Recombinant Danio rerio Transmembrane protein 111 (tmem111)

What is Transmembrane protein 111 (tmem111) in Danio rerio?

Transmembrane protein 111 (tmem111) is a protein encoded by the tmem111 gene in Danio rerio (zebrafish), also known as Brachydanio rerio. It is alternatively named Protein pob with ORF name zgc:63727. The protein is characterized by multiple transmembrane domains and plays important roles in cellular membrane organization. The protein has UniProt accession number Q7SXW4 and consists of 261 amino acids in its full-length form . While there might be some confusion with Tmem11, which has been extensively studied in zebrafish, tmem111 is a distinct protein with its own functional characteristics that appear to be related to the endoplasmic reticulum membrane protein complex (EMC) .

How does tmem111 compare to its orthologs in other species?

Zebrafish tmem111 appears to be the ortholog of EMC3 in mammals, which is encoded by the Tmem111 gene in mice. The EMC complex is highly conserved across eukaryotes, from yeast to humans, suggesting critical cellular functions. In mouse, EMC3 (encoded by the Tmem111 gene) functions as a subunit in the highly conserved ER membrane protein complex (EMC), first identified in Saccharomyces cerevisiae as a 6-subunit complex required for protein folding in the ER .

The zebrafish tmem111 protein shares functional similarities with its mammalian counterparts, particularly in terms of its role in membrane protein biogenesis. While sequence conservation might vary between species, the functional domains and mechanisms appear to be preserved, allowing for translational research between zebrafish models and mammalian systems .

What is the primary function of tmem111 in zebrafish?

Based on the available research, tmem111 in zebrafish appears to function similarly to its mammalian ortholog as a component of the EMC complex. The EMC plays crucial roles in the biogenesis of transmembrane proteins, particularly in facilitating the insertion of transmembrane domains into the lipid bilayer .

The primary functions likely include:

• Facilitating proper membrane protein folding in the ER

• Mediating the insertion of transmembrane domains into lipid bilayers

• Working with other EMC components to form a functional complex that maintains ER homeostasis

• Potentially assisting in the assembly of multipass membrane proteins, as seen with EMCs in other species that are essential for assembling complex membrane proteins such as nicotinic acetylcholine receptors and rhodopsin

How does tmem111 contribute to the endoplasmic reticulum membrane protein complex (EMC)?

As an ortholog of EMC3, zebrafish tmem111 likely serves as a core component of the EMC. In other organisms, EMC3 is considered part of the "core" EMC subunits along with EMC1, EMC2, EMC5, and EMC6, as the loss of any of these subunits strongly impairs the integrity of the remaining complex .

The EMC complex directly mediates the insertion of transmembrane domains (TMDs) into the lipid bilayer by reducing the energetic barrier that would otherwise be imposed by the hydrophilic head groups of membrane lipids. This function is facilitated through a proposed conduit within the EMC similar to models for TMD insertion by other protein complexes like YidC, the Sec61 translocon, and the Get1/Get2 complex . Through this mechanism, tmem111/EMC3 contributes to the proper biogenesis and assembly of various membrane proteins.

What cellular processes are affected by tmem111 dysfunction?

Based on studies of EMC3 (the mammalian ortholog of tmem111) dysfunction, several critical cellular processes are likely affected when tmem111 is dysfunctional:

• Accumulation of misfolded membrane proteins, leading to ER stress and potential induction of the unfolded protein response (UPR)

• Impaired differentiation and function of secretory lineages, as seen in mice with Emc3 depletion

• Compromised assembly of multipass membrane proteins, potentially affecting various cellular signaling and transport systems

• Possible disruption of intestinal homeostasis, as Emc3 has been shown to be essential for maintaining proper goblet cell density and function in mice

In mouse models, Emc3 depletion results in reduced goblet cell density and size, and downregulation of typical mucus components. Similarly, Paneth cell function is affected, and these cellular defects can be partially rescued by treatment with the ER stress inhibitor TUDCA, suggesting that ER stress contributes to the observed phenotypes .

What are the optimal conditions for handling recombinant Danio rerio tmem111?

