Recombinant Mouse Promethin (Tmem159)

What is Promethin (Tmem159) and what cellular processes does it regulate?

Promethin, formally known as Transmembrane protein 159 (Tmem159), is a membrane-bound protein implicated in several critical cellular functions. This protein appears to function primarily as a regulator of cell membrane dynamics, with significant roles in membrane trafficking and protein sorting pathways within cells. The protein contains transmembrane domains that anchor it within cellular membranes, enabling it to participate in these dynamic cellular processes . Understanding Promethin's role in membrane organization provides insights into fundamental cellular architecture and compartmentalization mechanisms that maintain proper cellular function.

What protein interaction networks is mouse Promethin (Tmem159) involved in?

Mouse Promethin participates in several protein-protein interaction networks that suggest functional importance in lipid metabolism and cellular regulation. STRING database analysis reveals that Tmem159 interacts with multiple proteins with high confidence scores, particularly Bscl2 (Seipin) with a confidence score of 0.849 . Seipin is a critical regulator of lipid catabolism essential for adipocyte differentiation and proper lipid storage maintenance. This strong interaction suggests Tmem159 may have complementary roles in lipid metabolism pathways. Additional interaction partners include Kctd9 (0.679 confidence), which functions as a substrate-specific adapter in E3 ubiquitin-protein ligase complexes, potentially linking Promethin to protein degradation pathways . The network also shows connections to Ccdc28a, Tmem256, Crim1, and other proteins that collectively suggest Promethin's involvement in diverse cellular processes beyond simple membrane organization.

How is mouse Promethin structurally characterized?

Mouse Promethin belongs to the TMEM159 family of proteins and is characterized by its transmembrane domains. While the search results don't provide the complete structural details for mouse Promethin specifically, related information suggests that human TMEM159 is a relatively small protein of approximately 19,987 Da molecular weight . The protein likely contains multiple membrane-spanning domains consistent with its role in membrane dynamics. For experimental purposes, researchers should note that recombinant approaches typically utilize expression of specific domains rather than the full-length protein to overcome challenges with membrane protein expression. When designing constructs for mouse Promethin studies, researchers should consider using predictive algorithms to identify soluble domains that maintain functional characteristics while improving expression yields.

What CRISPR/Cas9 strategies are most effective for genetic manipulation of mouse Tmem159?

For CRISPR/Cas9-mediated genetic manipulation of mouse Tmem159, researchers should prioritize guide RNA sequences specifically designed for high target specificity and minimal off-target effects. The Feng Zhang laboratory at the Broad Institute has developed optimized gRNA sequences that uniquely target the Tmem159 gene within the mouse genome . These sequences were designed using rigorous selection criteria to ensure efficient Cas9 binding while minimizing off-target effects elsewhere in the genome. For researchers planning knockout studies, it is recommended to order at least two different gRNA constructs per target gene to increase success rates . When implementing these constructs, several methodological considerations are crucial: 1) verify the gRNA sequences against your specific mouse strain's genomic sequence, 2) ensure the gRNAs target functionally important exons, 3) consider targeting multiple sites simultaneously if complete gene ablation is desired, and 4) implement appropriate screening methods to verify editing efficiency. Additionally, careful consideration of PAM site accessibility within the chromatin context can significantly impact editing efficiency in different cell types.

How can Promethin-Seipin interactions be effectively studied in the context of lipid metabolism?

Given the strong interaction between Promethin (Tmem159) and Seipin (Bscl2) with a confidence score of 0.849 in STRING database analysis , investigating this relationship requires sophisticated methodological approaches. To effectively study this interaction in lipid metabolism contexts, researchers should implement a multi-faceted experimental strategy combining protein-protein interaction assays with functional metabolic analyses. Begin with co-immunoprecipitation studies using either endogenous proteins or carefully designed tagged constructs that preserve functional domains. For higher resolution analysis, proximity ligation assays can demonstrate in situ interactions within cellular compartments, particularly at lipid droplet interfaces where Seipin is known to function.

