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The ubiquitous mitochondrial protein unfoldase CLPX regulates erythroid heme synthesis by control of iron utilization and heme synthesis enzyme activation and turnover

2021; Elsevier BV; Volume: 297; Issue: 2 Linguagem: Inglês

10.1016/j.jbc.2021.100972

ISSN

1083-351X

Autores

Catherine M. Rondelli, Mark Perfetto, Aidan Danoff, Hector A. Bergonia, Samantha Gillis, Leah M. O'Neill, Laurie K. Jackson, Gaël Nicolas, Hervé Puy, Richard West, John D. Phillips, Yvette Y. Yien,

Tópico(s)

Hemoglobin structure and function

Resumo

Heme plays a critical role in catalyzing life-essential redox reactions in all cells, and its synthesis must be tightly balanced with cellular requirements. Heme synthesis in eukaryotes is tightly regulated by the mitochondrial AAA+ unfoldase CLPX (caseinolytic mitochondrial matrix peptidase chaperone subunit X), which promotes heme synthesis by activation of δ-aminolevulinate synthase (ALAS/Hem1) in yeast and regulates turnover of ALAS1 in human cells. However, the specific mechanisms by which CLPX regulates heme synthesis are unclear. In this study, we interrogated the mechanisms by which CLPX regulates heme synthesis in erythroid cells. Quantitation of enzyme activity and protein degradation showed that ALAS2 stability and activity were both increased in the absence of CLPX, suggesting that CLPX primarily regulates ALAS2 by control of its turnover, rather than its activation. However, we also showed that CLPX is required for PPOX (protoporphyrinogen IX oxidase) activity and maintenance of FECH (ferrochelatase) levels, which are the terminal enzymes in heme synthesis, likely accounting for the heme deficiency and porphyrin accumulation observed in Clpx−/− cells. Lastly, CLPX is required for iron utilization for hemoglobin synthesis during erythroid differentiation. Collectively, our data show that the role of CLPX in yeast ALAS/Hem1 activation is not conserved in vertebrates as vertebrates rely on CLPX to regulate ALAS turnover as well as PPOX and FECH activity. Our studies reveal that CLPX mutations may cause anemia and porphyria via dysregulation of ALAS, FECH, and PPOX activities, as well as of iron metabolism. Heme plays a critical role in catalyzing life-essential redox reactions in all cells, and its synthesis must be tightly balanced with cellular requirements. Heme synthesis in eukaryotes is tightly regulated by the mitochondrial AAA+ unfoldase CLPX (caseinolytic mitochondrial matrix peptidase chaperone subunit X), which promotes heme synthesis by activation of δ-aminolevulinate synthase (ALAS/Hem1) in yeast and regulates turnover of ALAS1 in human cells. However, the specific mechanisms by which CLPX regulates heme synthesis are unclear. In this study, we interrogated the mechanisms by which CLPX regulates heme synthesis in erythroid cells. Quantitation of enzyme activity and protein degradation showed that ALAS2 stability and activity were both increased in the absence of CLPX, suggesting that CLPX primarily regulates ALAS2 by control of its turnover, rather than its activation. However, we also showed that CLPX is required for PPOX (protoporphyrinogen IX oxidase) activity and maintenance of FECH (ferrochelatase) levels, which are the terminal enzymes in heme synthesis, likely accounting for the heme deficiency and porphyrin accumulation observed in Clpx−/− cells. Lastly, CLPX is required for iron utilization for hemoglobin synthesis during erythroid differentiation. Collectively, our data show that the role of CLPX in yeast ALAS/Hem1 activation is not conserved in vertebrates as vertebrates rely on CLPX to regulate ALAS turnover as well as PPOX and FECH activity. Our studies reveal that CLPX mutations may cause anemia and porphyria via dysregulation of ALAS, FECH, and PPOX activities, as well as of iron metabolism. Heme is a prosthetic group comprising a central iron chelated by a tetrapyrrole ring. It is critical for many life-essential redox processes, such as detoxification, oxygen transport, circadian rhythm, and control of transcription and translation (1Feng D. Lazar M.A. Clocks, metabolism, and the epigenome.Mol. Cell. 2012; 47: 158-167Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 2Girvan H.M. Munro A.W. Heme sensor proteins.J. Biol. Chem. 2013; 288: 13194-13203Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 3Martínková M. Kitanishi K. Shimizu T. Heme-based globin-coupled oxygen sensors: Linking oxygen binding to functional regulation of diguanylate cyclase, histidine kinase, and methyl-accepting chemotaxis.J. Biol. Chem. 2013; 288: 27702-27711Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Most of the body's heme is synthesized in differentiating red cells, whose main function is to transport oxygen via hemoglobin (4Chen C. Paw B.H. Cellular and mitochondrial iron homeostasis in vertebrates.Biochim. Biophys. 