Recombinant Danio rerio Protein transport protein Sec31A (sec31a), partial

What is the function of SEC31A in zebrafish (Danio rerio)?

SEC31A functions as a critical component of the coat protein complex II (COPII) which promotes the formation of transport vesicles from the endoplasmic reticulum (ER). In zebrafish, as in other vertebrates, SEC31A plays an essential role in ER-to-Golgi trafficking of newly synthesized proteins . The protein is particularly crucial during early development, as studies have shown that mutations in sec31a or related COPII components in zebrafish can lead to developmental defects . SEC31A works in concert with other COPII proteins including SEC23, SEC24, SEC13, and SAR1 to facilitate proper vesicle formation and cargo selection .

How does zebrafish SEC31A compare structurally to its human ortholog?

Zebrafish (Danio rerio) SEC31A shares significant structural homology with its human ortholog, reflecting the evolutionary conservation of the COPII trafficking machinery across vertebrates. Both proteins contain similar functional domains including regions that interact with SEC23 and other COPII components. The conservation between human and zebrafish SEC31A makes zebrafish an excellent model organism for studying SEC31A-related trafficking defects . Specifically, the SEC23-SEC31 interface that plays a critical role in cargo selection is well-preserved between species, allowing researchers to study mutations at this interface in zebrafish and extrapolate findings to human conditions such as cranio-lenticulo-sutural dysplasia (CLSD) .

What phenotypes are observed in zebrafish with SEC31A deficiency?

Zebrafish with SEC31A deficiency exhibit developmental abnormalities similar to those observed in other COPII component mutations. These include defects in craniofacial development, cartilage formation, and notochord development . At the cellular level, SEC31A deficiency causes extensive enlargement of the ER cisternae due to inefficient COPII vesicle formation and cargo export . These phenotypes are consistent with those observed in other vertebrate models with mutations in COPII components, including SEC23A mutations in humans that cause CLSD. The phenotypic similarities between zebrafish and human COPII-related disorders further validate zebrafish as a model for studying these pathways .

What expression systems are optimal for producing recombinant Danio rerio SEC31A protein?

For producing recombinant Danio rerio SEC31A protein, several expression systems have proven effective:

• E. coli expression system: This is the most commonly used system for producing recombinant Danio rerio SEC31A fragments. For optimal expression, BL21(DE3) strain with pET expression vectors can be used with IPTG induction at 25°C rather than 37°C to enhance protein solubility .

• Baculovirus expression system: For full-length SEC31A, which is a large protein (approximately 140 kDa), the baculovirus expression system may provide better folding and yield compared to bacterial systems.

• Mammalian cell expression: For functional studies requiring proper post-translational modifications, HEK293T cells can be transfected with SEC31A expression vectors .

The choice of expression system should be guided by the specific experimental requirements, particularly whether partial fragments or the full-length protein is needed, and whether post-translational modifications are essential for the study.

What are the most effective purification strategies for recombinant Danio rerio SEC31A?

Effective purification strategies for recombinant Danio rerio SEC31A include:

• Affinity chromatography: Adding a His-tag to the N- or C-terminus of SEC31A allows for efficient purification using nickel or cobalt affinity resins. This approach has been successfully used for purifying both full-length and partial SEC31A proteins .

• GST fusion purification: For functional studies, SEC31A can be expressed as a GST fusion protein and purified using Glutathione-Sepharose under native conditions, followed by overnight dialysis (50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM DTT, and 20% glycerol) .

• Size exclusion chromatography: As a secondary purification step, size exclusion chromatography can be used to isolate properly folded SEC31A protein and separate it from aggregates or degradation products.

• Ion-exchange chromatography: This can be used as an additional purification step to obtain highly pure protein based on charge differences.

To verify protein purity and identity, SDS-PAGE analysis followed by Western blotting using anti-His-tag or specific anti-SEC31A antibodies is recommended .

How can researchers assess the functional activity of purified recombinant Danio rerio SEC31A?

