Recombinant Mouse Transmembrane protein 186 (Tmem186)

What post-translational modifications have been identified in mouse TMEM186?

Several post-translational modifications have been identified in mouse TMEM186:

These modifications may play critical roles in regulating TMEM186 function, particularly in its role within mitochondrial complexes . S-nitrosylation at C156 could be especially relevant given the importance of redox regulation in mitochondrial function.

What is the primary function of TMEM186 in mitochondria?

Mouse TMEM186 functions as part of the MCIA (Mitochondrial Complex I Assembly) complex and is required for the efficient assembly of mitochondrial complex I . Research indicates that TMEM186 is enriched with other complex I assembly factors including DMAC1, COA1, and the mitochondrial insertase OXA1L .

Complex I (NADH:ubiquinone oxidoreductase) is the first enzyme in the mitochondrial respiratory chain, crucial for oxidative phosphorylation and energy production. The assembly of this large multisubunit complex requires numerous assembly factors, with TMEM186 playing a specific role in this process .

Analysis of protein interaction networks shows that TMEM186 associates with complex I subunits and assembly machinery, demonstrating its importance in maintaining mitochondrial function .

How does TMEM186 relate to disease pathology?

While direct evidence linking mouse TMEM186 to specific diseases is limited in the provided search results, research on related proteins suggests potential implications in mitochondrial disorders. For instance, mutations in TMEM126A, which interacts in complexes with TMEM186, are associated with autosomal-recessive optic atrophy (AROA) .

Given that TMEM186 participates in mitochondrial complex I assembly, dysfunction may potentially contribute to mitochondrial disorders characterized by complex I deficiency. These disorders often present with neurological symptoms, cardiomyopathy, and other manifestations related to energy metabolism disruption .

What knockout systems are available for studying mouse TMEM186 function?

Several CRISPR/Cas9-based tools are available for generating TMEM186 knockout models:

• CRISPR/Cas9 KO Plasmids: Systems like the TMEM186 CRISPR/Cas9 KO Plasmid (m) consist of a pool of three plasmids, each encoding the Cas9 nuclease and a target-specific 20 nt guide RNA designed for maximum knockout efficiency .

• Lentiviral Particles with gRNA: Available options include:

  • Individual gRNA against Tmem186 in lentiviral particles with Cas9 in a single vector (Vector backbone: pLenti-U6-sgRNA-SFFV-Cas9-2A-Puro)

  • Individual gRNA against Tmem186 in lentiviral particles requiring Cas9 separately (Vector backbone: pLenti-U6-sgRNA-PGK-Neo)

These systems allow for stable integration and expression of the CRISPR/Cas9 machinery for efficient gene knockout. The choice between systems depends on experimental needs, including whether Cas9 is already expressed in the target cells and the preferred selection marker (puromycin vs. neomycin) .

What detection methods are available for mouse TMEM186?

Various tools are available for detecting and quantifying mouse TMEM186:

• ELISA Kits: Mouse Transmembrane protein 186 (TMEM186) ELISA Kit with the following specifications:

  • Test Range: 0.156 ng/ml - 10 ng/ml

  • Detection Method: Colorimetric

  • Sample Types: Tissue homogenates, cell lysates, and other biological fluids

  • Storage conditions: Ship at 4°C, with specific storage instructions provided in the kit manual

• Recombinant Proteins: Available as standards or controls for assay development:

  • Full-length protein (AA 1-216) with His tag expressed in HEK-293 cells

  • Partial recombinant proteins expressed in E. coli with >85% purity (SDS-PAGE)

These tools enable quantitative analysis of TMEM186 in experimental systems, providing researchers with options for protein detection across different applications .

How do environmental factors affect TMEM186 expression?

Research indicates that TMEM186 expression is affected by various chemicals and environmental factors. A comprehensive list of documented interactions includes:

These findings suggest that TMEM186 expression is responsive to various environmental toxins, pharmaceuticals, and metabolites, potentially impacting mitochondrial function in exposed organisms .

How can protein-protein interactions of TMEM186 be effectively studied?

To investigate TMEM186 protein-protein interactions, researchers have employed several approaches:

• Co-immunoprecipitation with epitope-tagged TMEM186: Studies have used C-terminal Flag-tagged TMEM186 to identify interaction partners. This approach revealed associations with complex I subunits and assembly factors .

• Blue Native PAGE (BN-PAGE): This technique has been used to analyze TMEM186-containing complexes, enabling visualization of higher molecular weight species (~720 kDa) that represent assembly intermediates of complex I .

• Comparative analysis using knockout cell lines: Research comparing TMEM186 complexes in control versus knockout cell lines for various complex I accessory subunits helps elucidate the specific interactions and dependencies within the assembly pathway .

• Mitochondrial isolation and fractionation: Prior to interaction studies, mitochondrial isolation ensures enrichment of the relevant organellar complexes containing TMEM186 .

These methodologies collectively provide insights into the functional interactions of TMEM186 within the mitochondrial complex I assembly pathway.

What approaches can be used to study the function of recombinant mouse TMEM186?

Several approaches can be implemented to study recombinant mouse TMEM186 function:

• Complementation studies in knockout cells: Reintroducing recombinant TMEM186 into knockout cell lines can verify if observed phenotypes are directly attributable to TMEM186 loss and identify which domains are crucial for function .

• In vitro complex I assembly assays: Using purified recombinant TMEM186 in reconstitution experiments to assess its direct role in complex I assembly.

• Structure-function analysis: Using site-directed mutagenesis of recombinant TMEM186 to identify critical residues, particularly at post-translational modification sites (S48, T51, C156) .

• Mitochondrial activity assays: Measuring complex I activity, oxygen consumption, and ATP production in systems with wild-type versus mutant recombinant TMEM186.

• Interaction studies with labeled recombinant protein: Using tagged recombinant TMEM186 (available with His tag) for pull-down assays or fluorescently labeled protein for interaction studies.

These methods allow for detailed examination of TMEM186's functional role in mitochondrial complex I assembly and related cellular processes.

How conserved is TMEM186 across different species?

TMEM186 appears to be conserved across multiple species, including:

• Mouse (Mus musculus): The primary focus of the search results, with UniProt ID Q9CR76

• Human: Referenced with UniProt ID Q96B77

• Amur tiger (Panthera tigris altaica): Gene ID 102970967

The conservation of this protein across diverse mammalian species suggests an evolutionarily important functional role, likely in mitochondrial biology given its involvement in complex I assembly .

While specific sequence homology percentages between species are not provided in the search results, the existence of commercial research tools targeting TMEM186 across multiple species indicates sufficient structural and functional conservation to enable cross-species research applications.

What are the optimal methods for expressing and purifying recombinant mouse TMEM186?

Based on the available commercial recombinant TMEM186 products, several expression systems and purification approaches have been established:

• Expression Systems:

  • HEK-293 cells for full-length protein (AA 1-216)

  • E. coli for partial protein fragments

• Purification Approaches:

  • His-tag affinity purification for HEK-293 expressed protein

  • Standard chromatography methods yielding >85% purity as determined by SDS-PAGE for E. coli expressed proteins

• Reconstitution and Storage:

  • For lyophilized proteins, reconstitution at concentrations of 0.1-1.0 mg/mL in deionized sterile water is recommended

  • Addition of 5-50% glycerol (final concentration) for long-term storage

  • Storage at -20°C/-80°C with avoidance of repeated freeze-thaw cycles

These methods provide a framework for researchers seeking to produce recombinant mouse TMEM186 for functional studies, structural analysis, or as standards for quantitative assays.