TMEM14C is required for erythroid mitochondrial heme metabolism

Abstract

The transport and intracellular trafficking of heme biosynthesis intermediates are crucial for hemoglobin production, which is a critical process in developing red cells. Here, we profiled gene expression in terminally differentiating murine fetal liver-derived erythroid cells to identify regulators of heme metabolism. We determined that TMEM14C, an inner mitochondrial membrane protein that is enriched in vertebrate hematopoietic tissues, is essential for erythropoiesis and heme synthesis in vivo and in cultured erythroid cells. In mice, TMEM14C deficiency resulted in porphyrin accumulation in the fetal liver, erythroid maturation arrest, and embryonic lethality due to profound anemia. Protoporphyrin IX synthesis in TMEM14C-deficient erythroid cells was blocked, leading to an accumulation of porphyrin precursors. The heme synthesis defect in TMEM14C-deficient cells was ameliorated with a protoporphyrin IX analog, indicating that TMEM14C primarily functions in the terminal steps of the heme synthesis pathway. Together, our data demonstrate that TMEM14C facilitates the import of protoporphyrinogen IX into the mitochondrial matrix for heme synthesis and subsequent hemoglobin production. Furthermore, the identification of TMEM14C as a protoporphyrinogen IX importer provides a genetic tool for further exploring erythropoiesis and congenital anemias.

Figure 7. The heme defect in Tmem14c-deficient MEL cells is complemented by the addition of DP.

(A) ⁵⁵Fe-metabolic labeling of differentiating MEL cells that were treated with either Fe-dextran (Fe) or Fe-dextran with DP (Fe+DP). Fe-dextran with DP supplementation, but not Fe-dextran alone, significantly increased heme synthesis in Tmem14c-deficient (CRISPR and shRNA) cells to similar quantities as untreated control cells. In contrast, Fe-dextran with DP does not complement the heme synthesis defect in Snx3-deficient cells. *P < 0.05. (B) Quantitation of total cellular iron by ⁵⁵Fe metabolic labeling. Fe-dextran and Fe-dextran with DP supplementation did not significantly alter total cellular iron content in control, Tmem14c-deficient, or Snx3-deficient cells. (C) Proposed model for the function of TMEM14C (brown cylinder) as a PPgenIX transporter, enabling access to PPOX and facilitating synthesis of PPIX and eventually heme in the mitochondria of developing erythroblasts. Heme synthesis enzymes are represented by light blue boxes, while heme and porphyrin intermediates are indicated in dark blue text.

Figure 6. TMEM14C is required for synthesis of PPIX.

HPLC analysis of porphyrin intermediates and heme in wild-type, CRISPR, control, and silenced cell lines and E12.5 fetal liver tissue. Porphyrinogens were oxidized to their corresponding porphyrins during isolation; porphyrin levels are a surrogate for porphyrinogen levels in vivo. (A) Uroporphyrin III (UROIII) levels are normal in differentiating Tmem14c-deficient cells in the absence and presence of exogenous ALA. (B) Coproporphyrin III (CPIII) levels are normal in the absence of exogenous ALA but are mildly elevated in ALA-supplemented CRISPR cells. Coproporphyrin III is significantly increased in Tmem14c^gt/gt fetal liver. (C) PPIX levels are reduced in Tmem14c-deficient cells in the absence and presence of exogenous ALA. PPIX levels are significantly decreased in Tmem14c^gt/gt fetal liver. As a control for our assay, PPIX is significantly elevated in Fech^Tm1Pas Fech^Tm1Pas (Fech) fetal liver. (D) Heme levels are significantly reduced in Tmem14c-deficient cells both in the absence and presence of exogenous ALA as well as in Tmem14c^gt/gt fetal liver tissue. (E) There is an increase in the level of total porphyrin excreted into the cell culture media of CRISPR cells. *P < 0.05.

Figure 5. TMEM14C is not required for mitochondrial iron homeostasis.

(A) Quantitation of cellular iron status by ⁵⁵Fe metabolic labeling shows that basal cellular iron import is not decreased in Tmem14c-deficient MEL cells. However, cellular iron content is decreased in Tmem14c-deficient erythroid cells during differentiation. (B) Deficiency in heme synthesis in Tmem14c-silenced cells is not due to a defect in mitochondrial iron content. Inductively coupled plasma analysis of mitochondrial iron shows that Tmem14c deficiency does not cause a defect in mitochondrial iron content either basally or during erythroid differentiation (left). This was confirmed by ⁵⁹Fe metabolic labeling and quantitation of mitochondrial iron from differentiating Tmem14c-silenced cells (right). (C) Normal activities for [2Fe-2S] cofactor–dependent mitochondrial aconitase, FECH, and cytosolic xanthine oxidase in Tmem14c-silenced cells exclude defects in [2Fe-2S] cluster assembly. *P < 0.05.

Figure 4. TMEM14C is required for heme synthesis in murine erythroid cells both basally and during terminal differentiation.