To assess the functional activity of purified recombinant Danio rerio SEC31A, researchers can employ several complementary approaches:

• In vitro vesicle budding assays: Reconstituting COPII vesicle formation using purified components (SEC23-SEC24, SEC13-SEC31A, and SAR1) with ER-enriched membrane fractions. Successful vesicle formation can be monitored by electron microscopy or by analyzing the incorporation of cargo proteins into vesicles .

• SAR1 GTPase activity assays: Since SEC31A stimulates the SEC23-mediated GAP activity toward SAR1, researchers can measure SAR1 GTPase activity in the presence of recombinant SEC31A using colorimetric assays that detect released phosphate from GTP hydrolysis .

• Binding assays with other COPII components: GST-pulldown assays can be used to verify that recombinant SEC31A properly interacts with its binding partners, particularly SEC23. The interaction between 35S-methionine-labeled proteins and GST-fusion proteins can be analyzed to confirm binding specificity .

• Cell-based rescue experiments: Functional activity can be confirmed by expressing recombinant SEC31A in SEC31A-depleted zebrafish cells and assessing rescue of phenotypes such as ER export defects or procollagen secretion .

How do mutations at the SEC23-SEC31A interface affect cargo selection and export from the ER?

Mutations at the SEC23-SEC31A interface have profound effects on cargo selection and export from the ER, as demonstrated by studies of disease-causing mutations. The M702V mutation in human SEC23A, located at the SEC31 binding site, causes selective retention of procollagen in the ER without compromising COPII assembly or vesicle size .

This selectivity occurs because:

• The SEC23-SEC31 interface influences the residence time of COPII on ER membranes by modulating SAR1 GTPase activity.

• M702V SEC23A activates SAR1B GTPase more strongly than wild-type SEC23A when SEC13-SEC31 is present, causing premature dissociation of COPII from membranes .

• Large cargo molecules like procollagen require longer residence times of COPII proteins on the membrane compared to smaller cargo molecules.

These findings suggest that the SEC23-SEC31 interface is finely tailored for selecting various cargo molecules, with different cargo types requiring different kinetics of coat assembly and disassembly. This mechanism explains why certain mutations can cause selective rather than general export defects, providing insight into the pathogenesis of diseases like cranio-lenticulo-sutural dysplasia .

What are the comparative advantages of using zebrafish versus mammalian models for studying SEC31A-related disorders?

Zebrafish offer several distinct advantages over mammalian models for studying SEC31A-related disorders:

Zebrafish mutants in COPII components, including SEC31A, display phenotypes that mirror human diseases, such as skeletal defects and cartilage abnormalities seen in CLSD patients . The ability to directly observe these phenotypes during development makes zebrafish particularly valuable for understanding the pathogenesis of SEC31A-related disorders and for screening potential therapeutic interventions .

How can transcriptomic analysis contribute to understanding SEC31A function in zebrafish?

Transcriptomic analysis provides valuable insights into SEC31A function in zebrafish through several approaches:

• Global gene expression changes: RNA-Seq analysis of SEC31A-deficient zebrafish embryos can reveal transcriptome-wide changes associated with disrupted COPII trafficking. This approach can identify compensatory mechanisms and downstream pathways affected by SEC31A deficiency .

• Temporal expression patterns: Analyzing SEC31A expression throughout zebrafish development can identify critical periods when SEC31A function is most essential, correlating with phenotypic manifestations of SEC31A deficiency .

• Tissue-specific expression: Single-cell RNA-Seq can map SEC31A expression patterns across different cell types, revealing tissue-specific roles and potentially explaining why certain tissues are more affected by SEC31A mutations .

• Stress response pathways: SEC31A deficiency triggers ER stress responses, and transcriptomic analysis can comprehensively characterize these responses, including unfolded protein response (UPR) activation and potential connections to cell death pathways .