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Rhee K.Y. Paw B.H. Baker T.A. Mitochondrial ClpX activates a key enzyme for heme biosynthesis and erythropoiesis.Cell. 2015; 161: 858-867Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). CLPX is a ring-shaped homo-hexamer and is best understood for its function in a proteasome-like enzyme complex with the peptidase CLPP (caseinolytic protease proteolytic subunit). The complex of CLPX and CLPP together forms the CLPXP ATP-dependent protease. The CLPXP protease is best understood to facilitate degradation of misfolded mitochondrial proteins. CLPX recognizes specific sequences in protein substrates and unfolds protein tertiary structures through its central pore, presenting the unfolded polypeptide chain to the CLPP proteolytic chamber (10Baker T.A. Sauer R.T. ClpXP, an ATP-powered unfolding and protein-degradation machine.Biochim. Biophys. Acta. 2012; 1823: 15-28Crossref PubMed Scopus (265) Google Scholar, 11Olivares A.O. Baker T.A. Sauer R.T. Mechanistic insights into bacterial AAA+ proteases and protein-remodelling machines.Nat. Rev. Microbiol. 2016; 14: 33-44Crossref PubMed Scopus (164) Google Scholar). Although CLPXP functions as a protease, detailed studies on yeast and vertebrate cells indicate that CLPX has functions that are distinct from its role in CLPXP. While Clpp−/− mice survive to adulthood (12Gispert S. Parganlija D. Klinkenberg M. Dröse S. Wittig I. Mittelbronn M. Grzmil P. Koob S. Hamann A. Walter M. Büchel F. Adler T. Hrabé de Angelis M. Busch D.H. Zell A. et al.Loss of mitochondrial peptidase Clpp leads to infertility, hearing loss plus growth retardation via accumulation of CLPX, mtDNA and inflammatory factors.Hum. Mol. Genet. 2013; 22: 4871-4887Crossref PubMed Scopus (112) Google Scholar, 13Jenkinson E.M. Rehman A.U. Walsh T. Clayton-Smith J. Lee K. Morell R.J. Drummond M.C. Khan S.N. Naeem M.A. Rauf B. Billington N. Schultz J.M. Urquhart J.E. Lee M.K. Berry A. et al.Perrault syndrome is caused by recessive mutations in CLPP, encoding a mitochondrial ATP-dependent chambered protease.Am. J. Hum. Genet. 2013; 92: 605-613Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 14Wang T. Babayev E. Jiang Z. Li G. Zhang M. Esencan E. Horvath T. Seli E. Mitochondrial unfolded protein response gene Clpp is required to maintain ovarian follicular reserve during aging, for oocyte competence, and development of pre-implantation embryos.Aging Cell. 2018; 17e12784Crossref PubMed Scopus (36) Google Scholar), Clpx−/− mouse embryos die before gastrulation (15Cheong A. Archambault D. Degani R. Iverson E. Tremblay K.D. Mager J. Nuclear-encoded mitochondrial ribosomal proteins are required to initiate gastrulation.Development. 2020; 147dev188714Crossref PubMed Scopus (9) Google Scholar). Saccharomyces cerevisiae express Clpx (Mcx1), but lack a Clpp homolog, suggesting that CLPX regulates protein unfolding independent of proteolysis (9Kardon J.R. Yien Y.Y. Huston N.C. Branco D.S. Hildick-Smith G.J. Rhee K.Y. Paw B.H. Baker T.A. Mitochondrial ClpX activates a key enzyme for heme biosynthesis and erythropoiesis.Cell. 2015; 161: 858-867Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Insights from model organisms show that CLPX can regulate heme synthesis by mediating activation (9Kardon J.R. Yien Y.Y. Huston N.C. Branco D.S. Hildick-Smith G.J. Rhee K.Y. Paw B.H. Baker T.A. Mitochondrial ClpX activates a key enzyme for heme biosynthesis and erythropoiesis.Cell. 2015; 161: 858-867Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) and degradation (16Kubota Y. Nomura K. Katoh Y. Yamashita R. Kaneko K. Furuyama K. Novel mechanisms for heme-dependent degradation of ALAS1 protein as a component of negative feedback regulation of heme biosynthesis.J. Biol. Chem. 2016; 291: 20516-20529Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) of the ALAS enzymes, which catalyze the committed step of heme synthesis. Yeast ALAS (Hem1) requires CLPX (Mcx1) for its full activation; as yeast lack a CLPP ortholog, CLPXP does not regulate Hem1 stability in yeast. Clpxa-deficient zebrafish were anemic, suggesting that CLPX was required for heme synthesis (9Kardon J.R. Yien Y.Y. Huston N.C. Branco D.S. Hildick-Smith G.J. Rhee K.Y. Paw B.H. Baker T.A. Mitochondrial ClpX activates a key enzyme for heme biosynthesis and erythropoiesis.Cell. 2015; 161: 858-867Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). These studies predicted that CLPX deficiency will cause accumulation of inactive ALAS protein and heme deficiency. However, erythroid cells expressing an ATPase inactive CLPX Gly298Asp mutant protein (ATPase activity being necessary for CLPX fucntion (9Kardon J.R. Yien Y.Y. Huston N.C. Branco D.S. Hildick-Smith G.J. Rhee K.Y. Paw B.H. Baker T.A. Mitochondrial ClpX activates a key enzyme for heme biosynthesis and erythropoiesis.Cell. 2015; 161: 858-867Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar)) had increased ALAS protein and ALAS activity. The elevated ALAS activity caused erythropoietic protoporphyria (EPP) resulting from accumulation of protoporphyrin IX (PPIX) (17Yien Y.Y. Ducamp S. van der Vorm L.N. Kardon J.R. Manceau H. Kannengiesser C. Bergonia H.A. Kafina M.D. Karim Z. Gouya L. Baker T.A. Puy H. Phillips J.D. Nicolas G. Paw B.H. Mutation in human CLPX elevates levels of delta-aminolevulinate synthase and protoporphyrin IX to promote erythropoietic protoporphyria.Proc. Natl. Acad. Sci. U. S. A. 2017; 114: E8045-E8052Crossref PubMed Scopus (41) Google Scholar) (Fig. 1A). These results suggested that regulation of heme synthesis by CLPX was not conserved across species, necessitating additional work to understand the role of CLPX in erythroid heme regulation and in hematologic diseases. To determine the role of CLPX in erythroid heme synthesis, we conducted loss-of-function studies in murine erythroid cells. As CLPX-interacting regions of yeast ALAS (Hem1), required for CLPX to interact with and activate ALAS (18Kardon J.R. Moroco J.A. Engen J.R. Baker T.A. Mitochondrial ClpX activates an essential biosynthetic enzyme through partial unfolding.Elife. 2020; 9e54387Crossref PubMed Scopus (9) Google Scholar), had little similarity to vertebrate ALAS2 (Fig. S1), we wanted to determine if CLPX was required for ALAS2 activation in mammalian cells. We used CRISPR/Cas9 to knock out the Clpx and Clpp genes in mouse erythroleukemia (MEL) cells and verified the loss of CLPX and CLPP protein and mRNA expression by western blot (Fig. 1B) and qRT-PCR (Fig. 1C). Benzidine staining of differentiated Clpx−/− and Clpp−/− MEL cells indicated that Clpx−/− cells were hemoglobin-deficient, consistent with published observations (Fig. 1D) (9Kardon J.R. Yien Y.Y. Huston N.C. Branco D.S. Hildick-Smith G.J. Rhee K.Y. Paw B.H. Baker T.A. Mitochondrial ClpX activates a key enzyme for heme biosynthesis and erythropoiesis.Cell. 2015; 161: 858-867Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). This was not caused by defects in mitochondrial activity (Fig. S2). Clpp−/− cells, predicted to accumulate active ALAS2 due to defective CLPXP-catalyzed protein degradation, had increased hemoglobin (Fig. 1D). To quantify the role of Clpx and Clpp in heme synthesis, we labeled newly synthesized heme with 55Fe. Clpx−/− MEL cells had decreased heme synthesis. In contrast, Clpp−/− MEL cells synthesized increased quantities of heme (Fig. 1E). As heme is an important transcriptional regulator of globin (19Kobayashi M. Kato H. Hada H. Itoh-Nakadai A. Fujiwara T. Muto A. Inoguchi Y. Ichiyanagi K. Hojo W. Tomosugi N. Sasaki H. Harigae H. Igarashi K. Iron-heme-Bach1 axis is involved in erythroblast adaptation to iron deficiency.Haematologica. 2017; 102: 454-465Crossref PubMed Scopus (14) Google Scholar, 20Suzuki H. Tashiro S. Hira S. Sun J. Yamazaki C. Zenke Y. Ikeda-Saito M. Yoshida M. Igarashi K. Heme regulates gene expression by triggering Crm1-dependent nuclear export of Bach1.EMBO J. 2004; 23: 2544-2553Crossref PubMed Scopus (157) Google Scholar, 21Tahara T. Sun J. Nakanishi K. Yamamoto M. Mori H. Saito T. Fujita H. Igarashi K. Taketani S. Heme positively regulates the expression of beta-globin at the locus control region via the transcriptional factor Bach1 in erythroid cells.J. Biol. Chem. 2004; 279: 5480-5487Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 22Zenke-Kawasaki Y. Dohi Y. Katoh Y. Ikura T. Ikura M. Asahara T. Tokunaga F. Iwai K. Igarashi K. Heme induces ubiquitination and degradation of the transcription factor Bach1.Mol. Cell. Biol. 2007; 27: 6962-6971Crossref PubMed Scopus (193) Google Scholar), we analyzed the effect of Clpx or Clpp deficiency on globin expression. Consistent with a heme defect in Clpx−/− cells, differentiating Clpx−/− cells experienced a significant decrease in Hbb-b1 and Hbb-b2 mRNA expression. Undifferentiated Clpx−/− cells also had a significant decrease in Hbb2 expression (Fig. 1F). These data confirm previous observations that CLPX is required for heme synthesis, while CLPP is not, and confirm that CLPX regulates heme independent of its role in the CLPXP proteolytic complex. Clpx deficiency also decreased globin mRNA expression, contributing to the hemoglobinization defect. To understand the role of Clpx in erythropoiesis in vivo, we characterized a zebrafish clpxb mutant (clpxbsa38141) obtained from the Zebrafish International Resource Center (ZIRC). Zebrafish