Recombinant Human Transmembrane protein 111 (TMEM111)

What is the molecular structure and cellular localization of TMEM111/EMC3?

TMEM111/EMC3 is a 261 amino acid, 30 kDa type II transmembrane protein that is primarily localized to the endoplasmic reticulum (ER) membrane . While its main localization is in the ER membrane, it can also be found in trans-Golgi networks, endosomes, and lysosomes . The protein is highly conserved across species and serves as a key subunit of the ER membrane protein complex (EMC) . The gene is located at chromosome 3 in humans, and its UniProt ID is Q9P0I2 . The protein's functional domains include transmembrane regions that facilitate its insertion into the ER membrane and interaction with client proteins.

How does TMEM111 function within the ER membrane protein complex?

TMEM111/EMC3 is an essential component of the endoplasmic reticulum membrane protein complex (EMC), which was first identified in Saccharomyces cerevisiae as a 6-subunit transmembrane protein complex required for protein folding in the ER . Within this complex, TMEM111/EMC3 enables the energy-independent insertion of newly synthesized membrane proteins into ER membranes . It preferentially accommodates proteins with transmembrane domains that are weakly hydrophobic or contain destabilizing features such as charged and aromatic residues . TMEM111/EMC3 forms a functional unit with other EMC components to mediate both cotranslational insertion of multi-pass membrane proteins and post-translational insertion of tail-anchored (TA) proteins in ER membranes . It also associates with ER-associated degradation (ERAD) pathway components including Ubac2 and Derlin-2, indicating a close functional link between the EMC and ERAD systems .

What are the established experimental models for studying TMEM111 function?

Several experimental models have been established to study TMEM111/EMC3 function:

• Cell culture models: Various human cell lines have been used, including HEK293 variants, A431, HuH-7, and MDA-MB cell lines .

• Mouse models: Knockout models have demonstrated that TMEM111/EMC3 is required for murine pulmonary surfactant synthesis and lung function at birth .

• Viral infection models: TMEM111/EMC3 has been studied in the context of flavivirus infections (DENV, YFV, ZIKV), where it acts as a proviral factor .

• Yeast models: The EMC was first characterized in Saccharomyces cerevisiae, providing fundamental insights into the complex's role in protein folding and membrane protein insertion .

These models employ various approaches including gene knockout/knockdown, overexpression systems, and protein-protein interaction studies to elucidate TMEM111/EMC3 functions in different biological contexts.

What are the most effective detection methods for studying endogenous TMEM111 expression?

For detecting endogenous TMEM111/EMC3 expression, researchers can employ the following methods:

• Western Blot (WB): Anti-TMEM111 antibodies have been validated for detecting the 30 kDa protein in various cell and tissue types. Recommended antibody dilutions range from 1:500 to 1:2000 . Positive control samples include A431 cells, HEK-293 cells, HuH-7 cells, MDA-MB-453s cells, and various mouse and rat tissues such as brain, stomach, and testis .

• Immunohistochemistry (IHC): IHC analysis can be performed using paraffin-embedded tissue sections with antibody dilutions of 1:200 to 1:800 . For optimal results, antigen retrieval with TE buffer pH 9.0 is recommended, although citrate buffer pH 6.0 can also be used as an alternative . Human ovary cancer tissue and intrahepatic cholangiocarcinoma tissue have shown positive detection .

• Immunofluorescence (IF)/Immunocytochemistry (ICC): For cellular localization studies, IF/ICC can be performed with antibody dilutions of 1:50 to 1:500 . A431 cells are recommended as positive controls .

• qPCR: Quantitative PCR can be used to measure TMEM111 mRNA expression levels, which is particularly useful when studying regulation of gene expression.

Each method should be validated with appropriate positive and negative controls to ensure specificity and sensitivity of TMEM111/EMC3 detection.

What approaches are recommended for overexpressing functional TMEM111 in mammalian cells?

Several approaches have been established for overexpressing functional TMEM111/EMC3 in mammalian cells:

• Adenoviral expression systems: Commercially available TMEM111 adenoviruses can achieve nearly 100% transduction efficiency, although expression is transient (approximately 7 days) . These systems are particularly useful for cells with low transfection efficiency and can achieve high-level gene expression in most mammalian cells .