The optimal conditions for handling recombinant Danio rerio tmem111 involve careful attention to storage and experimental parameters:

Storage Conditions:

• Store at -20°C for regular use

• For extended storage, conserve at -20°C or -80°C

• Repeated freezing and thawing is not recommended

• For working aliquots, store at 4°C for up to one week

Buffer Composition:

• Typically maintained in Tris-based buffer with 50% glycerol, optimized for protein stability

• Buffer composition may need adjustment based on specific experimental requirements

Handling Recommendations:

• When thawing, allow the protein to reach room temperature gradually

• Work with small aliquots to avoid multiple freeze-thaw cycles

• For functional studies, consider the native membrane environment of the protein

What experimental approaches can be used to study tmem111 localization and interactions?

Several experimental approaches can be effectively employed to study tmem111 localization and interactions:

For Subcellular Localization:

• Fluorescent tagging: N-terminal EGFP tagging has been successfully used with related proteins like Tmem11 in zebrafish. Results showed mitochondrial localization for Tmem11, while tmem111/EMC3 would be expected to localize to the ER .

• Immunostaining with specific antibodies against tmem111 or epitope tags.

• Subcellular fractionation followed by Western blotting to determine the membrane compartment where tmem111 resides.

For Protein-Protein Interactions:

• Co-immunoprecipitation (Co-IP) assays to identify binding partners.

• Yeast two-hybrid screening, which has been used to explore interactions between related proteins and their binding partners .

• Proximity labeling methods such as BioID or APEX to identify proteins in close proximity to tmem111 in vivo.

• Crosslinking mass spectrometry to capture transient interactions.

For Functional Assays:

• Membrane insertion assays using reconstituted liposomes to assess the ability of tmem111/EMC to facilitate TMD insertion.

• ER stress reporter assays to monitor UPR activation in response to tmem111 dysfunction.

• CRISPR-Cas9 gene editing to generate knockout or knock-in zebrafish models for in vivo studies.

How can researchers effectively produce and purify recombinant tmem111 for functional studies?

Producing and purifying recombinant transmembrane proteins like tmem111 presents specific challenges due to their hydrophobic nature. Here is a methodological approach:

Expression Systems:

• Bacterial expression (E. coli): Can be used for partial domains but often results in inclusion bodies for full-length transmembrane proteins.

• Yeast expression (P. pastoris): Better suited for membrane proteins with proper folding.

• Insect cell expression (Sf9, Sf21): Provides eukaryotic processing and often yields properly folded membrane proteins.

• Mammalian cell expression (HEK293, CHO): Provides the most native-like environment but with lower yields.

Purification Strategy:

• Solubilization: Carefully select detergents (DDM, LMNG, or digitonin) that maintain protein structure.

• Affinity chromatography: Utilize His-tag, FLAG-tag, or other affinity tags for initial purification.

• Size-exclusion chromatography: Further purify based on size and remove aggregates.

• Detergent exchange or reconstitution into nanodiscs or liposomes for functional studies.

Quality Control:

• SDS-PAGE and Western blotting to confirm purity and identity.

• Circular dichroism to assess secondary structure.

• Thermal stability assays to evaluate protein folding.

• Mass spectrometry for accurate molecular weight determination and post-translational modification analysis.

How can CRISPR-Cas9 gene editing be applied to study tmem111 function in zebrafish?

CRISPR-Cas9 gene editing offers powerful approaches to study tmem111 function in zebrafish through various strategic modifications:

Knockout Strategies:

• Complete gene knockout: Design gRNAs targeting early exons to create frameshift mutations.

• Domain-specific disruption: Target specific functional domains to understand their contributions.

• Conditional knockout: Utilize Cre-loxP systems for tissue-specific or temporally controlled deletion.

Knockin Approaches:

• Fluorescent reporter fusion: Insert fluorescent tags (GFP, mCherry) to monitor protein localization and dynamics in vivo.

• Point mutations: Introduce specific mutations to study structure-function relationships.

• Epitope tagging: Add small epitope tags for biochemical studies without disrupting function.

Methodological Considerations:

• Design multiple gRNAs with minimal off-target effects.

• Screen F0 mosaic embryos for phenotypes before establishing stable lines.

• Use appropriate controls including wild-type siblings and non-targeting gRNA injections.

• Validate mutations by sequencing and protein expression analysis.

• Perform rescue experiments by co-injecting wild-type mRNA to confirm specificity.

