Recombinant Xenopus laevis Transmembrane protein 214-A (tmem214-a)

What is Xenopus laevis TMEM214-A and what is its function?

TMEM214-A is one of the duplicated alleles of transmembrane protein 214 found in the allotetraploid genome of Xenopus laevis. Based on research in human systems, TMEM214 is a critical mediator of endoplasmic reticulum (ER) stress-induced apoptosis. It localizes to the outer membrane of the ER and constitutively associates with procaspase 4, which is critical for ER stress-induced apoptotic pathways .

In Xenopus laevis, TMEM214-A likely serves similar functions, acting as an anchor for recruitment of the Xenopus equivalent of procaspase 4 to the ER and facilitating its subsequent activation during stress conditions. Overexpression studies in human cells have demonstrated that TMEM214 can induce morphological changes characteristic of apoptosis, including positive annexin V staining and DNA fragmentation .

The "-A" designation refers to one of the two homeologous copies present in Xenopus laevis due to its evolutionary history of genome duplication. This duplication resulted from the hypothesized hybridization of two ancestral species, creating an allotetraploid organism with duplicated genes that often show divergent functions or expression patterns .

Table 1: Comparative Features of Human and Xenopus laevis TMEM214

While human TMEM214 shows clear involvement in ER stress-induced apoptosis through its interaction with procaspase 4, Xenopus laevis TMEM214-A may have evolved subtle functional differences due to the unique developmental needs of amphibians. The presence of a duplicate copy (TMEM214-B) in the Xenopus genome may have allowed for subfunctionalization or neofunctionalization of these paralogs .

Unlike human TMEM214, which appears independent of CHOP and JNK pathways in mediating apoptosis, Xenopus TMEM214-A might have developed alternative regulatory mechanisms within the amphibian ER stress response network . These potential differences highlight the value of comparative studies between human and Xenopus systems.

Why use Xenopus laevis for studying TMEM214 function?

Xenopus laevis offers several unique advantages for studying TMEM214 function that make it an excellent complement to mammalian models:

First, Xenopus laevis provides large, abundant eggs and readily manipulated embryos with conserved cellular, developmental, and genomic organization shared with mammals . This allows researchers to examine TMEM214 function in the context of a vertebrate developmental system.

Second, the Xenopus system has a long history of contribution to understanding basic cellular processes. Research using this model has defined key principles of gene regulation, signal transduction, embryonic induction, morphogenesis, and cell cycle regulation . This rich background provides context for new studies on TMEM214.

Third, Xenopus laevis embryos yield about five-fold more material per embryo than their diploid relative Xenopus tropicalis, making them particularly advantageous for biochemical work requiring substantial protein amounts . This is especially valuable for membrane proteins like TMEM214 that may be expressed at relatively low levels.

Fourth, the Xenopus system allows versatile experimental approaches. Gain-of-function experiments can be readily performed by mRNA injection into embryos, while loss-of-function can be achieved through morpholino oligonucleotides or CRISPR/Cas9 genome editing .

Finally, the allotetraploid nature of Xenopus laevis, while sometimes challenging, offers unique opportunities to study gene duplication and functional divergence through comparison of the TMEM214-A and TMEM214-B paralogs .

Table 2: Predicted Structural Domains of Xenopus laevis TMEM214-A

While specific structural information for Xenopus TMEM214-A is not directly available in the current research literature, we can infer its domain organization based on human TMEM214 studies. The human protein's N-terminal region (amino acids 1-110) has been used successfully for antibody generation, suggesting this region is immunogenic and likely represents an accessible cytoplasmic domain .

The transmembrane topology of TMEM214-A would facilitate its localization to the ER membrane, positioning it to sense stress conditions within the ER lumen while maintaining cytoplasmic domains that can interact with apoptotic machinery like procaspases .

Understanding these structural elements is crucial for experimental design, particularly when creating truncation mutants or fusion proteins for functional studies. Research on human TMEM214 has successfully employed FLAG- or cherry-tagged constructs and various truncated mutants to investigate domain-specific functions , suggesting similar approaches would be valuable for Xenopus TMEM214-A characterization.