• Lentiviral expression systems: For stable expression, lentiviral systems can be employed, which integrate into the genome with relatively high transduction efficiency . These are particularly useful for long-term studies and for cells that are difficult to transfect.

• Tetracycline-inducible expression systems: For controlled expression, tetracycline-inducible promoters (CMV/TetO2) have been used successfully for membrane proteins in HEK293 variants . This approach allows for regulated expression levels to prevent potential toxicity from overexpression.

• Transient transfection: Standard transfection methods using lipid-based reagents can be employed for short-term studies in readily transfectable cell lines.

When overexpressing TMEM111/EMC3, it's important to verify proper localization to the ER membrane and functional integration into the EMC complex. Inclusion of epitope tags should be carefully considered as they may interfere with protein function or complex assembly.

How can researchers effectively knockdown or knockout TMEM111 to study its function?

For functional studies requiring TMEM111/EMC3 depletion, researchers can employ several approaches:

• siRNA/shRNA-mediated knockdown: Small interfering RNAs targeting TMEM111/EMC3 have been successfully used to reduce expression levels . This approach provides a transient reduction in protein levels and is useful for short-term functional studies.

• CRISPR/Cas9-mediated knockout: For complete elimination of TMEM111/EMC3 expression, CRISPR/Cas9 genome editing can be employed. This approach has been used to study the role of EMC components in various cellular processes .

• Conditional knockout systems: For temporal control of gene disruption, especially in in vivo models, conditional knockout systems using Cre-lox technology can be utilized to study tissue-specific functions of TMEM111/EMC3.

When designing knockdown or knockout experiments, several considerations should be taken into account:

• Validation of knockdown/knockout efficiency using multiple methods (WB, qPCR)

• Inclusion of appropriate controls to account for off-target effects

• Assessment of potential compensatory mechanisms, particularly involving other EMC subunits

• Careful phenotypic analysis considering the multiple functions of TMEM111/EMC3 in different cellular contexts

Knockdown studies have demonstrated that depletion of TMEM111/EMC3 can inhibit flavivirus infections (DENV, YFV, ZIKV) and affect membrane protein insertion and quality control .

How does TMEM111/EMC3 contribute to protein quality control in the ER membrane?

TMEM111/EMC3 plays multiple sophisticated roles in protein quality control within the ER membrane:

• Membrane protein insertion: TMEM111/EMC3, as part of the EMC, enables the energy-independent insertion of newly synthesized membrane proteins with specific characteristics into ER membranes . It preferentially accommodates proteins with transmembrane domains that are weakly hydrophobic or contain destabilizing features such as charged and aromatic residues .

• Topological control: By mediating the proper cotranslational insertion of N-terminal transmembrane domains in an N-exo topology (with translocated N-terminus in the lumen of the ER), TMEM111/EMC3 controls the topology of multi-pass membrane proteins including G protein-coupled receptors .

• Tail-anchored protein insertion: TMEM111/EMC3 is required for the post-translational insertion of tail-anchored (TA) proteins in ER membranes . This function is particularly important for TA proteins with relatively hydrophilic transmembrane domains that are not optimal substrates for the GET (guided entry of TA proteins) pathway .

• Association with ERAD: TMEM111/EMC3 forms a complex with ER-associated degradation (ERAD) pathway components including Ubac2 and Derlin-2, indicating a role in the recognition and processing of misfolded membrane proteins .

• Dynamic membrane protein partitioning: Recent evidence suggests that TMEM111/EMC3 participates in kinetically driven cycles of protein insertion and extraction that determine the proper partitioning of membrane proteins between different cellular compartments . Perturbation of this cycle can result in aberrant accumulation of proteins at incorrect membranes .

These functions collectively ensure that membrane proteins are properly inserted, folded, and localized within cellular membranes, thereby maintaining proteostasis and organelle identity.

What is the role of TMEM111 in viral infection pathways, particularly for flaviviruses?

TMEM111/EMC3 plays significant roles in viral infection pathways, particularly for flaviviruses:

• Proviral factor for flaviviruses: TMEM111/EMC3 has been identified as a host dependency factor required for efficient dengue virus-2 (DENV2), yellow fever virus (YFV), and Zika virus (ZIKV) infections . Interestingly, West Nile virus infection was unchanged in cells lacking EMC subunits, suggesting virus-specific dependencies .

