Recombinant Bovine Transmembrane protein 111 (TMEM111)

What is TMEM111 and what are its primary cellular functions?

TMEM111, more commonly referenced in scientific literature as EMC3, is a subunit of the highly conserved endoplasmic reticulum membrane protein complex (EMC). This transmembrane protein plays critical roles in several cellular processes:

First, TMEM111/EMC3 functions as an integral component of the protein quality control machinery in the endoplasmic reticulum. It contributes to the proper folding of membrane proteins, preventing the accumulation of misfolded proteins that could trigger cellular stress responses. Loss of EMC subunits, including TMEM111/EMC3, has been shown to cause accumulation of misfolded membrane proteins and induction of the unfolded protein response (UPR) in yeast models .

Second, TMEM111/EMC3 is essential for the biogenesis of certain membrane proteins, particularly in the insertion of transmembrane domains. The EMC complex, which includes TMEM111/EMC3, directs the insertion of transmembrane domains consistent with a role in protein biogenesis . This function is especially important for tail-anchored (TA) proteins, which rely on post-translational insertion mechanisms due to their C-terminal transmembrane domain topology .

Third, TMEM111/EMC3 coordinates surfactant protein and lipid homeostasis required for pulmonary function, particularly at birth. Research has demonstrated that TMEM111 was required for murine pulmonary surfactant synthesis and lung function at birth, highlighting its importance in respiratory physiology .

Additionally, TMEM111/EMC3 serves as part of the endoplasmic reticulum-associated secretory pathway and has been implicated in the life cycle of several flaviviruses, including dengue virus, yellow fever virus, and Zika virus .

How is TMEM111 structurally organized and detected in experimental settings?

TMEM111/EMC3 is a membrane-bound protein with distinct structural characteristics that influence its detection and analysis in laboratory settings:

The protein has a molecular weight of approximately 30 kDa as detected by Western blot analysis . This characteristic band is useful for confirming the presence and relative abundance of TMEM111/EMC3 in experimental samples. The protein contains specific domains, including the segment corresponding to human TMEM111 amino acids 139-261, which is commonly used as an immunogen for antibody production .

For experimental detection, researchers typically employ several complementary approaches:

Western blotting remains the gold standard for detecting TMEM111/EMC3 expression levels. Using specific antibodies, such as recombinant rabbit monoclonal antibodies, provides high specificity and sensitivity for detecting the characteristic 30 kDa band . Immunocytochemistry techniques allow visualization of the subcellular localization of TMEM111/EMC3, typically showing ER membrane localization patterns consistent with its known function .

In addition, researchers can identify TMEM111/EMC3 through its association with other EMC complex components. The protein is part of a heterodecameric complex composed of subunits EMC1-7, 8a, 8b, and 10 . Co-immunoprecipitation experiments can reveal these interactions and help confirm the identity and integrity of TMEM111/EMC3 in experimental samples.

Modern proteomic approaches using mass spectrometry provide another powerful tool for detecting and quantifying TMEM111/EMC3 in complex biological samples, allowing for unbiased identification based on peptide sequences rather than antibody specificity.

What is the relationship between TMEM111 and the ER membrane protein complex (EMC)?

TMEM111 is specifically identified as EMC3, a core subunit of the endoplasmic reticulum membrane protein complex (EMC), which plays fundamental roles in membrane protein biogenesis and quality control. This relationship has significant implications for understanding TMEM111's function:

TMEM111/EMC3 forms specific interactions with other EMC components, particularly with EMC1 and EMC2, establishing a functional core of the complex. Research has shown that Emc1, Emc2, and Emc3 form associations with ER-associated degradation (ERAD) pathway components, including Ubac2 and Derlin-2, indicating a close link between the EMC and ERAD machinery . This interaction network suggests that TMEM111/EMC3 functions as a bridge between protein insertion and quality control processes at the ER membrane.