Table 3: Expression Systems for Recombinant TMEM214-A Production

The selection of an appropriate expression system for recombinant Xenopus laevis TMEM214-A depends on experimental goals. For structural or biochemical studies requiring larger protein quantities, mammalian or insect cell expression systems would likely be preferable to maintain proper folding and post-translational modifications of this multi-pass membrane protein.

Based on published research with human TMEM214, mammalian expression using FLAG- or fluorescent protein-tagged constructs has proven successful . These systems allow proper membrane insertion and folding while providing convenient tags for detection and purification.

For functional studies, the Xenopus oocyte system offers unique advantages as it represents the native cellular environment. The large size of Xenopus oocytes makes them excellent "expression chambers" for membrane proteins, as demonstrated with numerous channels and transporters . This approach enables electrophysiological or fluorescence-based functional assays in a near-native context.

Cell-free expression systems derived from Xenopus egg extracts represent another viable option, particularly for initial screening or small-scale studies . These systems retain many of the cellular components needed for proper membrane protein folding while offering flexibility in experimental design.

Table 4: Detergent Screening for TMEM214-A Purification

Purification of recombinant Xenopus laevis TMEM214-A presents challenges typical of multi-pass membrane proteins. A systematic approach to optimization involves several key considerations:

First, affinity tag selection is crucial. Research with human TMEM214 has successfully employed FLAG-tagged constructs , suggesting this approach may work well for the Xenopus protein. Alternative tags (His, GST, MBP) should be evaluated at both N- and C-termini to determine optimal placement that doesn't interfere with protein folding or function.

Second, membrane extraction requires careful optimization of detergent conditions. While specific detergents for TMEM214-A extraction haven't been reported, a screening approach testing multiple detergents (DDM, LMNG, digitonin) at various concentrations is advisable. The goal is to identify conditions that efficiently extract the protein while maintaining its structural integrity and functional properties.

Third, chromatography strategies typically benefit from a multi-step approach. Initial affinity purification using the chosen tag can be followed by size exclusion chromatography to remove aggregates and achieve higher purity. For challenging preparations, ion exchange chromatography may provide additional resolution.

Fourth, buffer optimization is essential for stability. Parameters to optimize include pH (typically 7.0-8.0), salt concentration (150-500 mM NaCl), and stabilizing additives (glycerol, cholesterol, specific lipids). For membrane proteins like TMEM214-A, including lipids that mimic the ER membrane composition may enhance stability.

Finally, quality control should employ multiple analytical methods including SDS-PAGE, Western blotting, size exclusion chromatography, and functional assays to confirm that the purified protein retains its native structure and activity.

What experimental approaches can be used to study TMEM214-A function in Xenopus embryos?

Xenopus embryos offer versatile platforms for investigating TMEM214-A function through multiple complementary approaches:

Gain-of-function studies can be performed through microinjection of synthesized TMEM214-A mRNA into embryos, a well-established technique in Xenopus research . This approach allows evaluation of overexpression phenotypes, which for TMEM214 might include increased sensitivity to ER stress or enhanced apoptotic responses in specific tissues . The large size of Xenopus embryos facilitates targeted injections into specific blastomeres to restrict expression to particular tissues.

Loss-of-function analyses can employ multiple strategies. Antisense morpholino oligonucleotides can be designed to block translation or splicing of TMEM214-A mRNA specifically . For genetic approaches, CRISPR/Cas9 genome editing has been successfully applied in Xenopus, enabling generation of targeted mutations in genes of interest . When designing such approaches for TMEM214-A, special consideration must be given to the allotetraploid nature of Xenopus laevis, which may require targeting both homeologous copies to observe phenotypes .

Subcellular localization studies can utilize fluorescent protein fusions, similar to the cherry-tagged TMEM214 constructs used in human cell studies . This approach can confirm the expected ER localization and potentially reveal dynamic changes in distribution during development or under stress conditions.

For biochemical analyses, co-immunoprecipitation experiments can identify interacting partners in Xenopus embryos or oocytes. Based on human studies, primary candidates would include procaspase homologs and components of the ER stress response machinery .

