Recombinant Danio rerio Transmembrane protein 14C (tmem14c)

What is TMEM14C and what is its primary function in cellular metabolism?

TMEM14C is an inner mitochondrial membrane protein that plays an essential role in erythroid mitochondrial heme metabolism. Research demonstrates that TMEM14C facilitates the import of protoporphyrinogen IX into the mitochondrial matrix for heme synthesis . This function is critical for erythropoiesis and proper red blood cell development. The protein is enriched in vertebrate hematopoietic tissues, suggesting its specialized role in blood cell formation .

In TMEM14C-deficient models, protoporphyrin IX synthesis is blocked, resulting in accumulation of porphyrin precursors . This disruption in heme synthesis leads to profound developmental consequences, including erythroid maturation arrest and embryonic lethality due to severe anemia in mouse models .

What is the molecular structure of Danio rerio TMEM14C protein?

Danio rerio TMEM14C is a transmembrane protein with the following amino acid sequence:
MAVDWAGYGYAALVASGGVIGYVKAGSVPSLAAGLVFGGLAGFGAYQTSQDPGNIWVSLAASGTALAIMGKRFYNSRKITPAGLIAGASVLMLAKLGAGMLQKPQKS .

The protein has a full-length expression region spanning amino acids 1-107 . As an inner mitochondrial membrane protein, TMEM14C contains hydrophobic domains that anchor it within the membrane, allowing it to function in the transport of porphyrin compounds. The protein is identified in UniProt under accession number Q0P436 and has synonyms including c6orf53 and zgc:153439 .

Why is zebrafish (Danio rerio) used as a model organism for TMEM14C research?

Zebrafish (Danio rerio) has emerged as a valuable model for studying TMEM14C function for several reasons:

• Zebrafish has been extensively used as a model organism for identifying genes required for early vertebrate development .

• The transparency of zebrafish embryos allows for direct visualization of developmental processes, including those related to hematopoiesis.

• Zebrafish develop tumors in a variety of tissues and are susceptible to chemical carcinogens and oncogenes in a manner similar to conventional mouse models .

• Many spontaneous and chemically-induced tumor types in zebrafish are histologically similar to their mammalian counterparts .

• The conservation of heme biosynthetic pathways between zebrafish and mammals makes it an excellent model for studying TMEM14C's role in erythropoiesis.

These characteristics make zebrafish an efficient and informative model system for investigating TMEM14C's biological functions and potential implications in disease states.

What techniques are most effective for studying TMEM14C function in erythroid cells?

Several complementary techniques have proven effective for investigating TMEM14C function in erythroid cells:

• Genetic Manipulation Approaches:

  • CRISPR/Cas9-mediated gene editing to generate stable compound heterozygote knockout cells

  • shRNA silencing to create stable knockdown models

  • Morpholino-based knockdown in zebrafish embryos

• Functional Assessment Methods:

  • 55Fe labeling to quantify heme synthesis rates in normal and TMEM14C-deficient cells

  • o-dianisidine staining to visualize hemoglobinized cells for qualitative assessment of heme synthesis

  • Flow cytometry analysis to evaluate erythroid differentiation using markers such as TER119 and CD71

• Biochemical Analysis:

  • Measurement of mitochondrial iron levels using inductively coupled plasma mass spectrometry

  • Assessment of enzyme activities including mitochondrial aconitase, ferrochelatase (FECH), and cytosolic xanthine oxidase to evaluate iron-sulfur cluster assembly

  • Quantification of porphyrin intermediates to identify metabolic blocks in the heme synthesis pathway

These methodological approaches provide complementary data that together build a comprehensive understanding of TMEM14C's role in erythroid development and heme metabolism.

How should researchers store and handle recombinant Danio rerio TMEM14C protein?

Proper storage and handling of recombinant Danio rerio TMEM14C protein is critical for maintaining its structural integrity and biological activity:

• Storage Conditions:

  • Store at -20°C for routine usage

  • For extended storage, conserve at -20°C or -80°C

  • Maintain in a Tris-based buffer with 50% glycerol, specifically optimized for this protein

• Handling Recommendations:

  • Avoid repeated freezing and thawing as this can compromise protein integrity

  • Store working aliquots at 4°C for up to one week to minimize freeze-thaw cycles

  • When preparing aliquots, use sterile technique to prevent contamination

• Quality Control Considerations:

  • Verify protein integrity by SDS-PAGE before experimental use

  • Confirm biological activity using appropriate functional assays

  • Consider including positive controls when evaluating TMEM14C function in experimental systems

Following these storage and handling protocols will help ensure consistent and reliable experimental results when working with recombinant Danio rerio TMEM14C.

What are the optimal approaches for generating TMEM14C knockout or knockdown models?