• Comparative analysis with human data: Comparing transcriptomic changes in zebrafish SEC31A models with those in human patient-derived cells can validate the zebrafish model and identify conserved disease mechanisms .

For meaningful transcriptomic analysis, researchers should consider developmental timing, sample preparation protocols that preserve RNA integrity, appropriate statistical approaches for differential expression analysis, and validation of key findings using techniques such as quantitative RT-PCR .

How does the interaction between SEC31A and SEC23 regulate COPII vesicle formation and cargo selection?

The interaction between SEC31A and SEC23 is central to regulating COPII vesicle formation and cargo selection through several mechanisms:

• GAP activity regulation: SEC31A enhances the GTPase-activating protein (GAP) activity of SEC23 toward SAR1, a critical step in controlling the timing of coat assembly and disassembly. This temporal regulation is essential for proper cargo selection and vesicle formation .

• Membrane curvature: The SEC23-SEC31A interaction facilitates membrane bending during vesicle formation. SEC31A, together with SEC13, forms rod-like structures that assemble into a cage-like lattice around the budding vesicle, providing the structural scaffold for membrane deformation .

• Cargo-specific adaptation: Different cargo molecules require different SEC23-SEC31A interaction dynamics. For example, procollagen, a large cargo molecule, requires prolonged residence of COPII components on the ER membrane, which is regulated by the SEC23-SEC31A interface .

• Mutation effects: Disease-causing mutations at the SEC23-SEC31A interface, such as the M702V mutation in SEC23A, enhance SAR1 GTPase activity through SEC31A, causing premature coat disassembly and selective retention of certain cargo molecules like procollagen .

This sophisticated regulatory mechanism allows the COPII machinery to adapt to various cargo molecules of different sizes and properties, ensuring efficient and selective protein transport from the ER to the Golgi apparatus.

What methodologies are most effective for studying SEC31A protein-protein interactions in Danio rerio models?

For studying SEC31A protein-protein interactions in Danio rerio models, several complementary methodologies have proven effective:

• GST pulldown assays: Using GST-fusion proteins expressed in E. coli and purified under native conditions to capture interaction partners from zebrafish lysates. This approach has been successfully used to study interactions between COPII components including SEC31A .

• Co-immunoprecipitation (Co-IP): Expressing tagged versions of SEC31A in zebrafish embryos or cell lines, followed by immunoprecipitation and Western blotting to detect interacting partners. This approach works in the native cellular environment where proper post-translational modifications occur .

• Yeast two-hybrid screening: For identifying novel interaction partners of SEC31A, yeast two-hybrid screening with zebrafish cDNA libraries can be employed, although this approach should be complemented with validation in zebrafish cells .

• Proximity labeling: BioID or APEX2 proximity labeling approaches, where SEC31A is fused to a biotin ligase, can identify proteins in close proximity to SEC31A in living zebrafish cells.

• Fluorescence microscopy: Techniques such as Förster resonance energy transfer (FRET) or fluorescence lifetime imaging microscopy (FLIM) can visualize SEC31A interactions with other COPII components in living zebrafish cells or embryos .

• Cross-linking mass spectrometry: Chemical cross-linking followed by mass spectrometry analysis can capture transient interactions and provide structural information about SEC31A complexes.

Each method has specific advantages and limitations, and combining multiple approaches provides the most comprehensive understanding of SEC31A interactions in the zebrafish model.

How does SEC31A paralog (SEC31B) function differ from SEC31A in vesicular transport?

The functional differences between SEC31A and its paralog SEC31B in vesicular transport include:

Understanding these differences is important for interpreting experimental results and developing targeted approaches to study specific aspects of COPII-mediated transport in zebrafish models. When working with recombinant proteins or designing genetic manipulations, researchers should consider the potential functional overlap between these paralogs .

What are the optimal methods for genetic manipulation of SEC31A in zebrafish?