To assess functional consequences of this interaction, design experiments using both loss-of-function approaches (CRISPR/Cas9 knockout or knockdown) and gain-of-function systems (overexpression of wild-type or mutant constructs). Specifically examine: 1) changes in lipid droplet morphology and distribution using fluorescent lipid dyes and high-resolution microscopy, 2) alterations in lipid composition using lipidomics approaches, 3) adipocyte differentiation efficiency in cellular models, and 4) changes in expression of lipid metabolism genes. The critical control should include rescue experiments where Promethin is reintroduced into knockout models to verify phenotype specificity. This comprehensive approach will elucidate both physical interaction parameters and functional significance of the Promethin-Seipin relationship in lipid metabolism regulation.

What strategies can overcome challenges in expressing functional recombinant mouse Promethin?

Expressing functional recombinant mouse Promethin presents significant challenges due to its transmembrane nature, which often results in protein aggregation, misfolding, or toxicity to expression hosts. A strategic approach begins with construct design optimization. Rather than attempting to express the full-length protein, create truncated constructs that exclude problematic hydrophobic regions while preserving functional domains of interest. For example, if investigating protein-protein interactions, focus on soluble domains predicted to mediate these interactions.

Expression system selection is crucial: mammalian expression systems (particularly HEK293 or CHO cells) often provide the most native-like post-translational modifications and membrane insertion machinery for transmembrane proteins. For these systems, consider using inducible promoters to control expression levels and prevent toxicity. Alternatively, insect cell expression (Sf9 or High Five cells) can produce higher yields while maintaining proper folding. Bacterial systems should be approached with caution, primarily for producing soluble domains with fusion partners like MBP or SUNET that enhance solubility.

For purification, detergent screening is essential when working with full-length or transmembrane-containing constructs. Begin with a panel of detergents including mild options like DDM, LMNG, or digitonin that preserve protein structure. Stability can be further enhanced by including lipids during purification or reconstituting the protein into nanodiscs or liposomes post-purification to provide a native-like membrane environment. Quality control should include SEC-MALS analysis to verify monodispersity and functional assays specific to Promethin's known activities in membrane dynamics or protein interactions.

What are the optimal detection methods for mouse Promethin in different experimental systems?

Detecting mouse Promethin across various experimental systems requires tailored approaches depending on whether you're working with endogenous protein or recombinant constructs. For immunodetection of endogenous Promethin, antibody selection is crucial. While our search results specifically mention an anti-human TMEM159 polyclonal antibody (PACO39550) , researchers working with mouse models should verify cross-reactivity or source mouse-specific antibodies. This particular antibody has been validated for immunofluorescence and ELISA applications with recommended dilutions of 1:50-1:200 for IF and 1:2000-1:10000 for ELISA .

For subcellular localization studies, co-staining with compartment markers (particularly ER, Golgi, and plasma membrane markers) will provide valuable context for Promethin's distribution. Super-resolution microscopy techniques like STORM or STED can resolve precise membrane localization patterns that conventional confocal microscopy might miss, particularly for studying dynamic membrane processes that Promethin likely influences.

How should researchers design experiments to study Promethin's role in membrane dynamics?

Investigating Promethin's role in membrane dynamics requires experimental designs that capture both static protein distribution and dynamic membrane processes. Begin with high-resolution localization studies using fluorescently-tagged Promethin constructs or immunofluorescence against the endogenous protein, combined with markers for specific membrane compartments. Time-lapse imaging with these constructs can reveal Promethin's behavior during processes like membrane trafficking, endocytosis, or response to cellular stressors.

For functional studies, implement both gain- and loss-of-function approaches. CRISPR/Cas9-mediated knockout using validated gRNA sequences provides a clean system for examining membrane alterations in Promethin's absence. Complement this with rescue experiments using wild-type or domain-mutated constructs to identify critical functional regions. For acute manipulation, consider optogenetic or chemical-genetic approaches that allow temporal control of Promethin activity.

Membrane dynamics can be quantitatively assessed through several specialized techniques: FRAP (Fluorescence Recovery After Photobleaching) to measure protein mobility within membranes, FLIP (Fluorescence Loss In Photobleaching) to examine continuity between membrane compartments, and pulse-chase experiments with lipid or protein markers to track trafficking rates. Combining these approaches with super-resolution microscopy provides both dynamic and high-resolution spatial information about Promethin's influence on membrane organization and trafficking.