• Meta-analysis identification: TMEM111/EMC3 was identified through a meta-analysis of YFV and DENV screens that identified 274 common proviral factors, including multiple EMC subunits (EMC2/TTC35, EMC3/TMEM111, and EMC5/TMEM32/MMGT1) .

• Viral replication complexes: The mechanism likely involves the EMC's role in biogenesis and insertion of viral transmembrane proteins that form replication complexes. Flaviviruses extensively remodel ER membranes to create replication organelles, a process that requires proper insertion of viral proteins into the ER membrane .

• Potential therapeutic target: The dependency of multiple flaviviruses on TMEM111/EMC3 suggests it could be a potential target for broad-spectrum antiviral strategies targeting host factors rather than viral components .

Research approaches to study TMEM111/EMC3 in viral infection include siRNA-mediated knockdown, CRISPR/Cas9 knockout, viral infection assays, and analysis of viral protein localization and replication complex formation in the presence or absence of functional EMC components.

How does TMEM111 coordinate surfactant protein and lipid homeostasis in pulmonary function?

TMEM111/EMC3 plays a critical role in coordinating surfactant protein and lipid homeostasis required for pulmonary function:

• Requirement for neonatal lung function: EMC3/TMEM111 coordinates the assembly of lipids and proteins in alveolar type II (AT2) cells that is necessary for surfactant synthesis and function at birth . Studies have demonstrated that TMEM111/EMC3 is essential for adaptation to respiration at birth by enabling the synthesis of pulmonary surfactant .

• Surfactant composition regulation: Pulmonary surfactant is a lipid-protein complex that reduces surface tension at the air-liquid interface in the alveoli and prevents lung collapse during the ventilatory cycle . TMEM111/EMC3 appears to be involved in the proper assembly of both the protein and lipid components of this complex .

• Membrane protein insertion: The role of TMEM111/EMC3 in inserting transmembrane proteins likely contributes to the proper localization and function of key surfactant-associated proteins in AT2 cells .

• ER-associated secretory pathway: As part of the endoplasmic reticulum-associated secretory pathway , TMEM111/EMC3 likely facilitates the processing and trafficking of surfactant proteins through the secretory pathway.

Research approaches to study this function include mouse knockout models, analysis of surfactant composition and function in the absence of TMEM111/EMC3, and examination of AT2 cell ultrastructure and function in models of EMC3 deficiency.

What is the relationship between TMEM111/EMC3 and mitophagy regulation?

Recent research has revealed an important relationship between TMEM111/EMC3 and mitophagy regulation:

• Restriction of mitophagy: The ER membrane protein complex, including TMEM111/EMC3, restricts mitophagy by controlling mitophagy receptor trafficking . This represents a novel role for the EMC in quality control of mitochondrial proteins.

• Regulation of mitophagy receptors: TMEM111/EMC3 affects the trafficking of established mitophagy receptors, BNIP3 and BNIP3L/NIX, which are constitutively delivered to lysosomes in an autophagy-independent manner .

• Tail-anchored protein quality control: As a tail-anchored (TA) protein, BNIP3 is subject to quality control mechanisms involving TMEM111/EMC3. The dynamic cycles of insertion and extraction regulated by the EMC help determine the proper localization of TA proteins between different cellular compartments, including mitochondria and the ER .

• Lysosomal degradation pathway: TMEM111/EMC3 participates in a mechanism where dimerization of mitophagy receptors like BNIP3 leads to stable protein complexes that are cleared from the ER through trafficking to lysosomes, representing a quality control pathway that supports proper localization of TA membrane proteins .

This function highlights the broader role of TMEM111/EMC3 in organelle communication and quality control beyond its established functions in membrane protein insertion and folding.

What evidence links TMEM111 to cancer progression and how might it be studied as a therapeutic target?

Several lines of evidence link TMEM111/EMC3 to cancer progression:

• Oncogenic functions in multiple cancers: TMEM111/EMC3 has been identified as playing a role in several cancer types including lung cancer, prostate cancer, pancreatic cancer, and melanoma . It appears to function primarily as an oncogene in these contexts .