encode two homologs of Clpx, clpxa, and clpxb. We previously investigated the role of the zebrafish clpxa gene in hematopoiesis, showing that clpxa deficiency caused a decrease in gata1-expressing erythroid progenitors and globin-expressing erythroid cells (9Kardon J.R. Yien Y.Y. Huston N.C. Branco D.S. Hildick-Smith G.J. Rhee K.Y. Paw B.H. Baker T.A. Mitochondrial ClpX activates a key enzyme for heme biosynthesis and erythropoiesis.Cell. 2015; 161: 858-867Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). As homologs in zebrafish arise by gene duplication, we wanted to determine if the clpxb homolog played redundant roles in erythropoiesis. The clpxbsa38141 mutant has an E129X nonsense mutation, which removes substantial portions of protein required for ATP binding. Homozygous mutant embryos express about half the clpxb mRNA as wild-type embryos due to nonsense mediated degradation (Fig. 2A). At 48 hpf, clpxb mutant embryos were anemic (Fig. 2B). To determine if the anemia resulted from erythroid differentiation defects, we quantitated erythroid cells in clpxb mutant Tg(globin-lcr:GFP) zebrafish (23Ganis J.J. Hsia N. Trompouki E. de Jong J.L. DiBiase A. Lambert J.S. Jia Z. Sabo P.J. Weaver M. Sandstrom R. Stamatoyannopoulos J.A. Zhou Y. Zon L.I. Zebrafish globin switching occurs in two developmental stages and is controlled by the LCR.Dev. Biol. 2012; 366: 185-194Crossref PubMed Scopus (85) Google Scholar). clpxb mutant embryos did not exhibit altered erythroid cell numbers, indicating that clpxb is not required for erythroid specification (Fig. 2C). Giemsa staining of sorted GFP+ cells revealed defects in clpxb erythroid cells, such as ruffled membranes and nonuniform morphology (Fig. 2D). In contrast to wild-type erythroid cells, clpxb mutant cells were larger and had more variable nuclear:cytoplasmic ratios (Fig. 2E) that were caused by increases in nuclear size (Fig. 2F) and decreases in cytoplasmic area (Fig. 2G). clpxb is hence required for erythroid heme synthesis and function but is not required for lineage determination or cell number. To determine if CLPX regulates ALAS2, we analyzed the expression of its steady-state mRNA and protein. qPCR analysis revealed that Alas2 mRNA decreased in undifferentiated Clpx−/− and Clpp−/− cells relative to wild-type cells. ALAS2 mRNA decreased in differentiated Clpx−/− cells, relative to wild-type cells, but remained unchanged in Clpp−/− cells (Fig. 3A, i). ALAS2 steady-state protein increased in both Clpx−/− and Clpp−/− cells (Fig. 3A, ii). These data suggested that CLPX and CLPP may regulate ALAS2 protein stability. In contrast to previous observations (24Seo J.H. Rivadeneira D.B. Caino M.C. Chae Y.C. Speicher D.W. Tang H.Y. Vaira V. Bosari S. Palleschi A. Rampini P. Kossenkov A.V. Languino L.R. Altieri D.C. The mitochondrial unfoldase-peptidase complex ClpXP controls Bioenergetics stress and metastasis.PLoS Biol. 2016; 14e1002507Crossref PubMed Scopus (72) Google Scholar), we did not observe statistically significant decreases in SDHB protein levels or activity in Clpx−/− and Clpp−/− MEL cells (Fig. S3). To determine if CLPXP regulates ALAS2 stability, we monitored ALAS2 turnover in WT, Clpx−/−, and Clpp−/− cells following inhibition of protein translation by cycloheximide (CHX) (17Yien Y.Y. Ducamp S. van der Vorm L.N. Kardon J.R. Manceau H. Kannengiesser C. Bergonia H.A. Kafina M.D. Karim Z. Gouya L. Baker T.A. Puy H. Phillips J.D. Nicolas G. Paw B.H. Mutation in human CLPX elevates levels of delta-aminolevulinate synthase and protoporphyrin IX to promote erythropoietic protoporphyria.Proc. Natl. Acad. Sci. U. S. A. 2017; 114: E8045-E8052Crossref PubMed Scopus (41) Google Scholar, 25Quadrini K.J. Bieker J.J. EKLF/KLF1 is ubiquitinated in vivo and its stability is regulated by activation domain sequences through the 26S proteasome.FEBS Lett. 2006; 580: 2285-2293Crossref PubMed Scopus (22) Google Scholar, 26Yien Y.Y. Bieker J.J. Functional interactions between erythroid Krüppel-like factor (EKLF/KLF1) and protein phosphatase PPM1B/PP2Cβ.J. Biol. Chem. 2012; 287: 15193-15204Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). ALAS2 was stabilized in Clpx−/− and Clpp−/− cells (Fig. 3B), echoing observations that CLPXP regulates ALAS1 turnover (16Kubota Y. Nomura K. Katoh Y. Yamashita R. Kaneko K. Furuyama K. Novel mechanisms for heme-dependent degradation of ALAS1 protein as a component of negative feedback regulation of heme biosynthesis.J. Biol. Chem. 2016; 291: 20516-20529Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). ALAS activity assays showed that Clpx−/− cells had significantly increased ALAS activity, indicating that CLPX is not required for ALAS activation (Fig. 4A). Strikingly, Clpp−/− cells had even higher