Recombinant Mouse Transmembrane and ubiquitin-like domain-containing protein 2 (Tmub2)

How is recombinant Mouse Tmub2 typically produced for research purposes?

Recombinant mouse Tmub2 can be produced using several expression systems, with the choice depending on the specific research requirements:

For mammalian expression (such as HEK-293 cells), the protein is typically purified via one-step affinity chromatography using the appropriate tag . Purity levels exceeding 90% can be achieved as determined by Bis-Tris PAGE, anti-tag ELISA, Western Blot, and analytical SEC (HPLC) .

When using cell-free protein synthesis systems like AliCE®, which is based on lysate obtained from Nicotiana tabacum, the purity typically ranges from 70-80% . This system contains protein expression machinery needed for difficult-to-express proteins requiring post-translational modifications.

Standard reconstitution procedures typically involve:

• Centrifuging the vial before opening

• Reconstituting at concentrations between 100-500 μg/mL in appropriate buffer (PBS pH 7.4 with 10% glycerol is common)

• Avoiding repeated freeze-thaw cycles

• Storage at -80°C for optimal stability

What is the difference between mouse and human TMUB2?

Mouse and human TMUB2 share significant homology but display several key differences that may influence experimental design and interpretation:

The sequence alignment shows particularly high conservation in the ubiquitin-like domain region, suggesting functional importance. The minor differences in the amino acid sequence may influence antibody selection for experimental procedures - antibodies raised against human TMUB2 may not always recognize mouse TMUB2 with equal affinity and specificity .

For studies transitioning between mouse models and human applications, researchers should validate that findings from mouse Tmub2 studies are applicable to human TMUB2 function, particularly when studying protein-protein interactions or regulatory pathways.

What are the important structural domains of Mouse Tmub2?

Mouse Tmub2 contains several distinct structural domains that are critical to its function:

The ubiquitin-like domain is particularly significant as it mediates interactions with components of the ubiquitin-proteasome system. This domain likely enables Tmub2 to function in protein quality control pathways, particularly in the context of ER-associated degradation (ERAD) .

The transmembrane domains anchor the protein to cellular membranes, primarily the endoplasmic reticulum membrane, positioning the ubiquitin-like domain for interactions with other ERAD components such as the RNF185/Membralin complex .

What experimental applications can recombinant Mouse Tmub2 be used for?

Recombinant mouse Tmub2 can be employed in various experimental applications:

When designing experiments:

• Consider using carrier-free (CF) recombinant Tmub2 for applications where BSA might interfere, such as certain cell culture experiments or when performing complex protein interaction studies .

• For ELISA applications, commercially available kits typically use sandwich ELISA format with detection ranges of 0.156-10 ng/mL, allowing for quantitation in cell culture supernatants, serum, plasma, and other biological fluids .

• For stability in experiments, reconstituted proteins have been shown to withstand four freeze-thaw cycles without significant loss of activity, though minimizing such cycles is recommended for optimal results16.

How does Tmub2 function in the ERAD (ER-associated degradation) pathway?

Tmub2 appears to function as part of the endoplasmic reticulum-associated degradation (ERAD) pathway, which is responsible for the recognition, retrotranslocation, and degradation of misfolded or unassembled proteins in the ER. Current research indicates several specific roles:

• Component of a specialized ERAD complex: Tmub2 forms part of a distinct ERAD branch defined by a complex composed of the ubiquitin ligase RNF185, TMEM259/Membralin, and TMUB1/2 proteins. This complex cooperates with UBE3C, a cytosolic ubiquitin ligase recently implicated in ERAD .

• Substrate recognition and processing: The ubiquitin-like domain (UBL) of Tmub2 likely participates in substrate recognition or processing within the ERAD pathway. UBL domains in other proteins have been shown to interact with the proteasome and other components of the ubiquitin-proteasome system.

• Regulation by RNF185: Evidence suggests that Tmub2 levels are regulated by the catalytic activity of RNF185, pointing to a potential feedback mechanism within the ERAD pathway .

Methodological approaches to study Tmub2's role in ERAD include:

• Pulse-chase experiments: To track the degradation of ERAD substrates in the presence or absence of Tmub2.

• Proteasome inhibition: Treating cells with MG132 or bortezomib to determine if Tmub2-dependent degradation is proteasome-mediated.