• Cellular processes regulated: TMEM111/EMC3 has been implicated in cancer-related cellular processes including:

  • Cell proliferation (in lung cancer, prostate cancer, gastric cancer, osteosarcoma)

  • Metastasis and invasion (in prostate cancer, gastric cancer, osteosarcoma)

  • Migration (in pancreatic cancer, gastric cancer, osteosarcoma)

  • Apoptosis regulation (in pancreatic cancer)

• Detection in cancer tissues: Immunohistochemical analysis has detected TMEM111/EMC3 in human ovary cancer tissue and intrahepatic cholangiocarcinoma tissue , suggesting potential diagnostic or prognostic applications.

Research approaches to study TMEM111/EMC3 as a potential therapeutic target include:

• Analysis of TMEM111/EMC3 expression levels across cancer types and correlation with clinical outcomes

• Functional studies using knockdown/knockout approaches in cancer cell lines to assess effects on proliferation, migration, invasion, and sensitivity to therapies

• Investigation of the molecular mechanisms by which TMEM111/EMC3 promotes cancer progression, potentially through its roles in membrane protein folding, quality control, or specific client proteins relevant to cancer biology

• Development of strategies to modulate TMEM111/EMC3 function or expression as potential therapeutic approaches

• Analysis of potential synthetic lethal interactions between TMEM111/EMC3 inhibition and other cancer therapies

How is TMEM111 implicated in innate immunity and inflammatory responses?

TMEM111/EMC3 has been implicated in innate immunity and inflammatory responses through several mechanisms:

• Role in innate immunity: TMEM111/EMC3 has been reported to play a role in innate immunity , although the specific mechanisms require further investigation.

• Viral infection response: As a host dependency factor for flavivirus infections (DENV, YFV, ZIKV) , TMEM111/EMC3 is intrinsically linked to host-pathogen interactions and immune responses to viral infections.

• Membrane protein quality control: Through its role in membrane protein insertion and quality control , TMEM111/EMC3 likely affects the proper expression and function of various immune receptors and signaling molecules embedded in cellular membranes.

• Potential link to inflammatory pathways: While direct evidence is limited in the provided search results, the association of TMEM111/EMC3 with various cancers suggests potential roles in inflammatory signaling pathways that contribute to cancer progression.

Research approaches to further investigate these functions include:

• Analysis of immune responses in TMEM111/EMC3-deficient models

• Investigation of specific immune receptors or signaling molecules that depend on TMEM111/EMC3 for proper expression or function

• Examination of inflammatory pathways in the context of TMEM111/EMC3 modulation

• Assessment of TMEM111/EMC3 expression and function in immune cells and inflammatory conditions

Has TMEM111 been implicated in genetic disorders or neurodegenerative diseases?

• Protein quality control: As a component of the ER membrane protein complex involved in protein folding and quality control , TMEM111/EMC3 could potentially be relevant to neurodegenerative diseases characterized by protein misfolding and aggregation.

• Membrane protein insertion: TMEM111/EMC3's role in membrane protein insertion suggests it could affect the expression and function of various neuronal membrane proteins, including receptors and transporters implicated in neurological disorders.

• Link to severe depression: One search result suggests a potential genetic linkage for severe depression on chromosome 3 , where the TMEM111 gene is located, although a direct functional connection is not established.

• Nicotinic acetylcholine receptors: EMCs are essential for the assembly of several multipass membrane proteins including nicotinic acetylcholine receptors (AChRs) in Caenorhabditis elegans , suggesting potential relevance to disorders involving cholinergic neurotransmission.

Future research directions could include:

• Genetic association studies examining TMEM111/EMC3 variants in neurodegenerative diseases

• Functional studies of TMEM111/EMC3 in neuronal cells and its effects on neuronal membrane proteins

• Investigation of TMEM111/EMC3's role in the processing and trafficking of proteins implicated in neurodegenerative diseases

• Analysis of TMEM111/EMC3 expression and function in models of neurodegenerative disorders

What are common technical challenges when working with recombinant TMEM111 and how can they be addressed?

Researchers working with recombinant TMEM111/EMC3 may encounter several technical challenges:

• Protein expression and solubility: As a transmembrane protein, TMEM111/EMC3 can be difficult to express in soluble, correctly folded form.
  Solution: Use mammalian expression systems rather than bacterial systems . HEK293 variants, particularly HEK293S-GnTI- or HEK293S-TetR, have been successfully used for membrane protein expression . Tetracycline-inducible systems allow for controlled expression levels to prevent toxicity .

