Recombinant Rat Transmembrane protein 111 (Tmem111)

Basic Research Questions

• What is Rat Transmembrane protein 111 (Tmem111) and what are its alternative designations?

  Rat Transmembrane protein 111 (Tmem111) is now more commonly known as ER membrane protein complex subunit 3 (Emc3). It is a protein encoded by the Emc3 gene (Gene ID: 312640) in rats with an ORF size of 786 bp (RefSeq# BC085846). The protein has several synonyms in scientific literature, including Pob and RGD1311566 . When working with this protein, it's important to search literature using all alternative designations to ensure comprehensive results, as older publications may primarily use the Tmem111 designation while newer research typically refers to it as Emc3.

• What is the fundamental role of Tmem111/Emc3 in cellular processes?

  Tmem111/Emc3 is a subunit of the highly conserved ER membrane protein complex (EMC), which was first identified in Saccharomyces cerevisiae as a 6-subunit transmembrane protein complex required for protein folding in the endoplasmic reticulum (ER) . The complex plays critical roles in:

  • Proper folding of membrane proteins

  • Prevention of misfolded protein accumulation

  • Coordination of the unfolded protein response (UPR)

  • Assembly of multipass membrane proteins

  • Lipid homeostasis and metabolism

  Functionally, EMC3 coordinates the assembly of lipids and proteins in alveolar type 2 (AT2) cells that is necessary for surfactant synthesis and function at birth . This makes it particularly relevant for researchers studying pulmonary development and function.

• How is the EMC complex structured and what is Tmem111/Emc3's position within it?

  The EMC was initially identified as a 6-subunit complex in yeast, but further research has characterized the mammalian EMC (mEMC) as more complex. Emc1, Emc2, and Emc3 (Tmem111) form a subcomplex that interacts with ER-associated degradation (ERAD) pathway components, including Ubac2 and Derlin-2 . This indicates a close functional link between the EMC and ERAD pathways.

  In rat experimental systems, EMC3 has been shown to be particularly important as it forms part of the core structural components of the complex. When designing experiments to study the EMC complex, it's advisable to focus on EMC3 interactions with both its EMC partners and ERAD components to understand the complete functional network.

• How is Tmem111/Emc3 expression regulated during cellular stress conditions?

  Tmem111/Emc3 expression is significantly upregulated during ER stress conditions. Research shows that while multiple EMC components are transcriptionally upregulated during ER stress induced by tunicamycin, Tmem111/Emc3 shows a particularly notable response . This is consistent with observations in yeast where EMC3 is specifically upregulated during the unfolded protein response (UPR) .

  For experimental design, this regulation pattern makes Tmem111/Emc3 a useful marker for monitoring ER stress responses. Researchers can use qPCR to track Tmem111/Emc3 upregulation as an indicator of UPR activation, alongside traditional markers like BiP and HERP.

Advanced Research Questions

• What are the optimal vectors and systems for overexpressing Rat Tmem111/Emc3 in experimental models?

  For researchers seeking to overexpress Rat Tmem111/Emc3, several validated vector systems are available:

  AAV-based expression systems:

  • Vector catalog: AAV-292333 (AAV-r-TMEM111)

  • Available serotypes: AAV1, AAV2, AAV3, AAV5, AAV6, AAV8, AAV9, AAV-DJ, AAV-DJ8, AAV-DJ9

  • Default promoter: CMV (with approximately 30 alternative promoter options)

  • Optional reporters: GFP, CFP, YFP, RFP, mCherry

  • Storage buffer: PBS/5% Glycerol

  Adenovirus-based expression systems:

  • Vector catalog: ADV-292333 (Ad-r-TMEM111)

  • Viral backbone: Human Adenovirus Type5 (dE1/E3)

  • Default promoter: CMV

  • Optional reporters: GFP, CFP, YFP, RFP, mCherry

  • Storage buffer: DMEM, 2% BSA, 2.5% Glycerol

  When selecting an expression system, consider the target tissue tropism (AAV serotypes have different tissue preferences) and the required expression duration (adenovirus provides strong but more transient expression compared to AAV).

• What methodological approaches are most effective for studying Tmem111/Emc3 interactions with ERAD components?

  To study Tmem111/Emc3 interactions with ERAD components, researchers have successfully employed several complementary approaches:

  Protein complex isolation and characterization:

  • Differential detergent solubilization: Using digitonin preserves more native interactions compared to Triton X-100

  • Immunoprecipitation: Can be performed with various EMC3/Tmem111 antibodies

  • Mass spectrometry: Particularly useful for identifying novel interaction partners

  Interaction network mapping:

  • CompPASS analysis: Used to identify high-confidence interacting proteins (HCIPs)

  • Cluster analysis: Identifies functionally related protein groupings

  Research has shown that EMC3 forms complexes with ERAD components including Ubac2 and Derlin-2. These interactions are better preserved using milder detergents like digitonin, while Triton X-100 disrupts many of these interactions .