• Co-immunoprecipitation: To identify interactions between Tmub2 and other ERAD components.

• CRISPR-Cas9 knockout/knockdown: To assess the impact of Tmub2 depletion on ERAD efficiency.

• Ubiquitination assays: Using TUBEs (Tandem Ubiquitin Binding Entities) to capture ubiquitinated proteins and assess the impact of Tmub2 on substrate ubiquitination .

What is the relationship between Tmub2 and the RNF185/Membralin complex?

Recent research has elucidated a functional relationship between Tmub2 and the RNF185/Membralin complex in the ERAD pathway:

• Complex formation: Tmub2 appears to be part of a multi-protein complex that includes the E3 ubiquitin ligase RNF185, the multi-spanning ER membrane protein TMEM259/Membralin, and the UBL-containing proteins TMUB1/2 .

• Substrate selectivity: This complex demonstrates remarkable specificity for membrane substrates, suggesting that multiple, perhaps combinatorial, determinants are involved in substrate selection .

• Distinct ERAD branch: The RNF185/Membralin/TMUB2 complex represents a previously uncharacterized ERAD branch that is distinct from the TEB4 (MARCH6) ERAD complex. While Erg11TM (a model ERAD substrate) was degraded by the TEB4 complex, CYP51A1TM was degraded by the RNF185/Membralin/TMUB2 complex, demonstrating that these two ERAD branches recognize distinct substrate features .

• Regulation of Tmub2 levels: Studies indicate that regulation of Tmub2 levels depends on the catalytic activity of RNF185, suggesting that Tmub2 might itself be a substrate of this E3 ligase .

Experimental approaches to study this relationship include:

• Proximity-based protein labeling (BioID, APEX) to identify proteins in close proximity to Tmub2 within the ER membrane.

• Reconstitution of the complex in vitro using purified components to study its biochemical properties.

• Structure-function analysis using domain deletions or point mutations to map interaction surfaces.

• Quantitative proteomics following genetic perturbation of complex components to identify substrates and regulatory networks.

• Live-cell imaging to track the dynamics of complex formation and substrate processing.

What methods can be used to study Tmub2 protein-protein interactions?

Investigating Tmub2 protein-protein interactions requires specialized approaches suitable for transmembrane proteins. The following methodologies can be effectively employed:

When studying Tmub2 interactions, consider these methodological aspects:

• Detergent selection: For membrane protein interactions, detergent choice is critical. Digitonin, DDM, or CHAPS often preserve membrane protein complexes better than harsher detergents like SDS or Triton X-100.

• Tag position: For tagged Tmub2 constructs, consider whether N- or C-terminal tagging might interfere with protein interactions or localization.

• Validation strategies: Confirm interactions using multiple orthogonal methods and include appropriate controls (tag-only, interaction-deficient mutants).

• Known interactions: Known interactions like those with Ubiquitin C (UBC), BCL2L13, SGTA, and UBQLN1 can serve as positive controls .

How can post-translational modifications of Tmub2 be characterized?

Characterizing post-translational modifications (PTMs) of Tmub2 requires a comprehensive strategy employing multiple complementary techniques:

For comprehensive PTM characterization of Tmub2:

• Enrichment strategies: Use tandem ubiquitin binding entities (TUBEs) to capture ubiquitinated forms of Tmub2 . For phosphorylation, employ IMAC (immobilized metal affinity chromatography) or titanium dioxide enrichment.

• Mass spectrometry approaches:

  • Bottom-up proteomics: Tryptic digestion followed by LC-MS/MS

  • Top-down proteomics: Analysis of intact protein to preserve PTM combinations

  • Middle-down: Limited proteolysis to generate large fragments that retain PTM context

• Site-directed mutagenesis: Mutate predicted PTM sites (e.g., lysines for ubiquitination, serines/threonines for phosphorylation) to confirm their functional significance.

• Temporal dynamics: Use pulse-chase experiments or kinetic studies to track how PTMs change in response to cellular stimuli or during protein maturation.

• Crosstalking PTMs: Investigate how different modifications influence each other, such as phosphorylation affecting subsequent ubiquitination.

What approaches can be used to study Tmub2 function in mouse models?

Investigating Tmub2 function in mouse models requires strategic approaches considering both genetic manipulation and phenotypic analysis:

For phenotypic analysis:

• Biochemical assessment: Analyze ubiquitination patterns in tissues; measure levels of known ERAD substrates; assess proteasome activity in various tissues.