Several genetic manipulation methods can be employed to study SEC31A function in zebrafish, each with specific advantages for different research questions:

• Morpholino knockdown: Antisense morpholino oligonucleotides can be designed to block SEC31A translation or splicing. This approach provides rapid results but should be validated with rescue experiments and compared with genetic mutants to rule out off-target effects .

• CRISPR/Cas9 genome editing: For generating stable SEC31A mutant lines, CRISPR/Cas9 is the current gold standard. Multiple guide RNAs targeting different exons should be designed, particularly those encoding functional domains such as the SEC23 interaction site .

• Transgenic approaches: Overexpression of wild-type or mutant SEC31A under tissue-specific promoters can help understand gain-of-function effects or dominant-negative mutations. The Tol2 transposon system is effective for generating stable transgenic lines .

• Inducible expression systems: Systems like the Gal4/UAS or Tet-On can provide temporal control over SEC31A expression or knockdown, allowing researchers to study stage-specific requirements .

• Cell-specific knockout: Combining CRISPR/Cas9 with tissue-specific promoters allows for cell type-specific SEC31A knockout, which is useful for dissecting tissue-autonomous versus non-autonomous functions .

• Homology-directed repair: For introducing specific point mutations mimicking human disease variants (like the equivalent of M702V in SEC23A), homology-directed repair with CRISPR/Cas9 can be employed .

The choice of method depends on the specific research question, with considerations for timing (developmental stage of interest), specificity (whole organism vs. tissue-specific), duration (transient vs. stable), and the nature of the manipulation (loss-of-function vs. gain-of-function).

How can zebrafish be used to screen for compounds that rescue SEC31A-related trafficking defects?

Zebrafish offer an excellent platform for screening compounds that rescue SEC31A-related trafficking defects through several approaches:

• Phenotype-based screening: SEC31A mutant or morphant zebrafish often display visible phenotypes such as craniofacial abnormalities or notochord defects. Compounds can be screened for their ability to rescue these macroscopic phenotypes .

• Fluorescent cargo trafficking assays: Transgenic zebrafish expressing fluorescently tagged cargo proteins (such as GFP-tagged collagen) can be used to visualize trafficking defects in SEC31A-deficient embryos. High-content imaging can then identify compounds that restore normal trafficking patterns .

• ER stress reporter lines: Since SEC31A deficiency triggers ER stress, transgenic zebrafish with fluorescent reporters for ER stress (e.g., XBP1 splicing reporters) can be used to screen for compounds that reduce ER stress in SEC31A mutants .

• Automated analysis workflows: For high-throughput screening, automated imaging and analysis pipelines can quantify rescue effects across multiple parameters, including morphological features, fluorescent reporter intensity, and localization patterns .

• Validation in human cells: Hits from zebrafish screens should be validated in human cell models with SEC31A mutations to confirm translational relevance. This can include patient-derived fibroblasts from individuals with CLSD or other SEC31A-related disorders .

The zebrafish platform combines the advantages of whole-organism physiology with the scalability needed for compound screening, making it particularly valuable for identifying therapeutic candidates for SEC31A-related disorders.

What are the considerations for analyzing tissue-specific effects of SEC31A dysfunction in zebrafish?

Analyzing tissue-specific effects of SEC31A dysfunction in zebrafish requires careful consideration of several factors:

• Developmental timing: SEC31A expression and requirement vary throughout development. Researchers should establish a detailed timeline of SEC31A expression in different tissues using techniques such as in situ hybridization or a SEC31A-GFP reporter line .

• Tissue-specific markers: Combining SEC31A manipulation with transgenic lines marking specific tissues (e.g., fli1a:GFP for endothelial cells or sox10:GFP for neural crest) allows for precise assessment of tissue-specific phenotypes .

• Cargo-specific effects: Different tissues secrete different cargo proteins that may have varying dependencies on SEC31A. Researchers should assess multiple cargo proteins (e.g., collagen for cartilage, albumin for liver) to understand tissue-specific vulnerabilities .