The functional significance of TMEM111/EMC3 within the EMC is highlighted by studies showing that disruption of this subunit impairs the complex's ability to insert certain transmembrane domains and maintain proper ER proteostasis. The EMC, including TMEM111/EMC3, has been shown to direct the insertion of transmembrane domains consistent with a role in protein biogenesis . Loss of EMC function leads to the accumulation of misfolded membrane proteins and activation of the unfolded protein response (UPR) .

How does TMEM111/EMC3 contribute to pulmonary surfactant synthesis and lung function?

TMEM111/EMC3 plays a critical role in coordinating surfactant protein and lipid homeostasis required for pulmonary function, particularly at birth. This function has significant physiological implications for respiratory health:

TMEM111/EMC3 coordinates the assembly of lipids and proteins in alveolar type II (AT2) cells that is necessary for surfactant synthesis and function at birth . Pulmonary surfactant, a complex mixture of lipids and proteins, reduces surface tension at the air-liquid interface in the alveoli and prevents lung collapse during the ventilatory cycle. The precise assembly and secretion of this substance are essential for the first breath and ongoing respiratory function.

Mechanistically, TMEM111/EMC3 appears to facilitate the proper folding and assembly of surfactant proteins in the endoplasmic reticulum of AT2 cells. Given that surfactant proteins are membrane-associated or transmembrane in nature, the role of TMEM111/EMC3 in membrane protein biogenesis directly impacts surfactant production. The EMC complex likely ensures the correct insertion and folding of surfactant proteins, preventing their aggregation or degradation before they can be incorporated into the functional surfactant complex.

Research in murine models has demonstrated that TMEM111/EMC3 is required for pulmonary surfactant synthesis and lung function at birth . Disruption of TMEM111/EMC3 function in these models likely leads to defects in surfactant production, resulting in respiratory distress syndrome or similar pathologies characterized by insufficient surfactant activity. This finding highlights the translational significance of TMEM111/EMC3 research for understanding and potentially treating neonatal respiratory conditions.

In experimental settings, researchers investigating this aspect of TMEM111/EMC3 function typically employ lung-specific knockout models, isolated AT2 cell cultures, and biochemical assays to measure surfactant composition and activity. Electron microscopy techniques are also valuable for visualizing lamellar bodies, the storage organelles for surfactant in AT2 cells.

What is the role of TMEM111/EMC3 in tail-anchored (TA) protein insertion and quality control?

TMEM111/EMC3, as a component of the ER membrane protein complex (EMC), plays a specialized role in the insertion and quality control of tail-anchored (TA) proteins, which represent a significant class of membrane proteins with unique biogenesis requirements:

Tail-anchored proteins are characterized by a single, C-terminal transmembrane domain (TMD) that mediates their post-translational insertion into membranes . This topology defines a diverse class of membrane proteins (approximately 50 in yeast and over 300 in humans) that rely exclusively on post-translational insertion mechanisms . Due to their structure, TA proteins cannot use the co-translational insertion pathways that most membrane proteins employ.

The EMC, including TMEM111/EMC3, serves as one of the primary pathways for inserting TA proteins into the ER membrane. While TA proteins can be inserted via either the 'guided entry of TA proteins' (GET) pathway or the EMC, there appears to be a preference based on the hydrophobicity of the transmembrane domain . TMEM111/EMC3 and the EMC complex preferentially handle TA proteins with relatively hydrophilic transmembrane domains, similar to those found in mitochondrial TA proteins .

In terms of quality control, the dynamic exchange of TA proteins between organelles requires constant monitoring and regulation. Rather than a single, high-fidelity insertion event, growing evidence suggests that kinetically driven cycles of insertion and extraction best explain the observed, steady-state partitioning of many membrane proteins . TMEM111/EMC3 participates in this cycle, with mislocalized or aberrant TA proteins being extracted by ATP13A1 (Spf1 in yeast) when they inappropriately localize to the ER .

