TMEM14C is required for erythroid mitochondrial heme metabolism
Yvette Y. Yien, … , Luanne L. Peters, Barry H. Paw
Yvette Y. Yien, … , Luanne L. Peters, Barry H. Paw
Published August 26, 2014
Citation Information: J Clin Invest. 2014;124(10):4294-4304. https://doi.org/10.1172/JCI76979.
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Research Article Hematology

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.

Authors

Yvette Y. Yien, Raymond F. Robledo, Iman J. Schultz, Naoko Takahashi-Makise, Babette Gwynn, Daniel E. Bauer, Abhishek Dass, Gloria Yi, Liangtao Li, Gordon J. Hildick-Smith, Jeffrey D. Cooney, Eric L. Pierce, Kyla Mohler, Tamara A. Dailey, Non Miyata, Paul D. Kingsley, Caterina Garone, Shilpa M. Hattangadi, Hui Huang, Wen Chen, Ellen M. Keenan, Dhvanit I. Shah, Thorsten M. Schlaeger, Salvatore DiMauro, Stuart H. Orkin, Alan B. Cantor, James Palis, Carla M. Koehler, Harvey F. Lodish, Jerry Kaplan, Diane M. Ward, Harry A. Dailey, John D. Phillips, Luanne L. Peters, Barry H. Paw

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Figure 1

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

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TMEM14C is enriched in differentiating murine erythroid cells and locali...
(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.