(A) qRT-PCR analysis shows a significant decrease in Tmem14c mRNA in both CRISPR and shRNA-silenced MEL cells compared with their respective controls. (B) Western blot analysis show deficiency of TMEM14C protein, but not the other heme synthesis enzymes, in CRISPR cells and shRNA-silenced MEL cells. (C) o-dianisidine staining shows a heme synthesis defect in CRISPR cells and shRNA-silenced cells (original magnification, ×20). (D) ⁵⁵Fe-Tf metabolic labeling demonstrates that basal heme synthesis (left) and heme synthesis in differentiating Tmem14c-deficient erythroid cells (right) are significantly decreased. *P < 0.05.

Figure 3. TMEM14C is specifically required for terminal differentiation of primary definitive murine erythroid cells.

(A) Primary murine fetal liver cells transduced with shRNA lentivirus, silencing the expression of Tmem14c mRNA, have significantly decreased hemoglobinization, compared with control cells. (B) E15.5 Tmem14c^gt/gt mice are profoundly anemic; their livers are deficient in hemoglobinization compared with wild-type littermates (original magnification, ×20). (C) Tmem14c mRNA expression is abrogated in fetal livers derived from E12.5 Tmem14c^gt/gt embryos. (D) Erythroid cells (original magnification, ×100) from E12.5 Tmem14c^gt/gt embryos are developmentally arrested. (E) Erythroid maturation arrest of E12.5 Tmem14c^gt/gt fetal liver population was confirmed by flow cytometry analysis of CD71 and TER119 expression. (F) Quantification of the R1–R5 subpopulations. Tmem14c^+/gt fetal liver erythropoiesis is similar to that in wild-type fetal livers. (G) Autofluorescent erythrocytes are visualized in Tmem14c^gt/gt and Fech^Tm1Pas Fech^Tm1Pas fetal livers under fluorescent light (original magnification, ×630) (λ,405 nm excitation and λ,620 nm emission; Supplemental Methods). Autofluorescent erythrocytes are not present in wild-type fetal livers. *P < 0.05.

Figure 2. Tmem14c is specifically required for maturation of the primitive erythroid lineage.

(A) Design of Tmem14c gene trap construct (E295C12) used to disrupt expression of Tmem14c. LTR, long term repeat; 6x opn, 6x osteopontin enhancer element; SA, splice acceptor sequence; β-Geo*, β-galactosidase gene fused with the neomycin resistance gene; pA, polyadenylation sequence; UTR, untranslated region. (B) Genomic PCR verifies disruption of Tmem14c locus. (C) Expression of Tmem14c mRNA is abrogated in a Tmem14c^gt/gt murine embryonic stem line. (D) The number of hemoglobinized cells is significantly reduced in Tmem14c^gt/gt cells, suggesting a defect of erythroid differentiation. (E) Primitive erythroid cells derived from Tmem14c^gt/gt embryoid bodies are developmentally arrested at the proerythroblast stage (original magnification, ×60). (F) The number of erythroid colonies formed is decreased in Tmem14c^gt/gt derived embryoid bodies. (G) Myeloid lineages derived from embryoid bodies are not affected by Tmem14c deficiency. *P < 0.05.

Figure 1. TMEM14C is enriched in differentiating murine erythroid cells and localizes to the inner mitochondrial membrane.

(A) RNAseq analysis of murine fetal liver cells sorted into 5 progressively differentiated erythroid subpopulations (R1–R5) shows that Tmem14c is upregulated during erythroid differentiation. (B) Tmem14c mRNA is expressed in hematopoietic organs, as shown by β-galactosidase staining (blue) of Tmem14c LacZ reporter expression in an E10.5 murine yolk sac (original magnification, ×63) and in situ hybridization of an E8.5 yolk sac (scale bar: 100 μm) and (C) fetal liver at E15.5 (pseudo-red; scale bar: 500 μm). (D) qRT-PCR shows Tmem14c mRNA is highly expressed in erythropoietic tissues and a MEL cell line. Tmem14c expression was normalized to Hprt levels. (E) Western blot analysis shows specific expression of TMEM14C protein in differentiating TER119⁺ murine fetal liver erythroid cells. HSP60 serves as a loading control. (F) Western blot analysis of fractionated Tmem14c-transfected HEK293T cells demonstrates localization of TMEM14C to the mitochondria. The control band indicates a protein that nonspecifically cross-reacts with MFRN1 antibody and migrates at a different molecular weight. (G) Confocal immunofluorescence microscopy (original magnification, ×63) shows that most of the transiently transfected FLAG-TMEM14C (fluorescein) colocalizes (merged, yellow) with HSP60 (rhodamine), a mitochondrial resident protein; nuclei were stained with DAPI (blue). (H) Transiently transfected FLAG-TMEM14C localizes to the inner mitochondrial membrane. TMEM14C, like TIM23, an inner mitochondrial protein, is sensitive to trypsin digestion when the outer mitochondrial membrane is disrupted by hypotonic swelling. The residual FLAG-TMEM14C that is trypsin resistant reflects mitochondria that are resistant to osmotic shock.