The experimental design should include appropriate controls: 1) comparison with other TMEM family proteins to identify Promethin-specific effects, 2) examination of multiple cell types to determine context-dependent functions, and 3) pharmacological manipulation of known membrane trafficking pathways to position Promethin within established cellular machinery.

What approaches are recommended for analyzing Promethin interactions with lipid metabolism proteins?

Based on the STRING database interaction data showing Promethin's strong association with lipid metabolism regulators, particularly Seipin (Bscl2) with a confidence score of 0.849 , several specialized approaches are recommended for analyzing these interactions. Begin with basic co-immunoprecipitation techniques to verify physical associations, but extend analysis using more sophisticated methods like proximity-dependent biotin identification (BioID) or APEX2 proximity labeling to capture transient or compartment-specific interactions within living cells.

For functional analysis of these interactions in lipid metabolism, implement metabolic labeling studies using radioactive or stable isotope-labeled fatty acids to track lipid synthesis, trafficking, and turnover rates in models with altered Promethin expression. Lipidomic profiling using LC-MS/MS can comprehensively examine how Promethin manipulation affects cellular lipid composition across different lipid classes.

A particularly valuable approach is visualization of lipid dynamics using fluorescent lipid analogs or lipid-binding protein domains combined with live-cell imaging in Promethin knockout or overexpression models. This allows direct observation of how Promethin influences lipid distribution, trafficking, and lipid droplet formation. These studies should be performed in metabolically relevant cell types such as adipocytes, hepatocytes, or macrophages where lipid processing is a primary function.

For analyzing the specific Promethin-Seipin interaction, implement mutations in predicted interaction domains of either protein followed by functional rescue experiments to determine which aspects of lipid metabolism require this specific protein partnership. The experimental design should consider both basal conditions and metabolic challenges (lipid loading, starvation, or differentiation induction) to comprehensively map Promethin's role in dynamic lipid metabolism processes.

How can inconsistent results in Promethin knockout studies be resolved?

Inconsistent results in Promethin knockout studies can stem from multiple sources that require systematic troubleshooting approaches. First, verify knockout efficiency using multiple methods: genomic sequencing to confirm CRISPR/Cas9 editing events, RT-qPCR to assess transcript levels, and Western blotting to confirm protein depletion. Partial knockouts or compensatory expression of splice variants can significantly impact phenotypic outcomes. When using CRISPR guide RNAs for Tmem159 targeting, verify complete editing by sequencing and consider using multiple guides as recommended by the Broad Institute protocols .

Genetic compensation mechanisms frequently confound knockout studies, particularly for membrane proteins where related family members may assume functional roles. Examine expression changes in related TMEM family proteins or functional partners like Seipin (Bscl2) following Promethin knockout. Consider acute depletion methods (inducible CRISPR or degron systems) to bypass compensatory adaptations that develop in constitutive knockout models.

Phenotypic inconsistencies often reflect cell type-specific or context-dependent functions. Systematically assess your knockout phenotypes across multiple cell types and under various conditions—particularly those that stress membrane dynamics or lipid metabolism pathways where Promethin likely functions. Include positive controls of known membrane trafficking or lipid metabolism disruptors to calibrate phenotypic expectations.

For mechanistic studies, implement rescue experiments with both wild-type Promethin and domain-specific mutants to map structure-function relationships. These rescue studies should include careful titration of expression levels, as both under-expression and over-expression can yield misleading results when interpreting membrane protein functions. Finally, consider the temporal aspects of your phenotypic analyses, as acute responses to Promethin loss may differ significantly from adaptive responses in stable knockout systems.

What quality control metrics should be applied to recombinant mouse Promethin preparations?

Quality control for recombinant mouse Promethin preparations requires rigorous multi-parameter assessment due to the challenges inherent in membrane protein production. For identity verification, implement both mass spectrometry analysis to confirm primary sequence and Western blotting with domain-specific antibodies or epitope tag detection. Size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) provides critical information about monodispersity, oligomeric state, and potential aggregation that can compromise functional studies.

For structural integrity assessment, circular dichroism spectroscopy can verify secondary structure content expected based on prediction algorithms. More advanced structural techniques like hydrogen-deuterium exchange mass spectrometry can provide domain-specific structural information without requiring crystallization. Thermal shift assays using differential scanning fluorimetry can assess protein stability under various buffer conditions, guiding optimization of storage buffers to maintain function.