Experimental evidence indicates that perturbing this cycle results in the aberrant accumulation of TA proteins at incorrect membranes. Constitutive delivery of model TA proteins to the ER is strongly dependent on the EMC, with the GET complex playing a lesser role . This observation is consistent with the finding that mitochondrial TA proteins and EMC substrates possess similarly hydrophilic TMDs, although the GET pathway can compensate when confronted with a significant buildup of non-optimal TA substrates .

How is TMEM111/EMC3 involved in viral infection processes?

TMEM111/EMC3 has been identified as a critical host factor for the efficient infection of several flaviviruses, revealing an unexpected connection between cellular membrane protein biogenesis and viral replication:

Research has demonstrated that TMEM111/EMC3 is required for the efficient infection of multiple flaviviruses, including dengue virus-2 (DENV2), yellow fever virus (YFV), and Zika virus (ZIKV) . Interestingly, not all flaviviruses show this dependency, as West Nile virus infection remains unchanged in cells lacking EMC components . This selective requirement suggests specific interactions between certain viral components and the EMC machinery.

The mechanism by which TMEM111/EMC3 supports viral infection likely relates to its role in membrane protein biogenesis and lipid metabolism. Flaviviruses extensively remodel host cell membranes to create suitable environments for viral replication complexes. The EMC, including TMEM111/EMC3, may facilitate the proper folding and insertion of viral transmembrane proteins necessary for these remodeling processes. Additionally, the EMC's involvement in lipid metabolism could contribute to the creation of specialized membrane domains that support viral replication.

Experimental approaches to study this phenomenon include RNAi-based loss-of-function screens, which have confirmed the role of the EMC in DENV2 replication . These genome-scale screens have consistently identified EMC components, including TMEM111/EMC3 (originally identified as TMEM111), as proviral factors . The meta-analysis of such screens has revealed a striking pattern, with EMC components appearing alongside other essential host factors such as ribosomal proteins and components of the ER protein translocation machinery.

This research area provides valuable insights into host-virus interactions and potential targets for antiviral interventions. By understanding how TMEM111/EMC3 supports viral infection, researchers may identify novel strategies to disrupt these processes without severely affecting normal cellular functions.

How do disruptions in TMEM111/EMC3 function affect cellular protein homeostasis and stress responses?

Disruptions in TMEM111/EMC3 function have profound effects on cellular protein homeostasis and trigger various stress responses, particularly the unfolded protein response (UPR) and ER-associated degradation (ERAD) pathways:

Loss of EMC subunits, including TMEM111/EMC3, causes the accumulation of misfolded membrane proteins and consequent induction of the unfolded protein response . This cellular stress response is activated when the protein folding capacity of the ER is overwhelmed, leading to the activation of three main signaling branches: PERK, IRE1, and ATF6. These pathways collectively work to reduce protein translation, increase chaperone production, and enhance ER-associated degradation to restore homeostasis.

TMEM111/EMC3 forms a complex with ER-associated degradation (ERAD) pathway components, including Ubac2 and Derlin-2, indicating a close functional link between the EMC and ERAD machinery . This association suggests that TMEM111/EMC3 may directly participate in the recognition or processing of misfolded membrane proteins destined for degradation. When TMEM111/EMC3 function is compromised, this quality control mechanism may be impaired, leading to the accumulation of potentially harmful protein species.

The consequences of disrupted TMEM111/EMC3 function extend beyond the ER to affect other cellular compartments and processes. For instance, the proper assembly and function of various multipass membrane proteins, including nicotinic acetylcholine receptors (AChRs) and rhodopsin, depend on intact EMC function . Disruptions in TMEM111/EMC3 can therefore lead to widespread defects in membrane protein localization and activity across multiple cellular systems.