The zebrafish model offers unique advantages for studying tmem111, including rapid development, optical transparency for imaging, and genetic tractability. Combined with the efficiency of CRISPR-Cas9, researchers can generate valuable models to investigate the developmental and physiological roles of tmem111.

What are the implications of tmem111/EMC3 for understanding human diseases related to ER stress?

The study of tmem111/EMC3 has significant implications for understanding human diseases related to ER stress and protein homeostasis:

Disease Connections:

• Secretory cell disorders: Given the role of mouse Emc3 in maintaining secretory lineages (goblet cells, Paneth cells), tmem111/EMC3 may be implicated in intestinal disorders characterized by mucus layer defects or antimicrobial peptide deficiencies .

• Neurodegenerative diseases: Protein misfolding in the ER is a hallmark of several neurodegenerative conditions, and EMC dysfunction could contribute to pathogenesis.

• Metabolic disorders: Proper membrane protein insertion is crucial for metabolic processes, and EMC dysfunction could impact metabolic homeostasis.

Therapeutic Implications:

• ER stress modulation: The finding that TUDCA treatment rescues phenotypes in Emc3-deficient mice suggests that targeting ER stress could be beneficial in EMC-related disorders .

• EMC function enhancement: Developing compounds that enhance EMC function might improve membrane protein biogenesis in disease states.

• Personalized medicine: Understanding how genetic variations in tmem111/EMC3 affect disease susceptibility could inform personalized therapeutic approaches.

Research Directions:

• Zebrafish disease models: Develop tmem111-mutant zebrafish as models for human diseases to facilitate drug screening and mechanistic studies.

• Functional conservation: Compare the functional conservation of zebrafish tmem111 with human EMC3 to validate translational relevance.

• Tissue-specific roles: Investigate tissue-specific functions of tmem111/EMC3 to understand why certain tissues are more affected in disease states.

How does tmem111 interact with the unfolded protein response (UPR) pathway?

The interaction between tmem111 and the unfolded protein response (UPR) pathway represents a critical area of investigation:

Mechanistic Connections:

• ER stress induction: Loss of EMC function, including EMC3 (tmem111 ortholog), leads to accumulation of misfolded membrane proteins, triggering the UPR .

• UPR sensor interactions: EMC components may directly interact with UPR sensors (IRE1, PERK, ATF6) to modulate their activation.

• Adaptive responses: The EMC complex may participate in adaptive responses to ER stress, potentially through altered gene expression or protein degradation pathways.

Experimental Evidence:

• In yeast, loss of EMC subunits causes UPR induction, indicating accumulated misfolded proteins .

• In mice, Emc3 depletion leads to ER stress, and treatment with the ER stress inhibitor TUDCA partially rescues the associated phenotypes, suggesting a causal relationship between Emc3 deficiency, ER stress, and cellular dysfunction .

• EMC1, EMC2, and EMC3 form a complex with ER-associated degradation (ERAD) pathway components Ubac2 and Derlin-2, indicating a close link between the EMC and ERAD systems that remove misfolded proteins .

Research Applications:

• Use tmem111-deficient zebrafish as a model to study UPR activation and its consequences in vivo.

• Investigate the transcriptional changes associated with tmem111 dysfunction to identify UPR target genes.

• Explore the potential for UPR modulation as a therapeutic strategy in conditions associated with tmem111/EMC dysfunction.

How does zebrafish tmem111 compare functionally to related proteins like Tmem11?

Despite their similar names, zebrafish tmem111 and Tmem11 represent distinct proteins with different subcellular localizations and functions:

Comparative Analysis:

Functional Distinctions:

• tmem111/EMC3 participates in membrane protein insertion and folding at the ER, while Tmem11 appears to regulate mitochondrial dynamics.

• While both are transmembrane proteins, their distinct localizations suggest different roles in cellular compartments.

• The conservation of both proteins across species indicates separate evolutionary paths and non-redundant functions.

Understanding these distinctions is important for researchers to avoid confusion and to properly interpret experimental results when studying either protein in zebrafish models.

What is known about the evolutionary conservation of tmem111 across different species?

The evolutionary conservation of tmem111/EMC3 across species provides insights into its fundamental importance in cellular function:

Conservation Profile:

• The EMC complex is highly conserved from yeast to humans, indicating ancient origins and essential functions .

• In yeast, the EMC was first identified as a 6-subunit complex, while in mammals it has expanded to include additional components .