Functional validation is essential and should be tailored to Promethin's known activities. Based on its proposed role in membrane dynamics and interactions with proteins like Seipin , appropriate functional assays might include liposome binding assays, protein-protein interaction studies with verified partners like Bscl2, or reconstitution into membrane mimetics followed by functional testing. If developing a Promethin preparation for antibody production, verify epitope accessibility in native conformations through non-denaturing immunodetection methods.

For batch-to-batch consistency, establish quantitative acceptance criteria for protein yield, purity (typically >90% by SDS-PAGE), aggregation levels (<10% by SEC), endotoxin content (<1 EU/mg for cell-based applications), and at least one functional parameter relevant to your research applications. Implementing these comprehensive quality control metrics will ensure that experimental outcomes reflect true biological functions rather than artifacts of protein preparation quality.

How might single-cell approaches advance understanding of Promethin function in heterogeneous tissues?

Single-cell technologies offer powerful approaches to unravel Promethin's context-dependent functions across heterogeneous cell populations that may be masked in bulk tissue analyses. Single-cell RNA-sequencing (scRNA-seq) can map Tmem159 expression patterns across development, differentiation trajectories, and in response to metabolic or environmental challenges. This approach is particularly valuable for identifying cell populations where Promethin expression is dynamically regulated, potentially indicating functional importance in those specific contexts.

Beyond transcriptomics, emerging spatial proteomics approaches like multiplexed ion beam imaging (MIBI) or co-detection by indexing (CODEX) can reveal Promethin protein distribution patterns while preserving tissue architecture context. These techniques can be especially informative for understanding Promethin's relationship with lipid droplet formation in tissues with heterogeneous metabolic activities like liver or adipose tissue.

For functional studies, CRISPR screening at single-cell resolution using the validated guide RNAs for Tmem159 combined with high-content imaging or flow cytometry phenotyping can uncover cell-type-specific dependencies on Promethin function. This approach would be particularly valuable for tissues where Promethin may have specialized functions in subpopulations, such as in adipose tissue where preadipocytes and mature adipocytes may utilize Promethin differently in membrane dynamics or lipid metabolism.

The single-cell approaches should be integrated with spatial transcriptomics or in situ sequencing to maintain tissue context information, providing insights into how Promethin function may be influenced by cell-cell interactions or tissue microenvironments. These advanced approaches will likely reveal functional heterogeneity in Promethin activity that conventional bulk analyses have missed, advancing our understanding of this protein's role in tissue physiology and potential pathological states.

What are the recommended approaches for developing therapeutic strategies targeting Promethin pathways?

While therapeutic targeting of Promethin pathways remains exploratory, several methodological approaches can establish foundations for potential interventions. The first step is comprehensive validation of Promethin as a therapeutic target through genetic association studies with human disease conditions and detailed phenotypic characterization of Promethin perturbation in animal models. The close interaction between Promethin and Seipin (Bscl2) suggests potential relevance to lipodystrophy or metabolic disorders where Seipin mutations are causative.

For direct targeting approaches, structure-based drug design will require detailed structural information about Promethin. While awaiting crystallographic or cryo-EM structures, computational modeling based on related membrane proteins can guide initial small molecule screening efforts. Functional domain mapping using mutagenesis combined with the interaction studies described earlier can identify specific "hotspots" most suitable for therapeutic targeting.

Alternative approaches may target Promethin's regulatory mechanisms or key interaction partners rather than the protein itself. Transcriptional or post-transcriptional regulators of Promethin expression could be identified through genomic and proteomic screening approaches. Given Promethin's strong interaction with Seipin in lipid metabolism pathways , targeting the Promethin-Seipin interface with small molecules or peptide mimetics may provide more specific intervention points than targeting either protein individually.

For any therapeutic development, establishing clear biomarkers of Promethin pathway activity is essential. These might include quantifiable aspects of membrane dynamics, lipid droplet morphology, or metabolite profiles that change predictably with Promethin modulation. These biomarkers would serve both as patient stratification tools and as pharmacodynamic markers in intervention studies. The therapeutic development pipeline should include careful consideration of potential complications from manipulating fundamental membrane processes where Promethin functions.