For experimental investigation of these effects, researchers typically employ a combination of approaches:

• Genetic knockouts or knockdowns of TMEM111/EMC3 using CRISPR/Cas9 or RNAi technologies

• Biochemical assays to measure UPR activation (e.g., XBP1 splicing, phosphorylation of eIF2α)

• Proteomics analyses to identify accumulated misfolded proteins

• Imaging techniques to visualize ER morphology and stress

• Functional assays to assess the activity of specific membrane proteins dependent on EMC function

What experimental approaches are most effective for studying TMEM111/EMC3 structure-function relationships?

Investigating the structure-function relationships of TMEM111/EMC3 requires a sophisticated multi-disciplinary approach that combines structural biology, biochemistry, cell biology, and genetics:

For structural studies, researchers should consider cryo-electron microscopy (cryo-EM) as a primary method, given the challenges of crystallizing membrane protein complexes like the EMC. Recent advances in cryo-EM have made it possible to resolve the structures of large membrane protein assemblies at near-atomic resolution. Complementary approaches include cross-linking mass spectrometry (XL-MS) to map protein-protein interactions within the EMC complex, hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify dynamic regions, and integrative modeling to combine multiple experimental data types.

Functional studies benefit from precise genetic manipulation techniques. CRISPR/Cas9-mediated genome editing allows for the generation of knockout cell lines and the introduction of specific mutations to test structure-function hypotheses. For example, researchers have used genome-wide CRISPR screens to identify factors influencing protein flux through various degradation pathways, revealing unexpected connections between TMEM111/EMC3 and downstream cellular processes .

Biochemical reconstitution represents another powerful approach. Purifying recombinant TMEM111/EMC3 and other EMC components for in vitro assembly and functional assays can provide direct insights into membrane protein insertion mechanisms. This typically involves expression in suitable host systems (e.g., insect cells), followed by detergent extraction, affinity purification, and reconstitution into liposomes or nanodiscs for functional studies.

Imaging approaches offer unique insights into TMEM111/EMC3 dynamics and localization. Super-resolution microscopy techniques such as STORM or PALM can visualize TMEM111/EMC3 distribution at the nanoscale, while Förster resonance energy transfer (FRET) can detect protein-protein interactions in living cells. Time-lapse imaging with fluorescently tagged TMEM111/EMC3 can reveal dynamic behaviors during various cellular processes.

Domain mapping and mutagenesis studies are essential for pinpointing functional regions within TMEM111/EMC3. By systematically introducing mutations or creating chimeric proteins with domains from related proteins, researchers can identify critical regions for interactions with other EMC subunits, substrate recognition, or membrane integration.

How does TMEM111/EMC3 coordinate with other cellular machinery to maintain ER and organelle homeostasis?

TMEM111/EMC3 operates within a complex network of cellular machinery to maintain ER and organelle homeostasis, coordinating with multiple quality control and biogenesis pathways:

The EMC, including TMEM111/EMC3, works in concert with the translocon (Sec61 complex) and other insertion machineries to ensure proper membrane protein biogenesis. While the Sec61 complex handles co-translational insertion of most membrane proteins, the EMC specializes in post-translational insertion of certain classes of proteins, particularly those with moderately hydrophobic transmembrane domains . This division of labor ensures efficient and accurate membrane protein biogenesis under various cellular conditions.

TMEM111/EMC3 coordinates with extraction machineries that remove mislocalized or aberrant membrane proteins. For instance, while the EMC inserts TA proteins into the ER membrane, mislocalized or aberrant TA proteins are extracted by ATP13A1 (Spf1 in yeast) . Similarly, in the outer mitochondrial membrane, TA proteins are inserted via MTCH1/MTCH2, while mislocalized proteins are extracted by ATAD1 (Msp1 in yeast) . This dynamic cycle of insertion and extraction maintains the proper distribution of membrane proteins across cellular compartments.

The connection between TMEM111/EMC3 and the ERAD machinery is particularly significant. Emc1, Emc2, and Emc3 form a complex with ERAD pathway components Ubac2 and Derlin-2, indicating a close functional link . This association suggests that TMEM111/EMC3 not only contributes to membrane protein insertion but also participates in the recognition and processing of misfolded proteins for degradation, providing a direct link between biogenesis and quality control.