ALAS enzyme activity than Clpx−/− cells. This dramatic increase in ALAS activity was most pronounced in differentiated Clpp−/− cells (Fig. 4A). The increase of ALAS activity in Clpp−/− relative to Clpx−/− cells cannot be attributed to differences in protein expression or stability (Fig. 3) suggesting that CLPP may play an inhibitory role in ALAS activation. An alternative explanation is that excess CLPX in Clpp−/− cells (i.e., CLPX that was not in a CLPXP complex) activated ALAS. The differential effect on ALAS activity did not translate to differences in ALA content as Clpx−/− and Clpp−/− cells had similar ALA levels (Fig. 4B). We quantitated PPIX, the terminal heme intermediate, to determine if porphyrin levels mirrored changes in ALA synthesis. Surprisingly, while Clpx−/− and Clpp−/− cells had similar increases in ALA levels, Clpp−/− cells contained significantly more PPIX than Clpx−/− cells. This was especially apparent in differentiated cells, suggesting that CLPXP may regulate heme synthesis downstream of ALA production (Fig. 4C). Hence, the heme defect in Clpx−/− erythroid cells was not a result of a decrease in ALAS activation or porphyrin production. Since CLPX is in the mitochondrial matrix (27Rhee H.W. Zou P. Udeshi N.D. Martell J.D. Mootha V.K. Carr S.A. Ting A.Y. Proteomic mapping of mitochondria in living cells via spatially restricted enzymatic tagging.Science. 2013; 339: 1328-1331Crossref PubMed Scopus (624) Google Scholar), we hypothesized that CLPX may regulate the matrix localized terminal heme synthesis enzymes, PPOX and FECH (27Rhee H.W. Zou P. Udeshi N.D. Martell J.D. Mootha V.K. Carr S.A. Ting A.Y. Proteomic mapping of mitochondria in living cells via spatially restricted enzymatic tagging.Science. 2013; 339: 1328-1331Crossref PubMed Scopus (624) Google Scholar) or mitochondrial iron metabolism (28Shaw G.C. Cope J.J. Li L. Corson K. Hersey C. Ackermann G.E. Gwynn B. Lambert A.J. Wingert R.A. Traver D. Trede N.S. Barut B.A. Zhou Y. Minet E. Donovan A. et al.Mitoferrin is essential for erythroid iron assimilation.Nature. 2006; 440: 96-100Crossref PubMed Scopus (411) Google Scholar). Western blot analysis showed that PPOX and FECH protein levels were slightly decreased in both knockout cell lines (Fig. 4D). However, only Clpx−/− cells had a decrease in PPOX activity relative to WT or Clpp−/− cells (Fig. 4E). FECH activity was decreased in Clpx−/− cells in nondifferentiating cells. In differentiating cells, both Clpx−/− and Clpp−/− cells had decreased FECH activity (Fig. 4F). As Clpp−/− cells did not exhibit a heme defect, it was unlikely that the FECH deficiency was the sole cause of the heme defect in Clpx−/− cells. We also considered the possibility that CLPX regulated mitochondrial iron utilization. We chemically complemented the heme defect in Clpx−/− cells with ferric ammonium citrate complexed with a lipophilic iron chelator, hinokitiol (29Grillo A.S. SantaMaria A.M. Kafina M.D. Cioffi A.G. Huston N.C. Han M. Seo Y.A. Yien Y.Y. Nardone C. Menon A.V. Fan J. Svoboda D.C. Anderson J.B. Hong J.D. Nicolau B.G. et al.Restored iron transport by a small molecule promotes absorption and hemoglobinization in animals.Science. 2017; 356: 608-616Crossref PubMed Scopus (59) Google Scholar). Treatment with Fe-hinokitiol increased heme content in Clpx−/− cells to levels close to wild-type. These results suggested that a mitochondrial iron defect contributed to the heme defect in Clpx−/− cells (Fig. 5A). We also treated zebrafish embryos spawned from frs or clpxb heterozygous incrosses with either vehicle (DMSO) or Fe-hinokitiol. frs mutant zebrafish lack expression of mfrn1, the mitochondrial iron transporter (28Shaw G.C. Cope J.J. Li L. Corson K. Hersey C. Ackermann G.E. Gwynn B. Lambert A.J. Wingert R.A. Traver D. Trede N.S. Barut B.A. Zhou Y. Minet E. Donovan A. et al.Mitoferrin is essential for erythroid iron assimilation.Nature. 2006; 440: 96-100Crossref PubMed Scopus (411) Google Scholar, 30Christenson E.T. Gallegos A.S. Banerjee A. In vitro reconstitution, functional dissection, and mutational analysis of metal ion transport by mitoferrin-1.J. Biol. Chem. 2018; 293: 3819-3828Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar) and served as a positive control for this experiment as their anemia was ameliorated by Fe-hinokitiol (29Grillo A.S. SantaMaria A.M. Kafina M.D. Cioffi A.G. Huston N.C. Han M. Seo Y.A. Yien Y.Y. Nardone C. Menon A.V. Fan J. Svoboda D.C. Anderson J.B. Hong J.D. Nicolau B.G. et al.Restored iron transport by a small molecule promotes absorption and hemoglobinization in animals.Science. 