Researchers have successfully employed several strategies to generate TMEM14C-deficient models:

• CRISPR/Cas9 Genome Editing:

  • This approach has been used to generate stable compound heterozygote knockout cells in MEL cell lines

  • The technique allows for precise genomic modifications while minimizing off-target effects

  • Validation should include confirmation of both genomic alterations and protein depletion

• shRNA Silencing:

  • Stable Tmem14c knockdown MEL clones have been generated using shRNA technology

  • This method provides the advantage of creating stable cell lines with significantly reduced TMEM14C expression

  • Western blot analysis should be used to confirm reduction in protein levels

• Knockout Cell Lines:

  • Commercial TMEM14C knockout cell lines (such as HEK293-based models) are available for research applications

  • These lines offer advantages including high transfection efficiency, ease of culture, and a human cellular context

When implementing these approaches, researchers should include appropriate controls and validate TMEM14C depletion at both mRNA and protein levels to ensure experimental rigor and reproducibility.

How does TMEM14C deficiency affect erythroid development and heme metabolism?

TMEM14C deficiency has profound effects on erythroid development and heme metabolism, as demonstrated in multiple experimental systems:

• Developmental Consequences:

  • In mouse models, TMEM14C deficiency results in embryonic lethality due to profound anemia

  • Flow cytometry analyses reveal a decrease in terminally differentiating TER119+ erythroid cells in Tmem14c-deficient fetal livers, while erythroid progenitor numbers remain unchanged

  • The defect is erythroid-specific, as other hematopoietic lineages develop normally in the absence of TMEM14C

• Metabolic Disruptions:

  • Protoporphyrin IX synthesis is blocked in TMEM14C-deficient erythroid cells, leading to accumulation of porphyrin precursors

  • Both basal and differentiation-induced heme synthesis are reduced in TMEM14C-deficient cells, as measured by 55Fe labeling

  • The heme synthesis defect is not secondary to an erythroid differentiation defect but reflects direct involvement of TMEM14C in heme homeostasis

• Cellular Functions:

  • TMEM14C does not regulate mitochondrial iron import, as mitochondrial iron levels remain normal in TMEM14C-deficient cells

  • Normal activity of mitochondrial aconitase, FECH, and cytosolic xanthine oxidase in TMEM14C-deficient cells indicates normal iron-sulfur cluster assembly

  • TMEM14C specifically facilitates the import of protoporphyrinogen IX into the mitochondrial matrix for heme synthesis

These findings establish TMEM14C as a critical component of the heme biosynthetic pathway, specifically involved in the transport of porphyrin intermediates across the mitochondrial membrane.

What is the relationship between TMEM14C function and mitochondrial porphyrin transport?

TMEM14C plays a specific role in mitochondrial porphyrin metabolism that distinguishes it from other aspects of mitochondrial function:

• Specific Role in Porphyrin Transport:

  • TMEM14C facilitates the import of protoporphyrinogen IX into the mitochondrial matrix where it can be converted to protoporphyrin IX by PPOX and subsequently to heme by FECH

  • In TMEM14C-deficient cells, PPOX and FECH protein levels remain normal, indicating that TMEM14C does not regulate the expression of these enzymes

  • The block in protoporphyrin IX synthesis and accumulation of porphyrin precursors in TMEM14C-deficient cells points to a transport function rather than an enzymatic role

• Independence from Iron Metabolism:

  • Mitochondrial iron levels, as measured by inductively coupled plasma mass spectrometry and 59Fe labeling, are similar in control and TMEM14C-deficient cells

  • Normal activity of iron-dependent enzymes in TMEM14C-deficient cells further confirms that TMEM14C does not directly affect iron metabolism

  • Cellular 55Fe uptake is decreased only in differentiating TMEM14C-deficient cells, likely reflecting the decreased demand for iron due to blocked heme synthesis rather than a direct effect on iron transport

These findings position TMEM14C as a specialized component of the heme biosynthetic pathway, with a specific role in facilitating the movement of porphyrin intermediates across the mitochondrial membrane for the final steps of heme synthesis.

How do heterozygous versus homozygous TMEM14C deficiencies differ in their phenotypic manifestations?

Research has revealed important distinctions between heterozygous and homozygous TMEM14C deficiencies:

• Homozygous Deficiency:

  • Results in embryonic lethality due to profound anemia in mouse models

  • Causes erythroid maturation arrest with a decrease in terminally differentiating TER119+ erythroid cells

  • Leads to significant disruption of heme metabolism with accumulation of porphyrin precursors

• Heterozygous State:

  • Heterozygous mice are viable and fertile

  • No detectable hematopoietic lineage defects are observed in heterozygous animals

  • Normal erythroid development proceeds in the presence of one functional TMEM14C allele

This pattern suggests that TMEM14C functions in a dose-dependent manner, with one functional allele producing sufficient protein to maintain normal heme synthesis and erythroid development. This differs from some other genes involved in erythropoiesis that display haploinsufficiency, such as certain ribosomal protein genes in zebrafish that predispose to cancer when heterozygous .

