Recombinant Mouse Transmembrane protein 151B (Tmem151b)

What is the molecular structure and biochemical properties of Mouse Transmembrane protein 151B?

Mouse Transmembrane protein 151B (Tmem151b) is characterized by two transmembrane domains. Based on its human ortholog, the protein has a length of 566 amino acids with a molecular weight of approximately 61 kDa and a theoretical isoelectric point of 6.72. Notable biochemical features include its low lysine and arginine content, with lysine comprising only 1.4% of amino acids and arginine making up merely 0.8% of the total protein. The mouse ortholog similarly exhibits this arginine-poor composition .

The protein is encoded by the Tmem151b gene, which is also known by the synonym Gm323 . The product is available in recombinant form expressed in E. coli systems, typically provided in liquid form containing glycerol to enhance stability .

Where is Tmem151b primarily expressed in mouse tissues?

RNA-seq gene expression profiling demonstrates that Tmem151b exhibits high expression in the brain, with additional notable expression in the testes. Within the brain, the Allen Brain Atlas shows particularly high expression in specific regions including the cerebellum, medulla, and olfactory bulb . This distinctive expression pattern suggests potential specialized functions in these neuroanatomical structures that may relate to sensory processing, motor coordination, or specific neuronal populations.

What is the genomic organization of the Tmem151b gene?

The Tmem151b gene is located on the positive strand of chromosome 6 at position 6p21.1, spanning from position 44270450 to 44279444, for a total length of 8,995 base pairs . The gene contains 3 exons and represents a complex locus that contains both Tmem151b and SPATS1 . The transcribed mRNA has a length of 4911bp, with the coding region containing 1701bp . Tmem151b has one known paralog: TMEM151A .

What are the known protein interactions of Tmem151b?

According to BioGRID data, Tmem151b interacts with one known protein partner: SREBF2 (Sterol Regulatory Element Binding Transcription Factor 2) . SREBF2 is a transcription factor precursor embedded in the endoplasmic reticulum membrane that activates genes involved in cholesterol biosynthesis . This interaction suggests that Tmem151b may play a role in regulating lipid metabolism, particularly cholesterol biosynthesis pathways, which could be especially relevant in brain tissues where cholesterol metabolism is tightly regulated.

What is the evolutionary conservation of Tmem151b across species?

The Tmem151b gene exhibits strong evolutionary conservation across most vertebrate species and appears to be conserved in some invertebrates as well . This high degree of conservation suggests fundamental biological importance. The presence of a paralog (TMEM151A) indicates that a gene duplication event likely occurred during evolution, potentially allowing for functional specialization between the two family members.

What experimental approaches are recommended for studying Tmem151b function in neural tissues?

Given the high expression of Tmem151b in specific brain regions, optimal experimental approaches include:

• Region-specific expression analysis: Quantitative immunohistochemistry combined with single-cell RNA sequencing to identify specific neuronal or glial populations expressing Tmem151b in the cerebellum, medulla, and olfactory bulb.

• Conditional knockout models: Cre-loxP systems targeting Tmem151b in specific brain regions or neuronal populations to avoid potential developmental effects of global knockout.

• Primary neuronal culture models: Isolation of neurons from high-expression regions for in vitro studies, including gene silencing or overexpression approaches.

• Functional assays: Electrophysiological recordings and calcium imaging in neurons expressing or lacking Tmem151b to assess potential roles in neuronal signaling.

• Proteomic approaches: Co-immunoprecipitation followed by mass spectrometry to identify the complete interactome of Tmem151b in neural tissues.

How might the interaction between Tmem151b and SREBF2 impact cholesterol biosynthesis pathways?

The interaction between Tmem151b and SREBF2 suggests a potential regulatory role in cholesterol metabolism . Given that SREBF2 activates genes involved in cholesterol biosynthesis, Tmem151b could function as:

• A regulator of SREBF2 processing or transport from the ER to the Golgi, which is a key step in SREBF2 activation.

• A modulator of SREBF2's interaction with other regulatory proteins like SCAP or Insig.

• A component of the machinery that senses cellular cholesterol levels.

• A factor that influences SREBF2's transcriptional activity or target gene specificity.

To investigate these possibilities, researchers should consider:

• Analyzing SREBF2 cleavage patterns and nuclear translocation in cells with modified Tmem151b expression

• Measuring expression of SREBF2 target genes (e.g., HMGCR, LDLR) in response to Tmem151b manipulation

• Assessing cholesterol synthesis rates and cellular cholesterol content in neuronal cells with Tmem151b knockout or overexpression

• Examining whether Tmem151b expression is itself regulated by cellular cholesterol levels

What are the challenges in expressing and purifying recombinant Tmem151b for functional studies?

As a transmembrane protein, Tmem151b presents several challenges for recombinant expression and purification:

• Expression system selection: While E. coli has been used successfully , transmembrane proteins often require eukaryotic expression systems for proper folding and post-translational modifications.

• Protein solubilization: Extraction from membranes requires careful optimization of detergent types and concentrations to maintain native structure.

• Purification strategy: Affinity tags may need to be positioned to avoid disrupting transmembrane domains or functional sites.

• Functional reconstitution: For activity studies, the protein may need to be reconstituted into liposomes or nanodiscs that mimic the native membrane environment.

• Stability maintenance: Once purified, storage conditions must be optimized to prevent aggregation or denaturation. According to product information, storage at -20°C or -80°C for extended periods is recommended, with working aliquots maintained at 4°C for up to one week .

How can researchers investigate potential post-translational modifications of Tmem151b?

