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Mitochondrial ClpX Activates a Key Enzyme for Heme Biosynthesis and Erythropoiesis - PubMed

  • ️Thu Jan 01 2015

Mitochondrial ClpX Activates a Key Enzyme for Heme Biosynthesis and Erythropoiesis

Julia R Kardon et al. Cell. 2015.

Abstract

The mitochondrion maintains and regulates its proteome with chaperones primarily inherited from its bacterial endosymbiont ancestor. Among these chaperones is the AAA+ unfoldase ClpX, an important regulator of prokaryotic physiology with poorly defined function in the eukaryotic mitochondrion. We observed phenotypic similarity in S. cerevisiae genetic interaction data between mitochondrial ClpX (mtClpX) and genes contributing to heme biosynthesis, an essential mitochondrial function. Metabolomic analysis revealed that 5-aminolevulinic acid (ALA), the first heme precursor, is 5-fold reduced in yeast lacking mtClpX activity and that total heme is reduced by half. mtClpX directly stimulates ALA synthase in vitro by catalyzing incorporation of its cofactor, pyridoxal phosphate. This activity is conserved in mammalian homologs; additionally, mtClpX depletion impairs vertebrate erythropoiesis, which requires massive upregulation of heme biosynthesis to supply hemoglobin. mtClpX, therefore, is a widely conserved stimulator of an essential biosynthetic pathway and uses a previously unrecognized mechanism for AAA+ unfoldases.

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Figures

Figure 1
Figure 1

MCX1 interacts chemically and genetically with the heme biosynthetic pathway. (A) The metabolic pathway for the first step of heme biosynthesis in non-plant eukaryotes. The genetic and chemical interaction profile of MCX1 is highly correlated with the profiles of yeast genes (HEM25, HEM1, and HEM2, shown in red) involved in the first steps of heme biosynthesis. Dashed lines indicate uncertainty in assigning Hem25 to glycine uptake or ALA export. Gray bars indicate mitochondrial membranes. See Fig. S1 for alignment showing conservation among ClpX homologs. (B) MCX1, HEM1, and HEM25 alleles exhibit synthetic phenotypes. Five-fold serial dilutions from cell suspensions with OD600 = 1 were pinned on YP + 2% agar, + 2% glucose or 3% glycerol. +ALA indicates 50 μg/mL ALA. Growth on glucose after 2 d and on glycerol after 3 d is shown.

Figure 2
Figure 2

Mcx1 promotes heme biosynthesis at the step of ALA synthesis. (A) Total porphyrin levels were measured by fluorescence in oxalic acid cell extracts (ex. 400 nm, em. 662 nm; p < 0.001 for difference between wt and MCX1 mutants). +ALA indicates supplementation of growth medium with 50 μg/ml ALA. See also Fig. S2. (B) Metabolites involved in the first step of heme biosynthesis (KG, α–ketoglutarate; SA, succinic acid; GLY, glycine; GLX, glyoxylate; SER, serine) were measured in extracts of the indicated yeast strains by LC-MS. P < 0.001 for ALA perturbation in mcx1Δ cells. (C) ALA levels in cell extracts were measured using modified Ehrlich’s reagent. p ≤ 10−5 for ALA reduction in MCX1 and HEM1 mutants. (D) Mcx1 was isolated with α-FLAG antibody-conjugated beads from cells harboring HEM1-3xMYC and MCX1-3xFLAG (wt or Mcx1EQ (EQ)) or untagged Mcx1 (−)) alleles at the genomic loci, and eluted with 3xFLAG peptide. The eluate was analyzed by Western blot for Mcx1 (α-FLAG) and Hem1 (α-Myc). See Extended Experimental Procedures for detailed procedure. (E) Cellular levels of Hem1-3xMyc were analyzed by Western blot, using alkaline cell extracts (von der Haar, 2007). Hem1-3xMyc intensity: in mcx1Δ = 1.1 ± 0.1 relative to wt, p = 0.35 for difference; in hem1-DAmP = 0.3 ± 0.1, p = 0.01. The mitochondrial protein Por1 was probed as a loading control. Error bars represent mean ± SD.

Figure 3
Figure 3

Mcx1 accelerates incorporation of PLP cofactor into Hem1. (A) Rate of apoHem1 activation by PLP. apoHem1 (3 μM) was incubated with PLP (50 μM) and ATP (2 μM), with (orange) or without Mcx1 (2 μM) (blue), and assayed for ALA synthase activity at indicated times using modified Ehrlich’s reagent. (B) ALAS activity resulting from 4 min incubation of apoHem1 +/− Mcx1, +/− ATP, assayed as in (A). p < 0.0001 for stimulation by Mcx1 + ATP. (C) PLP binding to apoHem1 was monitored by pyridoxyllysine fluorescence (ex. 434 nm, em. 515 nm). (D) Rates of PLP binding to apoHem1 determined by linear fits to fluorescence increase between 100-200 s in (C). p < 0.0001 for stimulation by Mcx1. Error bars represent mean ± SD. See also Fig. S3 for holoHem1 activity measurements.