• The core functions in membrane protein biogenesis appear to be preserved across evolutionary lineages, suggesting strong selective pressure.

Functional Conservation:

• EMC components in diverse species (yeast, worms, flies, fish, mammals) share the ability to facilitate membrane protein insertion and assembly .

• The EMC3 subunit specifically appears to be part of the "core" EMC in various organisms, with loss of this subunit severely compromising complex integrity .

• The involvement of EMC3 orthologs in ER stress responses appears to be conserved from yeast to mammals .

Research Applications:

• Comparative genomics approaches can reveal conserved domains and residues crucial for tmem111/EMC3 function.

• Zebrafish models offer a vertebrate system to study conserved aspects of tmem111 function that may be relevant to human biology.

• Evolutionary analysis can help identify species-specific adaptations in tmem111 structure and function.

What are common challenges in working with recombinant tmem111 and how can they be addressed?

Working with recombinant transmembrane proteins like tmem111 presents several challenges that require specific technical approaches:

Common Challenges and Solutions:

Experimental Approach Recommendations:

• Start with small-scale expression trials to optimize conditions before scaling up.

• Consider expressing individual domains separately if the full-length protein proves challenging.

• Utilize GFP fusion constructs to monitor expression, solubility, and folding in real-time.

• Implement quality control at each purification step to ensure protein integrity and homogeneity.

How can researchers design effective assays to measure tmem111/EMC function?

Designing effective assays to measure tmem111/EMC function requires consideration of its role in membrane protein biogenesis:

Functional Assay Approaches:

• Membrane insertion assays:

  • Reconstitute purified tmem111/EMC components with liposomes

  • Use fluorescently labeled substrate proteins to monitor insertion

  • Measure protection from protease digestion as evidence of successful insertion

  • Compare insertion efficiency with and without functional EMC components

• Cell-based reporter systems:

  • Generate reporter constructs with known EMC-dependent membrane proteins

  • Couple successful insertion/folding to fluorescent protein expression or enzymatic activity

  • Measure reporter output in tmem111 wild-type versus knockout/knockdown backgrounds

• ER stress monitoring:

  • Utilize UPR reporter constructs containing ER stress response elements

  • Measure activation in response to tmem111 manipulation

  • Test rescue with wild-type tmem111 expression

• Interaction verification:

  • Employ split-protein complementation assays to verify interactions with other EMC components

  • Use FRET-based approaches to monitor interactions in live cells

  • Apply crosslinking followed by mass spectrometry to identify interaction sites

Methodological Considerations:

• Include appropriate positive and negative controls in all assays

• Validate results using multiple orthogonal approaches

• Consider both in vitro and in vivo assays to comprehensively assess function

What advanced imaging techniques are most suitable for studying tmem111 dynamics and localization?

Advanced imaging techniques offer powerful tools for investigating tmem111 dynamics and localization:

Recommended Imaging Approaches:

• Super-resolution microscopy:

  • Stimulated emission depletion (STED) microscopy for nanoscale resolution of ER structures

  • Structured illumination microscopy (SIM) for 3D visualization of tmem111 distribution

  • Single-molecule localization microscopy (PALM/STORM) for precise localization mapping

• Live-cell imaging techniques:

  • Fluorescence recovery after photobleaching (FRAP) to measure mobility within membranes

  • Fluorescence correlation spectroscopy (FCS) to analyze diffusion properties

  • Single-particle tracking to follow individual tmem111 complexes over time

• Proximity detection methods:

  • Förster resonance energy transfer (FRET) to detect interactions with partner proteins

  • Bimolecular fluorescence complementation (BiFC) to visualize protein complexes

  • Split-GFP complementation to verify membrane topology

• Correlative techniques:

  • Correlative light and electron microscopy (CLEM) to combine functional imaging with ultrastructural details

  • Expansion microscopy to physically enlarge specimens for enhanced resolution

  • Cryo-electron tomography to visualize tmem111/EMC in near-native states

Implementation Strategy:

• Use N-terminal fluorescent protein tags, as C-terminal tags may interfere with proper membrane integration (as observed with Tmem11)

• Validate that tagged constructs retain functionality through complementation assays

• Combine multiple imaging modalities for comprehensive characterization

• Consider the dynamic nature of ER membranes when interpreting localization data