2017; 356: 608-616Crossref PubMed Scopus (59) Google Scholar). As expected, the numbers of severely anemic zebrafish embryos in frs incrosses were sharply reduced when treated with Fe-hinokitiol. Fe-hinokitiol significantly decreased numbers of embryos from clpxb heterozygote incrosses in both "severely anemic" and "anemic" categories, but the results were not as clear-cut as the frs mutant zebrafish (Fig. 5, B and C). These intermediate results indicate that iron deficiency plays a role in anemia in the clpxb mutant embryos (Fig. 5, B and C), but is not the sole cause of the anemia. Neither Clpx−/− nor Clpp−/− cells had a defect in MFRN1 expression (Fig. S4A) or mitochondrial iron levels (Fig. S4B), suggesting the defect is not in mitochondrial iron transport. Our results indicate that the primary mechanism by which CLPX regulates ALAS in vertebrate cells is by control of its turnover, rather than its activity. We have also shown that CLPX is required for maximal PPOX and FECH activity and regulates mitochondrial iron metabolism in differentiating erythroid cells. The porphyrin accumulation observed in these cells is augmented by increased ALAS2 activity in these cells (Fig. 5D). Although the requirement for Clpx in heme synthesis is conserved from yeast to vertebrates, several differences in CLPX function exist. The role of CLPX in heme regulation was originally discovered in S. cerevisiae yeast, which do not possess a CLPP ortholog. In yeast, CLPX functions exclusively as a mitochondrial protein unfoldase that unfolds Hem1/ALAS, facilitating the incorporation of its PLP cofactor, leading to ALAS/Hem1 activation (9Kardon J.R. Yien Y.Y. Huston N.C. Branco D.S. Hildick-Smith G.J. Rhee K.Y. Paw B.H. Baker T.A. Mitochondrial ClpX activates a key enzyme for heme biosynthesis and erythropoiesis.Cell. 2015; 161: 858-867Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Vertebrates, however, possess a CLPP ortholog, which forms the CLPXP protease with CLPX. In vertebrate cells, CLPXP regulates ALAS turnover (16Kubota Y. Nomura K. Katoh Y. Yamashita R. Kaneko K. Furuyama K. Novel mechanisms for heme-dependent degradation of ALAS1 protein as a component of negative feedback regulation of heme biosynthesis.J. Biol. Chem. 2016; 291: 20516-20529Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Previous studies on vertebrate CLPX did not examine the requirement of CLPX for ALAS activity or heme synthesis. Our current studies bridged this gap by assaying the effects of Clpx and Clpp loss of function on the activities of heme synthetic enzymes and ALAS2 stability, as CLPX was previously shown to be required for erythroid hemoglobinization in zebrafish (9Kardon J.R. Yien Y.Y. Huston N.C. Branco D.S. Hildick-Smith G.J. Rhee K.Y. Paw B.H. Baker T.A. Mitochondrial ClpX activates a key enzyme for heme biosynthesis and erythropoiesis.Cell. 2015; 161: 858-867Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Clpx−/− erythroid cells have elevated ALA content and ALAS activity, unlike in yeast. In contrast to previous predictions in yeast, the heme defect in Clpx−/− erythroid cells was not due to defects in ALAS activity (Fig. 4, A and B). However, ALAS activity in Clpp−/− cells is higher than that of Clpx−/− cells even though its steady-state protein levels in Clpx−/− and Clpp−/− cells are similar (Fig. 3A). This suggests that CLPX may play a nonessential role in ALAS activation, leading to Clpp−/− cells exhibiting a "superactivated" ALAS phenotype as CLPX activates accumulated ALAS2. PPIX content was elevated in Clpx−/− and Clpp−/− cells, caused by a combination of elevated ALA production with downstream heme synthesis and iron metabolism defects (Fig. 4C). Clpx−/− cells had decreased PPOX and FECH activity, partially accounting for the heme defect (Fig. 4, E and F). The increase in PPIX levels was due to increased protoporphyrinogen IX (PPgenIX) caused by ALA overproduction and defects in PPOX and FECH activity. PPgenIX auto-oxidises to PPIX (31Poulson R. Polglase W.J. The enzymic conversion of protoporphyrinogen IX to protoporphyrin IX. Protoporphyrinogen oxidase activity in mitochondrial extracts of Saccharomyces cerevisiae.J. Biol. Chem. 1975; 250: 1269-1274Abstract Full Text PDF PubMed Google Scholar) and leads to increased measurable PPIX even with decreased PPOX activity in Clpx−/− cells. In differentiating MEL cells, FECH activity was decreased in Clpp−/− cells, which exacerbated PPIX accumulation, although this did not cause heme deficiency. The heme defect in Clpx−/− cells was exacerbated by an iron defect, which was ameliorated by exogenous iron (Fig. 5, A and B). While CLPX is best known for its proteolytic function (11Olivares A.O. Baker T.A. Sauer R.T. Mechanistic insights into bacterial AAA+ proteases and protein-remodelling machines.Nat. Rev. Microbiol. 