Functional assays should include examination of ER stress responses and apoptotic pathways. Treatment with established ER stress inducers like thapsigargin (TG) or brefeldin A (BFA) can reveal whether TMEM214-A knockdown protects against stress-induced apoptosis in Xenopus, as observed in human cells .

Table 5: Predicted Developmental Expression Pattern of Xenopus TMEM214-A

While specific expression data for TMEM214-A during Xenopus development is not directly available in the current literature, we can formulate hypotheses based on its function in ER stress-induced apoptosis .

As a protein involved in apoptotic pathways, TMEM214-A expression would likely be dynamically regulated during developmental stages requiring programmed cell death. These include metamorphosis, when extensive tissue remodeling occurs as the tadpole transforms into a froglet, and during earlier morphogenetic events such as neural tube formation.

The expression pattern might follow both temporal and spatial regulation. Temporally, expression levels could increase during stages with active cell death and remodeling. Spatially, expression might be enriched in tissues undergoing selective apoptosis, such as the regressing tail during metamorphosis.

To experimentally determine the developmental expression profile, several complementary approaches would be valuable:

• RT-PCR or quantitative PCR across developmental stages to establish temporal expression patterns

• Whole-mount in situ hybridization to visualize spatial expression domains

• Western blotting using stage-specific protein extracts to quantify protein levels

• Immunohistochemistry to detect tissue-specific localization at different stages

Considering the allotetraploid nature of Xenopus laevis, it would be critical to design primers or probes that can distinguish between TMEM214-A and its homeologous copy TMEM214-B, as these may show divergent expression patterns .

How do the duplicated alleles of TMEM214 in Xenopus laevis differ in function and expression?

Xenopus laevis possesses an allotetraploid genome resulting from hybridization of two ancestral species, yielding duplicated genes that have had approximately 50 million years to diverge functionally and regulatorily . This genome duplication likely resulted in two homeologous copies of TMEM214, designated as TMEM214-A and TMEM214-B.

Research on other duplicated genes in Xenopus laevis has revealed several evolutionary fates that may apply to TMEM214 homeologs:

First, sequence divergence between homeologs is typically substantial. The search results indicate that "the duplicated alloalleles differ substantially in sequence" , suggesting TMEM214-A and TMEM214-B may have accumulated significant differences. These sequence differences can serve as the basis for distinguishing between the homeologs experimentally.

Second, expression pattern divergence is common. The homeologs may show differential temporal or spatial expression during development, with one copy potentially being expressed earlier or in a more restricted tissue pattern than the other. This represents a form of subfunctionalization, where the ancestral expression pattern is partitioned between the duplicates.

Third, functional specialization may have occurred. While both copies likely retain the core function in ER stress-induced apoptosis, they may have evolved to respond to different stress stimuli or interact with different downstream effectors, analogous to the distinction between different human caspases.

Fourth, regulatory divergence often accompanies duplicated genes. The promoters and enhancers controlling TMEM214-A and TMEM214-B expression have likely accumulated differences, resulting in distinct responses to developmental or physiological signals.

Experimental approaches to characterize these differences would include homeolog-specific RT-PCR, morpholino knockdown targeting each copy separately and in combination, and biochemical characterization of protein-protein interactions unique to each homeolog.

What are the methodological challenges in studying membrane proteins like TMEM214-A in Xenopus systems?

Studying membrane proteins like TMEM214-A in Xenopus laevis presents several methodological challenges that require specialized approaches:

The first major challenge stems from the allotetraploid genome of Xenopus laevis, which contains duplicated genes including TMEM214 . This genomic complexity necessitates careful design of experimental tools that can either target both homeologs simultaneously or distinguish between them. For knockdown experiments, morpholino oligonucleotides must be designed with sufficient specificity to target TMEM214-A without affecting TMEM214-B if homeolog-specific functions are being investigated.

The second challenge involves protein solubilization and purification. As an integral membrane protein localized to the ER , TMEM214-A requires careful selection of detergents and buffer conditions to maintain its native conformation during extraction from membranes. The high lipid and yolk content of Xenopus embryos can further complicate purification protocols, requiring additional clarification steps.

