cells, erythroid cells from clpxb mutant embryos were more variable in appearance, had smaller cytoplasmic areas and larger nuclei. E, clpxb erythroid cells had higher nuclear:cytoplasmic ratios (p-value < 0.0001). The nuclear:cytoplasmic ratios of clpxb erythroid cells exhibited higher variability (F test: p < 0.05). F, The increased nuclear:cytoplasmic ratio was caused by increased nuclear area in clpxb erythroid cells (p-value < 0.0001) and G, decreased cytoplasmic area (p-value<0.0001). ∗p < 0.05, Student's t test; error bars indicate SD.

CLPX regulates the stability of ALAS2 in MEL cells.A, i, Alas2 mRNA expression was decreased or unaltered in the absence of CLPX and CLPP, (ii) but its protein levels are increased in Clpx^−/− and Clpp^−/− cells. The proteolytically cleaved, mitochondrial matrix localizing form of ALAS2 (38, 39, 40, 41, 42), which interacts with CLPX and catalyzes ALA formation (16), is marked with an asterisk (N = 3). B, ALAS2 stability is increased in both Clpx^−/− and Clpp^−/− cells. This is quantitated in (i), data shown relative to HSP60 expression. ∗p < 0.05. Error bars denote SEM (N = 4). Representative gels are shown in (ii).