2016; 14: 33-44Crossref PubMed Scopus (164) Google Scholar), it has roles that are distinct from CLPXP-mediated proteolysis. Our data support the premise that CLPX has CLPXP-independent functions in heme synthesis. While PPOX protein levels are slightly decreased in Clpx−/− and Clpp−/− MEL cells, PPOX activity was decreased in only Clpx−/− cells, suggesting that CLPX plays a role in PPOX activation. Further, iron complementation studies suggest that Clpx−/−, but not Clpp−/−, erythroid cells have an iron metabolism defect. Our studies suggest that CLPX regulates the function of mitochondrial matrix proteins that regulate heme synthesis in erythroid cells. The interactions between CLPX, CLPP, and erythroid heme synthesis are complex and are likely to be tissue and developmental stage specific. Several CLPXP-binding proteins are iron–sulfur (Fe-S) cluster proteins that are involved in oxidative phosphorylation or Fe-S cluster assembly (32Fischer F. Langer J.D. Osiewacz H.D. Identification of potential mitochondrial CLPXP protease interactors and substrates suggests its central role in energy metabolism.Sci. Rep. 2015; 5: 18375Crossref PubMed Scopus (42) Google Scholar). A mouse model of Friedreich ataxia with a striated-muscle specific frataxin knockout showed an increase in expression of the CLPP and LON proteases associated with a decrease in mitochondrial Fe-S proteins. These included FECH, NDUFS3, and SDHB, which are involved in heme synthesis and oxidative phosphorylation (33Guillon B. Bulteau A.L. Wattenhofer-Donzé M. Schmucker S. Friguet B. Puccio H. Drapier J.C. Bouton C. Frataxin deficiency causes upregulation of mitochondrial Lon and ClpP proteases and severe loss of mitochondrial Fe-S proteins.FEBS J. 2009; 276: 1036-1047Crossref PubMed Scopus (59) Google Scholar). These data contrast with the decrease in FECH activity that we observed in our Clpx−/− and Clpp−/− erythroid cell lines, although the decreased FECH activity may be caused by defects in iron metabolism in Clpx−/− cells (Fig. 4, A and C). Unlike previous studies on prostate adenocarcinoma cells (24Seo J.H. Rivadeneira D.B. Caino M.C. Chae Y.C. Speicher D.W. Tang H.Y. Vaira V. Bosari S. Palleschi A. Rampini P. Kossenkov A.V. Languino L.R. Altieri D.C. The mitochondrial unfoldase-peptidase complex ClpXP controls Bioenergetics stress and metastasis.PLoS Biol. 2016; 14e1002507Crossref PubMed Scopus (72) Google Scholar), SDHB protein levels and activity were relatively unchanged in Clpx−/− and Clpp−/− MEL cells (Fig. S3). Lastly, CLPXP interacts with and degrades ALAS1 in the presence of heme, but dissociates from ALAS1 when heme synthesis is suppressed; in contrast, ALAS2 turnover is not increased during erythroid differentiation, when there are increased levels of heme (Fig. 3B). This suggests that unlike ALAS1, CLPXP does not increase the degradation of ALAS2 under high heme conditions. It is likely that the specific roles of CLPXP are regulated by cell-specific proteins. During terminal differentiation, the function of erythroid mitochondria shifts toward heme production for hemoglobin synthesis (8Yien Y.Y. Shi J. Chen C. Cheung J.T.M. Grillo A.S. Shrestha R. Li L. Zhang X. Kafina M.D. Kingsley P.D. King M.J. Ablain J. Li H. Zon L.I. Palis J. et al.FAM210B is an erythropoietin target and regulates erythroid heme synthesis by controlling mitochondrial iron import and ferrochelatase activity.J. Biol. 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Invest. 2014; 124: 4294-4304Crossref PubMed Scopus (40) Google Scholar, 37Zhang J. Socolovsky M. Gross A.W. Lodish H.F. Role of Ras signaling in erythroid differentiation of mouse fetal liver cells: Functional analysis by a flow cytometry-based novel culture system.Blood. 2003; 102: 3938-3946Crossref PubMed Scopus (312) Google Scholar). It is therefore revealing that CLPXP regulates heme synthesis at multiple points in the pathway, but is not essential for regulating Fe-S proteins in erythroid cells. Our data shed light on how a dominant CLPXG298D heterozygous mutation caused erythroid protoporphyria (17Yien Y.Y. Ducamp S. van der Vorm L.N. Kardon J.R. Manceau H. Kannengiesser C. Bergonia H.A. Kafina M.D. Karim Z. Gouya L. Baker T.A. Puy H. Phillips J.D. Nicolas G. Paw B.H. Mutation in human CLPX elevates levels of delta-aminolevulinate synthase and protoporphyrin IX to promote erythropoietic protoporphyria.Proc. Natl. Acad. Sci. U. S. A. 2017; 114: E8045-E8052Crossref PubMed Scopus (41) Google Scholar). CLPXG298D lacked ATPase activity and hetero-oligomerized with wild-type CLPX, decreasing the ATPase activity of the CLPX hexamer. We previously proposed that the WT/G298D hetero-hexamer could activate, but not degrade ALAS, causing accumulation of active ALAS and overproduction of PPIX, leading to EPP. Our new data indicate that CLPX is not required for activation