• Compensatory mechanisms: Some tissues may activate paralogous genes (like SEC31B) or alternative trafficking pathways to compensate for SEC31A dysfunction. RNA-seq analysis of isolated tissues can reveal these compensatory mechanisms .

• Cell autonomous vs. non-autonomous effects: Using genetic mosaics or tissue-specific knockout approaches can distinguish between cell-autonomous defects and secondary effects caused by altered intercellular signaling .

• Quantitative phenotyping: Developing quantitative metrics for tissue-specific phenotypes ensures objective assessment and enables statistical analysis across experimental conditions. This may include morphometric measurements, fluorescence intensity quantification, or behavioral assays for neural tissue .

By systematically addressing these considerations, researchers can develop a comprehensive understanding of how SEC31A dysfunction affects different tissues, potentially explaining the tissue-specific manifestations observed in human SEC31A-related disorders.

What statistical approaches are most appropriate for analyzing phenotypic data from SEC31A-manipulated zebrafish?

When analyzing phenotypic data from SEC31A-manipulated zebrafish, several statistical approaches are appropriate depending on the type of data and experimental design:

• For categorical phenotypic data (e.g., presence/absence of specific defects):

  • Chi-square test or Fisher's exact test for comparing frequencies between experimental groups

  • Logistic regression for analyzing the influence of multiple factors on binary outcomes

  • Relative risk or odds ratio calculations to quantify the strength of associations

• For continuous measurements (e.g., body length, cartilage measurements):

  • Student's t-test (for comparing two groups) or ANOVA (for multiple groups) if data is normally distributed

  • Mann-Whitney U test or Kruskal-Wallis test for non-normally distributed data

  • Linear mixed models when there are repeated measurements or nested data structures

• For time-series data (e.g., developmental progression):

  • Repeated measures ANOVA or mixed-effects models

  • Survival analysis techniques like Kaplan-Meier curves and log-rank tests for time-to-event data

  • Growth curve analysis for comparing developmental trajectories

• For imaging-based quantitative data:

  • Image segmentation followed by parametric or non-parametric statistical tests

  • Machine learning approaches for complex pattern recognition and classification

• Multiple testing corrections:

  • Bonferroni correction (most stringent)

  • False Discovery Rate (FDR) methods like Benjamini-Hochberg (recommended for -omics datasets)

• Power analysis:

  • A priori power calculations to determine adequate sample sizes

  • Post-hoc power analysis to interpret negative results

The choice of statistical approach should be determined by the study design, data distribution, and specific hypotheses being tested. Consulting with a biostatistician during experimental design can ensure appropriate statistical approaches are planned from the outset .

How can researchers effectively control for off-target effects when studying SEC31A function in zebrafish?

Controlling for off-target effects when studying SEC31A function in zebrafish requires a multi-faceted approach:

• Use multiple independent targeting strategies:

  • Design and validate multiple morpholinos targeting different regions of SEC31A mRNA

  • Generate CRISPR/Cas9 mutants with different guide RNAs

  • Compare phenotypes across different knockdown/knockout methods

• Perform rescue experiments:

  • Co-inject morpholinos with morpholino-resistant SEC31A mRNA

  • Express wild-type SEC31A in mutant backgrounds

  • Use structure-function rescue (with different SEC31A domains) to validate specificity

• Include appropriate controls:

  • Use standard control morpholinos

  • Include Cas9-only or non-targeting guide RNA controls for CRISPR experiments

  • Generate and characterize multiple independent mutant lines

• Validate target knockdown/knockout:

  • Quantify SEC31A mRNA levels using qRT-PCR

  • Verify protein reduction by Western blot or immunofluorescence

  • Confirm altered splicing patterns for splice-blocking morpholinos

• Monitor p53 activation:

  • Co-inject with p53 morpholino to distinguish specific phenotypes from non-specific p53-mediated effects

  • Assess p53 pathway activation markers

• Examine closely related genes:

  • Monitor expression of SEC31B or other COPII components to check for compensatory changes

  • Validate that phenotypes are not due to altered expression of related genes

• Validate with human disease models:

  • Compare zebrafish phenotypes with those observed in human patients with SEC31A mutations

  • Test zebrafish models for their ability to recapitulate specific molecular defects seen in human cells

By implementing these controls systematically, researchers can distinguish genuine SEC31A-specific effects from off-target artifacts, enhancing the reliability and translational relevance of their findings .