Third, generating specific antibodies presents difficulties. Antibodies must distinguish between the highly similar homeologous proteins while also recognizing epitopes that may be partially obscured by membrane association. The search results mention that rabbit anti-TMEM214 antiserum was raised against recombinant human TMEM214 (amino acids 1-110) , suggesting that the N-terminal region might be suitable for antibody generation.

Fourth, functional redundancy between homeologs may mask phenotypes in single-gene perturbation experiments. As the search results note, gene duplicates in Xenopus laevis "would often preclude study of mutant phenotypes" . This necessitates simultaneous targeting of both copies or finding conditions where one copy is predominantly expressed.

Finally, visualizing subcellular localization in the large, yolk-rich cells of Xenopus embryos presents technical challenges. While fluorescent protein fusions have been successfully used with TMEM214 in other systems , the autofluorescence and opacity of Xenopus cells may require optimized imaging parameters.

Table 6: CRISPR/Cas9 Optimization Parameters for TMEM214-A Targeting

CRISPR/Cas9 genome editing in Xenopus laevis requires special considerations due to its allotetraploid genome and the presence of homeologous copies of genes like TMEM214 . While zinc-finger nucleases have been successfully used for targeted mutations in Xenopus tropicalis , CRISPR/Cas9 has emerged as the preferred genome editing tool due to its flexibility and efficiency.

The most critical aspect of CRISPR design for TMEM214-A is ensuring specificity. Guide RNAs should target sequences unique to TMEM214-A that differ from TMEM214-B to prevent unintended editing of both homeologs. This requires careful sequence alignment and selection of regions containing sufficient nucleotide differences, particularly in the seed region and PAM-adjacent sequences of the guide RNA.

Delivery method optimization is essential for successful editing. Microinjection of Cas9 protein pre-complexed with synthetic guide RNAs into one-cell stage embryos typically provides the highest efficiency with minimal toxicity. This ribonucleoprotein (RNP) approach offers immediate editing activity compared to mRNA-based methods, which require translation.

Validation strategies should employ multiple approaches. The T7 endonuclease assay provides a rapid screen for editing efficiency, while direct sequencing confirms the exact nature of induced mutations. For protein-level validation, Western blotting can confirm reduced expression, while functional assays related to ER stress response can demonstrate physiological consequences.

Breeding strategies must account for the mosaic nature of F0 embryos. Founder animals should be outcrossed, and F1 progeny screened for germline transmission of the desired mutations. The long fertility period of Xenopus (ten years or more) facilitates maintenance of valuable lines during this multi-generational process.

Table 7: Predicted Interacting Partners of TMEM214-A in Xenopus laevis

In human cells, TMEM214 has been identified as a critical mediator of ER stress-induced apoptosis through its constitutive association with procaspase 4 . This interaction is essential for recruiting procaspase 4 to the ER and facilitating its activation during stress conditions.

While specific interacting partners of TMEM214-A in Xenopus laevis have not been directly identified in the current literature, we can make informed predictions based on the conservation of ER stress response pathways across vertebrates.

The primary candidate interactor would be the Xenopus homolog of human procaspase 4. In amphibians, this would likely be procaspase-3, which serves analogous functions in inflammatory and ER stress responses. The interaction would presumably occur through cytoplasmic domains of TMEM214-A, similar to the human protein.

Additional predicted interactors would include components of the ER stress machinery. These might include chaperones like BiP/GRP78, which was examined in studies of TMEM214 overexpression , and possibly ER stress sensors like IRE1α, PERK, and ATF6. While direct interactions with these proteins might not occur, functional interactions within the ER stress response network are likely.

To experimentally identify TMEM214-A interacting partners in Xenopus, several approaches could be employed:

• Co-immunoprecipitation using tagged TMEM214-A followed by mass spectrometry

• Proximity labeling approaches (BioID, APEX) to identify proteins in close proximity to TMEM214-A in the ER membrane

• Yeast two-hybrid screening using cytoplasmic domains of TMEM214-A as bait

• In vitro binding assays with recombinant Xenopus proteins

These approaches would help establish whether the interaction network of TMEM214-A in Xenopus is conserved with humans or has evolved unique components.