What are common technical challenges when working with recombinant TMEM14C protein?

Researchers working with recombinant TMEM14C may encounter several technical challenges:

• Protein Stability Issues:

  • As a membrane protein, TMEM14C can be difficult to maintain in a properly folded, functional state

  • Repeated freezing and thawing can compromise protein integrity

  • Storage in optimized buffer conditions (Tris-based buffer with 50% glycerol) is critical for maintaining stability

• Expression and Purification Challenges:

  • Membrane proteins like TMEM14C often express at lower levels than soluble proteins

  • The hydrophobic nature of transmembrane domains can complicate purification procedures

  • The tag type used may affect protein folding and function, necessitating careful consideration during production

• Functional Assessment Difficulties:

  • Evaluating transport function in vitro requires appropriate membrane reconstitution systems

  • Distinguishing direct effects on porphyrin transport from secondary effects on erythroid differentiation requires careful experimental design

  • Measuring porphyrin intermediates can be challenging due to their chemical properties

To address these challenges, researchers should carefully optimize storage conditions, consider using specialized expression systems for membrane proteins, and employ multiple complementary approaches to assess TMEM14C function.

How can researchers distinguish between primary effects on heme synthesis versus secondary effects on erythroid differentiation?

Distinguishing primary metabolic effects from secondary developmental consequences requires strategic experimental approaches:

• Temporal Analysis:

  • Time-course experiments examining the sequence of biochemical and developmental changes can help establish causality

  • Quantification of heme synthesis rates and porphyrin intermediates at early time points before differentiation defects appear

• Comparative Cell Systems:

  • Analysis of TMEM14C function in both undifferentiated and differentiating cells

  • Studies have shown that TMEM14C affects heme synthesis both basally and during terminal differentiation, indicating a direct role in heme metabolism

• Rescue Experiments:

  • Complementation with wild-type TMEM14C to determine if metabolic and developmental phenotypes are both rescued

  • Evaluation of whether bypassing the metabolic block can restore normal differentiation

• Molecular Analysis:

  • Assessment of whether TMEM14C deficiency affects expression of heme synthetic enzymes

  • Research has shown that TMEM14C-deficient mitochondria contain similar amounts of PPOX and FECH proteins as controls, indicating that TMEM14C does not regulate protein levels of heme synthetic enzymes

These approaches collectively enable researchers to establish the primary function of TMEM14C in heme metabolism and differentiate it from secondary effects on erythroid development.

What are promising avenues for investigating TMEM14C in disease contexts?

Several promising research directions could expand our understanding of TMEM14C in disease contexts:

• Hematological Disorders:

  • Investigation of TMEM14C mutations or expression changes in congenital sideroblastic anemias

  • Exploration of TMEM14C's role in porphyrias and other disorders of heme metabolism

  • Assessment of whether TMEM14C dysfunction contributes to acquired anemias

• Cancer Biology:

  • Examination of TMEM14C in cancer contexts, given that zebrafish develop malignant peripheral nerve sheath tumors and other tumor types

  • Investigation of how altered heme metabolism might contribute to cancer progression

  • Exploration of TMEM14C as a potential therapeutic target in cancers dependent on elevated heme synthesis

• Mitochondrial Disorders:

  • Analysis of TMEM14C in broader mitochondrial dysfunction syndromes

  • Investigation of potential interactions between TMEM14C and other mitochondrial transporters

  • Exploration of whether TMEM14C function is altered in conditions of mitochondrial stress

These research avenues could yield important insights into the role of TMEM14C in human disease and potentially identify new therapeutic approaches for disorders involving disrupted heme metabolism.

How might understanding TMEM14C function contribute to therapeutic development?

Understanding TMEM14C function could inform therapeutic development in several ways:

• Novel Drug Target Identification:

  • TMEM14C represents a potential therapeutic target for modulating heme synthesis in disorders of erythropoiesis

  • Small molecules that enhance TMEM14C function might benefit conditions with impaired heme synthesis

  • Conversely, inhibitors of TMEM14C could potentially address conditions with pathological overproduction of heme

• Biomarker Development:

  • Patterns of porphyrin intermediate accumulation resulting from TMEM14C dysfunction could serve as biomarkers for certain hematological disorders

  • Changes in TMEM14C expression or function might indicate altered erythropoiesis in disease states

• Gene Therapy Approaches:

  • For congenital disorders involving TMEM14C mutations, gene therapy approaches could potentially restore normal protein function

  • TMEM14C knockout cell models serve as valuable platforms for testing such therapeutic approaches

• Drug Screening Applications:

  • TMEM14C knockout cell lines provide models for high-throughput screening of compounds that might bypass or compensate for TMEM14C deficiency

  • Such screens could identify molecules that enhance alternative pathways for porphyrin transport or metabolism

These potential therapeutic applications highlight the importance of continued research into TMEM14C's structure, function, and role in health and disease.