• Incorporation into native complexes: TMEM111/EMC3 functions as part of the multi-subunit EMC.
  Solution: Consider co-expression with other EMC components to facilitate proper complex assembly. Verify incorporation into the native complex by co-immunoprecipitation or blue native PAGE.

• Post-translational modifications: TMEM111/EMC3 may require specific post-translational modifications for proper function.
  Solution: Use mammalian expression systems that provide appropriate post-translational modifications. Consider cell lines with altered glycosylation (e.g., HEK293S-GnTI-) if glycosylation is a concern .

• Detection specificity: Ensuring specific detection of TMEM111/EMC3.
  Solution: Use validated antibodies with appropriate controls . For recombinant protein, inclusion of epitope tags can aid in detection, but consider their potential impact on protein function. Verification with control fragments may be useful .

• Functional assays: Establishing appropriate assays to assess TMEM111/EMC3 function.
  Solution: Develop assays based on known functions such as membrane protein insertion, viral infection support, or effects on surfactant synthesis. Include appropriate positive and negative controls.

How can researchers optimize transfection or transduction efficiency when working with TMEM111 expression systems?

Optimizing expression of TMEM111/EMC3 in experimental systems requires careful consideration of several factors:

• Expression system selection:

  • For transient expression: Adenoviral systems can achieve nearly 100% transduction efficiency but work transiently (about 7 days) .

  • For stable expression: Lentiviral systems can integrate into the genome with relatively high transduction efficiency .

  • For controlled expression: Tetracycline-inducible systems allow regulation of expression levels .

• Cell line selection:

  • HEK293 variants are commonly used for membrane protein expression .

  • Cell-specific factors may affect expression efficiency; consider the biological context of your research.

• Optimization strategies:

  • For viral transduction: Optimize the multiplicity of infection (MOI) and include polybrene to enhance efficiency.

  • For transfection: Optimize DNA:transfection reagent ratios and cell density.

  • Include appropriate controls to assess transfection/transduction efficiency (e.g., GFP expression).

• Verification of expression:

  • Multiple methods should be used to confirm successful expression, including WB, qPCR, or fluorescence microscopy if using tagged constructs .

  • Expression can typically be observed from 48 hours up to 5 days after infection .

• Troubleshooting low efficiency:

  • If efficiency is below 80%, consider optimizing cell culture conditions, reagent quality, or protocol parameters .

  • Providers of expression systems often require evidence of >80% transfection efficiency with a positive control for guarantee purposes .

What are the best practices for analyzing TMEM111 interactions with other proteins in the EMC complex?

Analyzing TMEM111/EMC3 interactions with other proteins in the EMC complex requires specialized approaches:

• Co-immunoprecipitation (Co-IP):

  • Use antibodies specific to TMEM111/EMC3 to pull down the protein and its interacting partners .

  • Alternative approach: Tag TMEM111/EMC3 with epitope tags (e.g., FLAG, HA) for pull-down using anti-tag antibodies.

  • Western blot analysis can identify co-precipitated EMC components or other interacting proteins.

  • Consider crosslinking approaches to stabilize transient interactions.

• Proximity labeling approaches:

  • BioID or APEX2 fusion proteins can label proteins in close proximity to TMEM111/EMC3 within cells.

  • These approaches can identify both stable and transient interactions in the native cellular environment.

• Blue Native PAGE:

  • This technique can separate intact protein complexes and identify TMEM111/EMC3 as part of the EMC complex.

  • Western blotting of BN-PAGE can confirm the presence of TMEM111/EMC3 in specific complexes.

• Fluorescence microscopy techniques:

  • Förster resonance energy transfer (FRET) or bimolecular fluorescence complementation (BiFC) can visualize protein-protein interactions in living cells.

  • Fluorescence co-localization studies can provide preliminary evidence for interactions.

• Mass spectrometry-based approaches:

  • After immunoprecipitation, mass spectrometry can identify interacting partners.

  • Quantitative approaches (e.g., SILAC) can distinguish specific from non-specific interactions.

• Functional validation:

  • Mutational analysis targeting specific domains or residues can identify regions required for interactions.

  • Knockdown/knockout of potential interacting partners can reveal functional relationships.

  • Analysis of client protein processing in the absence of specific EMC components can provide insights into functional interactions.

When analyzing TMEM111/EMC3 interactions, it's important to consider the membrane-embedded nature of the protein and the complex, which may require specialized detergents for extraction while maintaining protein-protein interactions.