Figure 4
Figure 4

Mcx1 requires the ClpX translocating pore loops to activate Hem1 and promote ALA production. (A) (left) Pore loops are highlighted on a cross-section diagram of a ClpX hexamer. RKH loops are shown in yellow, pore-1 loops in dark orange, and pore-2 loops in blue. See also Fig. S1 for pore loop sequences. (right) ALA levels in cell extracts were measured by modified Ehrlich’s reagent and normalized to wildtype. Cells harbored indicated mutations at the genomic MCX1 locus. p < 0.0001 for ALA reduction in all MCX1 mutants. (B) Co-immuoprecipitation of apo- and holoHem1 with Mcx1-3xFLAG variants was tested, using purified proteins. Proteins were separated by SDS-PAGE and stained with Sypro Orange. Lower panel (“Hem1 rescaled”) shows Hem1 bands, rescaled to maximum Hem1 intensity. Mcx1 variants are indicated as follows: wt = WT; Walker B E206Q = EQ; pore-1 Y174A = YA. p < 0.001 for more apoHem1 than holoHem1 bound by each Mcx1 variant. (C) ALAS activity resulting from 4 min incubation of apoHem1 with 50 μM PLP, +/− Mcx1Y174A, +/− ATP, assayed as in Fig. 3A. p < 0.05 for suppression of Hem1 by Mcx1Y174A both with and without ATP. Error bars represent mean ± SD.

Figure 5
Figure 5

Mammalian mtClpX stimulates PLP activation of apoALAS2, and does not direct apoALAS2 for degradation by mtClpP. (A) apoALAS2 activation by PLP in vitro. Recombinant human apoALAS2 (5 μM) was incubated with PLP, with mouse mtClpX (2 μM hexamer) and human mtClpP (3 μM 14-mer) as indicated, and activation between 4 and 10 minutes was measured by an NAD-coupled assay. p < 0.01 for acceleration of apoALAS2 activation by mtClpX, and p < 0.05 for acceleration by mtClpXP. See also Fig. S4, for ATP dependence of acceleration. (B, C) mtClpXP degradation test. apoALAS2 or α-casein (5 μM each) were incubated with mouse mtClpX (0.3 μM hexamer), human mtClpP (0.8 μM 14-mer) and ATP regenerating system (including 4 mM ATP, except where noted) at 30°C, and aliquots were withdrawn and quenched with SDS at indicated timepoints. Proteins were separated by SDS-PAGE and stained with Sypro Orange. Quantitation of degradation is shown in (B), and gel is shown in (C). (D) Mouse mtClpX (0.3 μM hexamer) ATPase was monitored by NADH-coupled assay, in the presence of human mtClpP (0.8 μM 14-mer) and apoALAS2 (“apoA2”, 10 μM) as indicated. p < 0.01 for suppression of ATPase by mtClpP, and p < 0.05 for stimulation of ATPase by apoALAS2. Error bars represent mean ± SD.

Figure 6
Figure 6

mtClpX is important for vertebrate heme biosynthesis and erythropoiesis. (A) Relative mRNA abundance for human CLPX, CLPP, ALAS2, and SLC25A38 (indicated as S25A38) throughout erythropoiesis as indicated in a microarray dataset described in (Novershtern et al., 2011). Erythroid development stages were defined by cell type specific markers: 1, CD34+ CD71+ GlyA−; 2, CD34− CD71+ GlyA−; 3, CD34− CD71+ GlyA+; 4, CD34− CD71low GlyA+; 5, CD34− CD71− GlyA+. See also Fig. S5A,B for expression in MEL cells and zebrafish embryos. (B) o-dianisidine staining (brown) for hemoglobinized red cells in zebrafish embyos. Embyros were grown from zygotes injected at the 1-2 cell stage with clpxa-targeting morpholinos or uninjected zygotes (control). (C) Erythrocyte development at 72 h post-fertiliztion (hpf) was quantified by flow cytometry, using dissociated cells from Tg(globin-LCR:eGFP) zebrafish. P ≤ 0.01 for erythrocyte reduction by clpxa knockdown with either morpholino. See also Fig. S5C-E for qPCR quantitation of clpxa knockdown and nontargeting morpholino injections. (D) Rescue of clpxa MOb-induced anemia by ALA supplementation. Tg(globin-LCR:eGFP) zebrafish embryos were supplemented with 2 mM ALA from 24 to 72 hpf, upon which GFP+ erythrocytes were quantified by flow cytometry. p = 0.025 for rescue of anemia in clpxa knockdown embryos by ALA supplementation (E) Heterozygous sauternes or frascati zebrafish were crossed, and progeny were grown for 72 hpf, with or without ALA supplementation as in (D). Anemia was assayed by o-dianisidine staining. p = 0.04 for rescue of anemia in sauternes+/− progeny by ALA. n=52 for sauternes −ALA; n=43 for sauternes +ALA; n=98 for frascati −ALA; n=122 for frascati +ALA. Error bars represent mean ± SD.

Figure 7
Figure 7

Model for mtClpX activation of ALAS. ALAS is unfolded by the mitochondrial import machinery, and refolds in the mitochondrial matrix. Newly folded ALAS binds PLP slowly on its own; partial unfolding by mtClpX renders the active site of ALAS more accessible to PLP, thus accelerating holoenzyme formation. Mitochondrial import machinery (light gray), ALAS (dark gray), mtClpX (purple), and PLP (green) are diagrammed.

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