Furthermore, TMEM111/EMC3 intersects with broader cellular degradation pathways. Research on mitophagy receptors like BNIP3 has revealed that the EMC influences the lysosomal turnover of certain tail-anchored proteins . This unexpected connection between mitophagy and TA protein quality control suggests that the endolysosomal system provides a critical axis for regulating cellular metabolism, with TMEM111/EMC3 playing a role in this process.

From a research perspective, studying these interconnections requires systems-level approaches:

• Proximity labeling methods (BioID, APEX) to map the interaction network of TMEM111/EMC3

• Global proteomics to identify changes in protein abundance and modifications upon TMEM111/EMC3 manipulation

• Genetic interaction screens to identify synthetic lethal or rescue relationships

• Live cell imaging of multiple organelle markers to assess morphology and contacts

• Lipidomics analyses to understand membrane composition changes

What are the optimal protocols for expressing and purifying recombinant TMEM111/EMC3 for functional and structural studies?

Expression and purification of recombinant TMEM111/EMC3 present several challenges due to its membrane-associated nature and participation in a large multi-subunit complex. The following methodological approaches optimize success:

Expression Systems Selection:
For functional studies of TMEM111/EMC3, insect cell expression systems (Sf9 or High Five cells) typically provide the best balance of protein yield and proper folding. These systems contain the necessary chaperones and membrane environments to support correct TMEM111/EMC3 folding. For structural studies requiring higher yields, consider co-expression of multiple EMC subunits (particularly EMC1, EMC2, and EMC3) to enhance stability and solubility of the complex .

Construct Design Considerations:
Careful construct design is critical for successful expression. Include a cleavable affinity tag (such as His8 or Twin-Strep) at either the N- or C-terminus, with a TEV or 3C protease cleavage site. For TMEM111/EMC3, N-terminal tagging is generally preferable as it avoids interference with the transmembrane regions. Consider creating truncated constructs that remove flexible regions while maintaining core functional domains, based on secondary structure predictions and evolutionary conservation analysis.

Purification Protocol Optimization:
A successful purification protocol for TMEM111/EMC3 typically involves:

• Membrane isolation through differential centrifugation

• Solubilization using mild detergents (DDM, LMNG, or GDN are good initial choices)

• Affinity chromatography using the engineered tag

• Size exclusion chromatography to isolate properly folded, monodisperse protein

For structural studies, consider detergent exchange to amphipols or reconstitution into nanodiscs during purification to maintain a native-like membrane environment. Addition of lipids, particularly those found in the ER membrane, during purification often enhances stability and activity.

Functional Verification:
Before proceeding to structural or detailed biochemical analyses, verify that the purified TMEM111/EMC3 is functional using:

• Binding assays with known interaction partners

• Membrane insertion assays using model substrates

• ATPase activity measurements if applicable

• Thermal stability assays to assess proper folding

The optimized protocol should yield protein that is >90% pure as assessed by SDS-PAGE, monodisperse as determined by size exclusion chromatography, and functionally active in appropriate assay systems.

What genetic manipulation strategies are most effective for investigating TMEM111/EMC3 function in cellular models?

Investigating TMEM111/EMC3 function requires sophisticated genetic manipulation strategies that balance complete removal of the protein with more nuanced approaches to dissect specific functions:

Inducible Knockdown Systems:
For temporal control over TMEM111/EMC3 depletion, consider tetracycline-inducible shRNA or doxycycline-regulated CRISPR interference (CRISPRi) systems. These approaches allow for gradual depletion of TMEM111/EMC3, making it possible to observe primary effects before secondary compensatory responses develop. The inducible nature also facilitates experiments in cell types where constitutive knockout might be lethal.