• How can researchers effectively detect and quantify endogenous Tmem111/Emc3 protein expression?

  Several validated antibodies and detection methods have been documented for Tmem111/Emc3:

  Western blotting:

  • Expected molecular weight: ~38 kDa (slightly higher than the predicted 31.4 kDa)

  • Sample preparation: Rat brain membrane homogenates show clear detection

  Immunocytochemistry:

  • Antibody validation: Antibodies show specific immunoreactivity against recombinant HA-tagged SynDIG4 but not related proteins

  qPCR for mRNA quantification:

  • Tmem111 has been successfully used as a reference gene in some experimental setups

  • Primers targeting specific exon junctions are recommended to avoid genomic DNA amplification

  When performing protein detection, it's advisable to use multiple antibodies targeting different epitopes to confirm specificity, as has been demonstrated with other transmembrane proteins .

• How does Tmem111/Emc3 coordinate surfactant protein and lipid homeostasis, particularly in pulmonary research?

  Tmem111/Emc3 plays a critical role in coordinating surfactant protein and lipid homeostasis in alveolar type 2 (AT2) cells, particularly important for lung function at birth . Research methodologies to study this function include:

  In vivo models:

  • Infant rat models can be used to study pulmonary development and function

  • EMC3 expression can be manipulated through viral vectors (AAV or Adenovirus)

  Analysis methods:

  • qPCR: To analyze expression changes in EMC3 and related surfactant proteins

  • Protein analysis: Western blotting and proteomic approaches

  • Lipid profiling: Mass spectrometry-based lipidomics to assess changes in lipid composition

  Research has demonstrated that EMC3 coordinates the assembly of both lipids and proteins in AT2 cells, making it a potentially important target for studying disorders of surfactant production or function .

• What are the implications of Tmem111/Emc3 upregulation during ER stress for neuroprotection research?

  The upregulation of Tmem111/Emc3 during ER stress has important implications for neuroprotection research, particularly in contexts like meningitis where inflammation and ER stress are significant factors . Methodological approaches include:

  Experimental models:

  • Pneumococcal meningitis infant rat model has been used to study ER stress responses

  • Dexamethasone treatment shows modulation of various genes, potentially including EMC components

  Analysis techniques:

  • Microarray analysis: To profile expressional changes

  • qPCR validation: Using reference genes like GAPDH, USO1, and Tmem111

  • Protein level confirmation: Through western blotting and Luminex® xMAP® Technology

  Data analysis methods:

  • ΔΔCq method for qPCR data analysis

  • Appropriate statistical analysis for multiple comparisons

  • Component analysis to visualize data structure based on parameters like infection, brain region, and treatment

  Understanding Tmem111/Emc3's role in ER stress responses could provide insights into neuroprotective mechanisms and potential therapeutic targets.

• What experimental design considerations are critical when using Tmem111 as a reference gene in qPCR studies?

  While Tmem111 has been used as a reference gene in some experimental contexts , researchers should consider several important factors:

  Selection criteria:

  • Expression stability: Verify that Tmem111 expression remains stable under your specific experimental conditions

  • Coefficient of variance: Genes with CV values greater than 4% of raw Cq values should be excluded

  • Multiple reference genes: Use a geometric mean of multiple references (e.g., GAPDH, USO1, Tmem111, Rpl24) for more robust normalization

  Technical considerations:

  • RT-negative controls: Important to assess genomic DNA contamination

  • Primer design: Consider exon-spanning primers to avoid genomic DNA amplification

  • Cut-off Cq: A threshold of 35 cycles is typically used

  Validation experiments:

  • Confirm reference gene stability across all experimental conditions

  • Perform pilot studies to assess reference gene suitability

  When properly validated, Tmem111 can serve as an effective reference gene in specific experimental contexts, but this should be verified for each new experimental design.

• What approaches can be used to investigate the functional differences of Tmem111/Emc3 across species for evolutionary research?

  Investigating cross-species functional differences in Tmem111/Emc3 requires integrated methodological approaches:

  Sequence analysis:

  • Alignment of Tmem111/Emc3 sequences across species

  • Within the ECD (extracellular domain), rat Tmem111/Emc3 shares varying levels of sequence identity with other species:

    • 94% with mouse

    • 69-76% with bovine, canine, cotton rat, equine, feline, human, porcine, and rhesus models

  Functional conservation studies:

  • The EMC was first identified in yeast as a 6-subunit complex

  • EMCs are essential for assembly of nicotinic acetylcholine receptors in C. elegans and rhodopsin in Drosophila and Danio

  Expression pattern analysis:

  • In rat models, Tmem111/Emc3 shows specific expression patterns in neuronal tissues

  • Comparative expression studies can reveal tissue-specific adaptations

  Understanding cross-species differences can provide insights into evolutionary adaptations of the ER protein folding machinery and identify conserved functional domains suitable as therapeutic targets.