Recombinant Human Transmembrane protein 143 (TMEM143)

What is TMEM143 and what is its basic structural organization?

TMEM143 (Transmembrane protein 143) is a dual-pass protein containing two transmembrane domains that is predicted to localize to the mitochondria. The protein contains a domain of unknown function (DUF3754) within which two transmembrane domains reside - one 24 amino acids in length and the other 16 amino acids in length, both helical in nature . The transmembrane domains encompass the uncharged region present at amino acids 278 to 302 . Additionally, TMEM143 contains a predicted mitochondrial target peptide at the N-terminus spanning 52 amino acids before the cleavage site . The protein is characterized by an isoelectric point of 9.7, suggesting a basic nature under physiological conditions .

Experimentally, researchers should note that when working with recombinant forms of this protein, the presence of the mitochondrial targeting sequence may affect protein solubility and localization in heterologous expression systems. Consider generating constructs both with and without this sequence depending on your experimental goals.

What are the known transcript variants and protein isoforms of TMEM143?

The TMEM143 gene encodes five transcript variants in humans, leading to four distinct protein isoforms with the following characteristics:

All N-terminally truncated isoforms (b, c, and d) are produced through alternative splicing mechanisms . When designing experiments targeting TMEM143, researchers should carefully consider which isoform(s) to focus on, with variant 1/isoform a being the most complete representation of the protein's function.

What is known about the genomic context and organization of the TMEM143 gene?

The TMEM143 gene is located on the negative (minus) strand of human chromosome 19, specifically at cytogenetic position 19q13.33 . The gene spans approximately 31,882 base pairs and contains 8 exons . Its chromosomal neighbors include the Coiled-coil domain containing 114 (CCDC114) gene and the ER lumen protein-retaining receptor 1 (KDELR1) gene .

When designing gene expression studies or genetic analyses, consider the following:

• Use strand-specific methods when analyzing transcription

• Be aware of the potential regulatory regions shared with neighboring genes

• Consider the 8-exon structure when designing primers for PCR or other amplification techniques

What is the tissue expression pattern of TMEM143?

TMEM143 shows a distinctive tissue expression pattern with particularly high expression in both human skeletal muscle and heart tissue . The Human Protein Atlas provides comprehensive tissue expression data that can guide researchers in selecting appropriate experimental models .

When designing experiments, prioritize cell lines or primary cultures derived from tissues with high endogenous expression for functional studies. For tissues with lower expression, consider whether this represents a physiologically relevant system or if overexpression approaches might be more informative.

What are the predicted functional roles of TMEM143?

TMEM143 is predicted to act upstream of or within hematopoietic progenitor cell differentiation processes . Additionally, protein interaction studies suggest that TMEM143 could potentially play a role in tumor suppression/expression and cancer regulation . Its mitochondrial localization indicates potential involvement in mitochondrial function, though specific metabolic pathways have not been fully elucidated.

When investigating these potential functions, researchers should design experiments that:

• Examine TMEM143 expression during hematopoietic differentiation stages

• Assess mitochondrial function parameters when TMEM143 is overexpressed or knocked down

• Screen for altered expression in cancer tissue samples compared to matched normal tissues

How can researchers effectively detect and quantify TMEM143 expression?

Detection and quantification of TMEM143 can be accomplished through several complementary approaches:

• Antibody-based methods: Commercial ELISA kits specific for TMEM143 are available for protein quantification in biological samples . These kits are designed to detect native, not recombinant, TMEM143 and are appropriate for undiluted body fluids and/or tissue homogenates and secretions .

• Transcript analysis: For mRNA expression analysis, design primers spanning exon-exon junctions to distinguish between the five transcript variants. Consider the following approach:

  • Forward primer in exon 1 and reverse primer in exon 3 to detect all variants

  • Primers flanking the alternatively spliced exons to distinguish between variants 1-4

  • Design a specific primer set for the non-coding variant 5

• Subcellular localization: Given its predicted mitochondrial localization, co-localization studies with established mitochondrial markers can provide important functional insights.

When implementing these methods, establish appropriate positive controls using tissues known to express TMEM143 highly (skeletal muscle or heart tissue) and negative controls where expression is minimal.

What experimental approaches are best suited for studying TMEM143's mitochondrial function?