of ALAS, suggesting that CLPX-EPP can be caused by stabilization of ALAS alone. We also show that CLPX and CLPP are required for optimal FECH activity. The decrease in FECH activity can exacerbate PPIX accumulation caused by ALA overproduction and may be compounded by an iron defect. CLPX mutations contribute to metabolic disorders in human patients and animal models (12Gispert S. Parganlija D. Klinkenberg M. Dröse S. Wittig I. Mittelbronn M. Grzmil P. Koob S. Hamann A. Walter M. Büchel F. Adler T. Hrabé de Angelis M. Busch D.H. Zell A. et al.Loss of mitochondrial peptidase Clpp leads to infertility, hearing loss plus growth retardation via accumulation of CLPX, mtDNA and inflammatory factors.Hum. Mol. Genet. 2013; 22: 4871-4887Crossref PubMed Scopus (112) Google Scholar, 13Jenkinson E.M. Rehman A.U. Walsh T. Clayton-Smith J. Lee K. Morell R.J. Drummond M.C. Khan S.N. Naeem M.A. Rauf B. Billington N. Schultz J.M. Urquhart J.E. Lee M.K. Berry A. et al.Perrault syndrome is caused by recessive mutations in CLPP, encoding a mitochondrial ATP-dependent chambered protease.Am. J. Hum. Genet. 2013; 92: 605-613Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 17Yien Y.Y. Ducamp S. van der Vorm L.N. Kardon J.R. Manceau H. Kannengiesser C. Bergonia H.A. Kafina M.D. Karim Z. Gouya L. Baker T.A. Puy H. Phillips J.D. Nicolas G. Paw B.H. Mutation in human CLPX elevates levels of delta-aminolevulinate synthase and protoporphyrin IX to promote erythropoietic protoporphyria.Proc. Natl. Acad. Sci. U. S. 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We propose that CLPX interacts with cell-specific factors to couple mitochondrial metabolism with cellular requirements. Unraveling the complexities of CLPX function will be key for designing therapies for metabolic diseases and mitochondriopathies. Methods are provided in the supporting information. Vertebrate animal studies were performed in compliance with Institutional Animal Care and Use Committee protocols at the University of Delaware. All data are contained in the manuscript. This article contains supporting information (8Yien Y.Y. Shi J. Chen C. Cheung J.T.M. Grillo A.S. Shrestha R. Li L. Zhang X. Kafina M.D. Kingsley P.D. King M.J. Ablain J. Li H. Zon L.I. Palis J. et al.FAM210B is an erythropoietin target and regulates erythroid heme synthesis by controlling mitochondrial iron import and ferrochelatase activity.J. Biol. Chem. 2018; 293: 19797-19811Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 17Yien Y.Y. Ducamp S. van der Vorm L.N. Kardon J.R. Manceau H. Kannengiesser C. Bergonia H.A. Kafina M.D. Karim Z. Gouya L. Baker T.A. Puy H. Phillips J.D. Nicolas G. Paw B.H. Mutation in human CLPX elevates levels of delta-aminolevulinate synthase and protoporphyrin IX to promote erythropoietic protoporphyria.Proc. Natl. Acad. Sci. U. S. A. 2017; 114: E8045-E8052Crossref PubMed Scopus (41) Google Scholar, 29Grillo A.S. SantaMaria A.M. Kafina M.D. Cioffi A.G. Huston N.C. Han M. Seo Y.A. Yien Y.Y. Nardone C. Menon A.V. Fan J. Svoboda D.C. Anderson J.B. Hong J.D. Nicolau B.G. et al.Restored iron transport by a small molecule promotes absorption and hemoglobinization in animals.Science. 2017; 356: 608-616Crossref PubMed Scopus (59) Google Scholar, 36Yien Y.Y. Robledo R.F. Schultz I.J. Takahashi-Makise N. Gwynn B. Bauer D.E. Dass A. Yi G. Li L. Hildick-Smith G.J. Cooney J.D. Pierce E.L. Mohler K. Dailey T.A. Miyata N. et al.TMEM14C is required for erythroid mitochondrial heme metabolism.J. Clin. Invest. 2014; 124: 4294-4304Crossref PubMed Scopus (40) Google Scholar). The authors declare that they have no conflicts of interest with the contents of this article. We thank Karl Schmitz and Molly Sutherland for helpful discussions, Amber Stratman for critical review of the manuscript, and Meilin Chen for technical assistance. C. M. R., M. P., A. D., G. N., H. P., J. D. P., and Y. Y. Y. conceptualization; C. M. R. and Y. Y. Y. data curation; C. M. R., M. P., A. D., and Y. Y. Y. formal analysis; C. M. R. and A. D. validation; C. M. R., M. P., A. D., H. P., S. G., L. O. N., L. J., G. N., H. P., R. W., J. D. P., and Y. Y. Y. investigation; C. M. R., M. P., A. D., H. B., S. G., G. N., H. P., R. W., J. D. P., and Y. Y. Y. writing-review and editing; H. B., R. W., and J. D. P. methodology; Y. Y .Y. supervision; Y. Y. Y. funding acquisition; Y. Y. Y. writing-original draft; Y. Y. Y. project administration. This work was supported by the Cooley's Anemia Foundation (Y. Y. Y.) and National Institutes of Health grants K01 DK106156 , R03 DK118307 , P01 HL032262 , P20GM104316 , R35 GM1133560 and a U. Utah CIHD pilot grant (Y. Y. Y.), and U54 DK083909 (J. D. P). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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