Domain-Specific Mutagenesis:
For structure-function studies, design point mutations or small deletions that target specific domains or functional motifs within TMEM111/EMC3. Selection of mutation sites should be guided by evolutionary conservation analysis, structural information if available, and knowledge of interaction interfaces with other EMC components. Common approaches include:

• Alanine scanning of conserved residues

• Charge reversal mutations at potential protein-protein interfaces

• Deletion of specific structural elements

Complementation Strategies:
After generating knockout or knockdown cells, reintroduce wild-type or mutant versions of TMEM111/EMC3 to assess rescue of phenotypes. This approach is particularly powerful for mapping functional domains. Construct a panel of TMEM111/EMC3 variants with different mutations or domain swaps, then assess their ability to restore normal cellular functions in the knockout background.

Cell Model Selection:
Different cell types may reveal different aspects of TMEM111/EMC3 function. Consider using:

• HEK293T cells for initial characterization due to high transfection efficiency

• AT2-like cell lines for investigating surfactant-related functions

• Primary cells or differentiated iPSCs for more physiologically relevant contexts

• Model organisms (mice, zebrafish) for in vivo functional studies

A successful genetic manipulation strategy employs multiple complementary approaches to build a comprehensive understanding of TMEM111/EMC3 function, combining both loss-of-function and structure-function studies.

What analytical techniques provide the most comprehensive characterization of TMEM111/EMC3 interactions with binding partners?

Comprehensively characterizing TMEM111/EMC3 interactions requires a multi-faceted analytical approach that captures both stable and transient interactions across different cellular contexts:

Affinity Purification-Mass Spectrometry (AP-MS):
For identifying the core interactome of TMEM111/EMC3, employ AP-MS using epitope-tagged TMEM111/EMC3 as bait. Consider both N- and C-terminal tags to account for potential interference with specific interactions. Critical experimental considerations include:

• Crosslinking with mild reagents (e.g., DSP) to capture transient interactions

• Optimization of detergent conditions to maintain membrane protein complexes

• Quantitative comparison with appropriate controls using SILAC, TMT, or label-free quantification

• Analysis of different cellular fractions to identify compartment-specific interactions

Proximity Labeling Approaches:
To capture dynamic and contextual interactions, employ proximity labeling techniques such as BioID or APEX2. Fusion of these enzymes to TMEM111/EMC3 enables labeling of proteins in close proximity in living cells. These methods are particularly valuable for identifying weak or transient interactions that may be lost during conventional immunoprecipitation. Comparative analysis across different cellular states (e.g., ER stress, viral infection) can reveal context-specific interaction networks.

Cross-linking Mass Spectrometry (XL-MS):
For detailed mapping of interaction interfaces, XL-MS provides residue-level resolution of protein-protein contacts. This approach involves:

• Treatment of purified complexes or cellular extracts with chemical cross-linkers

• Digestion of cross-linked proteins and enrichment of cross-linked peptides

• MS/MS analysis to identify linked residues

• Integration with structural models to map interaction surfaces

Fluorescence-based Interaction Assays:
For validating and characterizing specific interactions, several fluorescence-based methods provide complementary information:

• Förster Resonance Energy Transfer (FRET) to measure direct protein-protein interactions in living cells

• Fluorescence Recovery After Photobleaching (FRAP) to assess the dynamics of TMEM111/EMC3 complexes

• Number and Brightness (N&B) analysis to determine the stoichiometry of complexes

Functional Interaction Mapping:
Beyond physical interactions, functional relationships can be mapped through:

• Genetic interaction screens (e.g., double knockout/knockdown studies)

• Suppressor screens to identify proteins that can rescue TMEM111/EMC3 deficiency

• Systematic mutagenesis coupled with binding assays to map critical interaction residues

A comprehensive characterization involves integrating data from multiple approaches to build a dynamic model of TMEM111/EMC3 interactions across different cellular contexts and functional states. This integration can reveal not only the composition of complexes but also their assembly pathways, regulatory mechanisms, and functional significance.