Given TMEM143's predicted mitochondrial localization and dual transmembrane domains, the following experimental approaches are recommended:

• Mitochondrial isolation and subfractionation: Determine the precise submitochondrial localization (outer membrane, inner membrane, intermembrane space, or matrix) using protease protection assays and detergent solubilization methods.

• Mitochondrial functional assessments:

  • Measure oxygen consumption rates using a Seahorse XF analyzer following TMEM143 knockdown or overexpression

  • Assess mitochondrial membrane potential with fluorescent probes (TMRM, JC-1)

  • Evaluate mitochondrial dynamics (fusion/fission) via live-cell imaging

• Deletion construct analysis: Generate truncation mutants lacking the mitochondrial targeting sequence to confirm its functionality and requirement for mitochondrial localization.

When designing these experiments, utilize a systematic experimental design approach that varies multiple parameters simultaneously to account for interactions between variables that might affect mitochondrial function .

How can researchers investigate the potential role of TMEM143 in cancer regulation?

To investigate TMEM143's suggested role in cancer regulation:

• Expression analysis in cancer datasets:

  • Query cancer genomics databases for TMEM143 expression changes across cancer types

  • Analyze correlation with patient survival and tumor stage

• Functional studies in cancer cell lines:

  • Generate stable knockdown and overexpression cell lines using CRISPR/Cas9 or lentiviral approaches

  • Assess proliferation, apoptosis, migration, and invasion capacity

  • Examine effects on known cancer signaling pathways

• Protein interaction studies:

  • Perform immunoprecipitation followed by mass spectrometry to identify binding partners

  • Validate key interactions using proximity ligation assays or FRET

  • Map interaction domains using deletion constructs

• In vivo tumor models:

  • Develop xenograft models using cells with modified TMEM143 expression

  • Monitor tumor growth, metastasis, and response to therapies

When designing these studies, incorporate both gain-of-function and loss-of-function approaches to establish causality rather than mere correlation.

What are the best approaches for producing and purifying recombinant TMEM143?

For effective production of recombinant TMEM143:

• Expression system selection:

  • Consider mammalian expression systems (HEK293, CHO) for full-length protein with post-translational modifications

  • For structural studies, bacterial systems may be used for domains lacking the transmembrane regions

  • Insect cell systems represent a compromise between proper folding and yield

• Construct design considerations:

  • Remove the N-terminal mitochondrial targeting sequence (first 52 amino acids) to prevent mitochondrial import during expression

  • Add appropriate purification tags (His, GST, or FLAG) preferably at the C-terminus

  • Consider expressing individual domains separately for solubility

• Purification strategy:

  • Use detergent screening to identify optimal solubilization conditions for the transmembrane domains

  • Implement a two-step purification process combining affinity chromatography with size exclusion

  • Consider nanodiscs or amphipols for maintaining the native structure of the transmembrane regions

• Quality control:

  • Verify protein folding using circular dichroism

  • Confirm oligomeric state by analytical ultracentrifugation

  • Assess functionality through binding assays with predicted interacting partners

Document all optimization steps methodically to establish reproducible protocols for future studies.

How should researchers design experiments to study the role of TMEM143 in hematopoietic progenitor cell differentiation?

To investigate TMEM143's role in hematopoietic differentiation:

• Expression profiling during differentiation:

  • Monitor TMEM143 expression throughout the differentiation process of hematopoietic stem cells into various lineages

  • Compare expression patterns between normal and pathological hematopoiesis

• Loss-of-function studies:

  • Use shRNA or CRISPR to knock down TMEM143 in hematopoietic stem/progenitor cells

  • Assess effects on lineage commitment, proliferation, and maturation

  • Analyze changes in differentiation markers using flow cytometry

• Mechanistic investigations:

  • Identify transcription factors regulating TMEM143 expression during differentiation

  • Determine downstream effectors using RNA-seq following manipulation of TMEM143 levels

  • Investigate potential roles in mitochondrial metabolism during differentiation

• Experimental design considerations:

  • Implement a multifactorial experimental design approach that systematically varies:

    • TMEM143 expression levels

    • Differentiation inducers

    • Timepoints for analysis

    • Environmental conditions (oxygen levels, cytokines)

This comprehensive approach will help establish whether TMEM143 has a causal role in differentiation or is merely a marker of the process.