• Cellular phenotypes: Examine ER morphology (by EM); quantify ER stress markers (XBP1 splicing, ATF6 cleavage, CHOP expression); assess sensitivity to ER stressors (tunicamycin, thapsigargin).

• Tissue-specific analysis: Given Tmub2's role in ERAD, focus on:

  • Liver: Susceptibility to drug-induced liver injury; hepatocyte ER stress

  • Brain: Neurodegeneration markers; protein aggregation (particularly in aging)

  • Pancreas: β-cell function; susceptibility to diabetes

• Behavioral testing: For neuronal phenotypes, examine learning and memory (hippocampal function) ; motor coordination (cerebellar function); anxiety and depression-like behaviors.

• Disease models: Challenge Tmub2-deficient mice with disease models where protein quality control is implicated:

  • Neurodegenerative disease models (e.g., polyQ proteins)

  • Metabolic stress models

  • Aging-related phenotypes

How does Tmub2 trafficking occur in neuronal cells?

Tmub2 trafficking in neuronal cells likely involves specialized mechanisms given their polarized morphology and complex protein sorting requirements. While specific data on Tmub2 neuronal trafficking is limited, several methodological approaches can elucidate its pathways:

• Live-cell imaging approaches:

  • Fluorescently-tagged Tmub2 (e.g., GFP-Tmub2 or Tmub2-mCherry) for real-time visualization

  • Pulse-chase protocols with photoactivatable/photoconvertible tags to track protein cohorts

  • Superresolution microscopy (STED, PALM, STORM) for nanoscale localization

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

• Compartment-specific analysis:

  • Fractionation of neuronal compartments (soma, dendrites, axons, synapses)

  • Proximity labeling (APEX, BioID) in specific compartments to identify local interactors

  • Local translation assessment using puromycin labeling or ribosome profiling

• Transport mechanisms:

  • Inhibitors of cytoskeletal motors (e.g., nocodazole for microtubules, latrunculin for actin)

  • Live imaging of co-transport with known vesicle markers

  • Analysis of mutants lacking binding sites for trafficking adaptors

Tmub2 likely undergoes regulated trafficking given its UBL domain, which in other proteins has been shown to play roles in receptor trafficking . Some ubiquitin-like domain-containing proteins regulate AMPA receptor cycling between intracellular compartments and the cell surface in the central nervous system .

Theoretical transport pathways to investigate include:

• ER-to-Golgi trafficking via COPII vesicles

• Endosomal sorting and recycling

• Activity-dependent redistribution to synapses

• Local degradation via lysosomes or the proteasome

Experimental approaches should include both developing neurons and mature circuits, as trafficking mechanisms may differ during development versus maintenance stages.

What are the emerging techniques for studying Tmub2's role in membrane dynamics?

Cutting-edge techniques are expanding our ability to study membrane proteins like Tmub2 in increasingly sophisticated ways:

Methodological approaches specific to membrane dynamics:

• Membrane organization:

  • Super-resolution microscopy techniques like PALM, STORM, or STED to visualize nanoscale membrane organization of Tmub2

  • Single-particle tracking to monitor diffusion behavior and confinement zones

  • Structured Illumination Microscopy (SIM) for live imaging with improved resolution

• Membrane remodeling:

  • CRISPR-based gene editing combined with high-content imaging to assess Tmub2's impact on ER morphology

  • FRET-based tension sensors to measure membrane deformation

  • Microfluidic devices to control membrane shape and study Tmub2 redistribution

• Interaction dynamics:

  • Single-molecule pull-down (SiMPull) assays to analyze stoichiometry of Tmub2 complexes

  • Fluorescence fluctuation spectroscopy (FFS) to measure oligomerization states in membranes

  • Mass photometry to determine complex size distributions

• Functional manipulation:

  • Acute protein degradation (e.g., Auxin-inducible degron or dTAG) to rapidly deplete Tmub2

  • Chemically-induced dimerization to artificially recruit Tmub2 to specific membranes

  • Protein complementation assays to visualize protein-protein interactions in real-time

These emerging techniques promise to advance our understanding of Tmub2's dynamic behavior in cellular membranes and its functional role in processes like ERAD and membrane protein quality control.