To characterize post-translational modifications (PTMs) of Tmem151b:

• Mass spectrometry analysis: Immunoprecipitate endogenous Tmem151b from brain tissues and analyze using high-resolution mass spectrometry to identify PTMs.

• Site-directed mutagenesis: Mutate predicted modification sites and assess impacts on localization, interaction with SREBF2, and function.

• PTM-specific antibodies: Use antibodies that recognize specific modifications (phosphorylation, glycosylation, etc.) to detect modified forms of Tmem151b.

• Inhibitor studies: Treat cells with inhibitors of specific modification enzymes and assess effects on Tmem151b function.

• In vitro modification assays: Incubate purified recombinant Tmem151b with modification enzymes to confirm susceptibility to specific modifications.

What functional genomics approaches would be most effective for characterizing Tmem151b in vivo?

For comprehensive functional characterization of Tmem151b in vivo:

• CRISPR/Cas9 genome editing: Generate knockout, knockin, or specific mutations to assess function. Consider conditional alleles for temporal control.

• Cell type-specific transcriptomics: Perform RNA-seq on specific neuronal populations expressing Tmem151b to identify co-expressed genes and potential pathways.

• Behavioral phenotyping: Design assays targeting functions associated with brain regions where Tmem151b is highly expressed (olfactory discrimination, motor coordination, etc.).

• In vivo imaging: Use techniques like fiber photometry or miniature microscopy to monitor neural activity in Tmem151b-expressing neurons during relevant behaviors.

• Metabolic profiling: Assess lipid metabolism, particularly cholesterol homeostasis, in brain regions with Tmem151b knockout or overexpression.

What are the optimal storage and handling conditions for recombinant Tmem151b?

According to product information, recombinant Mouse Tmem151b should be handled as follows :

• Storage temperature: Store at -20°C for routine storage, or at -80°C for extended storage periods.

• Form: The protein is provided in liquid form containing glycerol, which helps prevent damage from freeze-thaw cycles.

• Aliquoting: Upon first thaw, divide into small working aliquots to avoid repeated freeze-thaw cycles, which can compromise protein integrity.

• Working conditions: Aliquots for immediate use can be stored at 4°C for up to one week.

• Thawing procedure: Thaw frozen aliquots rapidly at room temperature, then place on ice until use.

• Buffer considerations: When diluting for experiments, consider using buffers containing stabilizing agents such as low concentrations of glycerol, detergents, or carrier proteins depending on the application.

What approaches can validate antibody specificity for Tmem151b detection in experimental systems?

For rigorous validation of antibodies against Tmem151b:

• Genetic controls: Test antibodies in tissues/cells from Tmem151b knockout models as negative controls.

• Expression pattern concordance: Verify that detected protein patterns match known mRNA expression profiles across tissues (high in brain and testes) .

• siRNA knockdown: Perform knockdown experiments and confirm reduced signal by immunoblotting or immunocytochemistry.

• Recombinant protein controls: Use purified recombinant Tmem151b as a positive control in Western blots.

• Immunoprecipitation-mass spectrometry: Confirm that immunoprecipitated proteins include Tmem151b peptides.

• Multiple antibodies: Use antibodies targeting different epitopes to confirm consistent results.

• Brain region specificity: Verify higher signal in cerebellum, medulla, and olfactory bulb compared to other brain regions .

What methods are recommended for investigating the membrane topology of Tmem151b?

To characterize how Tmem151b is oriented within cellular membranes:

• Protease protection assays: Treat intact cells, permeabilized cells, or microsomes with proteases and analyze protected fragments to determine which domains are accessible.

• Glycosylation site mapping: Introduce artificial N-glycosylation sites at various positions and assess glycosylation status, which occurs only on luminally exposed domains.

• Fluorescence-based approaches: Create fusion constructs with pH-sensitive fluorescent proteins to determine orientation relative to cellular compartments.

• Cysteine accessibility methods: Introduce cysteine residues at various positions and assess their accessibility to membrane-impermeable sulfhydryl reagents.

• Computational prediction validation: Compare experimental results with predictions from algorithms like TMHMM or Phobius that predict transmembrane domains.

What are the recommended cell models for studying Tmem151b function?

Based on Tmem151b's expression pattern, the following cell models are recommended:

• Primary neurons from high-expression regions: Cultures from cerebellum, medulla, or olfactory bulb provide the most physiologically relevant context .

• Neuronal cell lines: Cell lines like Neuro2A, SH-SY5Y, or PC12 can be used for overexpression or knockdown studies in a neuronal context.

• Brain slice cultures: Organotypic slices from relevant brain regions maintain tissue architecture and cell-cell interactions.

• HEK293 or CHO cells: For basic biochemical and interaction studies, especially when co-expressing SREBF2 .

• Testicular cell lines: Given the expression in testes, appropriate testicular cell lines may also be valuable models .

How can researchers design experiments to investigate Tmem151b's role in cholesterol metabolism?

To explore the potential role of Tmem151b in cholesterol metabolism through its interaction with SREBF2 :

• Reporter assays: Use luciferase reporters driven by SREBF2-responsive elements to assess transcriptional activity in the presence/absence of Tmem151b.

• Sterol-regulated processing: Monitor SREBF2 cleavage and nuclear translocation under various sterol conditions with normal or altered Tmem151b levels.

• Target gene expression: Quantify expression of key cholesterol biosynthesis enzymes (HMGCR, FDFT1, etc.) following Tmem151b manipulation.

• Cholesterol quantification: Measure cellular and subcellular cholesterol content using enzymatic assays or filipin staining in models with altered Tmem151b expression.

• Brain region-specific analysis: Compare lipid profiles in brain regions with high versus low Tmem151b expression.