What are the most informative experimental controls for assessing recombinant Danio rerio SEC31A protein functionality?

To rigorously assess the functionality of recombinant Danio rerio SEC31A protein, several experimental controls are essential:

• Protein quality controls:

  • Size-exclusion chromatography to confirm proper oligomerization state

  • Circular dichroism to verify secondary structure integrity

  • Thermal shift assays to assess protein stability

  • Limited proteolysis to evaluate proper folding

• Negative controls:

  • Heat-denatured SEC31A protein to confirm that activity requires native conformation

  • SEC31A with mutations in critical functional domains (e.g., SEC23 binding interface)

  • Buffer-only controls for all functional assays

• Positive controls:

  • Commercially available mammalian SEC31A for comparative analyses

  • Well-characterized SEC31A from other species with known activity levels

  • Co-purified SEC31A-SEC13 complex as a reference for native activity

• Interaction verification controls:

  • Control proteins that should not interact with SEC31A (e.g., GST alone)

  • Known SEC31A binding partners (SEC13, SEC23) to confirm interaction capacity

  • Competition assays with peptides derived from interaction domains

• Functional rescue controls:

  • Wild-type SEC31A for rescue of SEC31A-depleted systems

  • SEC31A activity titration to establish dose-response relationships

  • Comparison with human SEC31A in equivalent assays

• System-specific controls:

  • For in vitro vesicle budding assays: membranes lacking ER components

  • For GTPase activation assays: SEC23 alone to establish baseline activity

  • For cellular assays: mock-transfected or irrelevant protein controls

By incorporating these controls, researchers can conclusively demonstrate that observed effects are specifically attributable to functional recombinant Danio rerio SEC31A protein rather than experimental artifacts or contaminating factors .

How has the SEC31A protein evolved across vertebrate species, and what are the implications for using zebrafish as a model?

SEC31A shows remarkable evolutionary conservation across vertebrate species, with important implications for using zebrafish as a model:

• Domain conservation:

  • The SEC23-binding interface of SEC31A is highly conserved from fish to mammals, supporting functional studies in zebrafish as relevant to human disease

  • The proline-rich region, which is important for enhancing the GAP activity of SEC23, shows strong conservation in sequence and spacing

• Paralog divergence:

  • While most vertebrates possess both SEC31A and SEC31B, the functional specialization between these paralogs varies across species

  • In zebrafish, the relative functions of sec31a and sec31b may differ from mammals, requiring careful interpretation when modeling human diseases

• Functional conservation:

  • Cross-species complementation experiments show that human SEC31A can rescue zebrafish sec31a deficiency phenotypes, confirming functional conservation

  • The core mechanisms of COPII trafficking are preserved from yeast to humans, with increasing complexity in multicellular organisms

• Species-specific adaptations:

  • Some regulatory mechanisms of SEC31A activity, particularly post-translational modifications, may differ between zebrafish and mammals

  • Tissue-specific expression patterns of SEC31A show some variation across species, potentially influencing the presentation of phenotypes in different models

This evolutionary context supports zebrafish as a valuable model for studying fundamental SEC31A functions while highlighting the need for validation of specific regulatory mechanisms in mammalian systems. The conservation of critical functional domains suggests that insights gained from zebrafish studies are likely relevant to human SEC31A biology and disease .

What techniques are most effective for comparing cargo selection mechanisms between zebrafish and mammalian SEC31A?

For comparing cargo selection mechanisms between zebrafish and mammalian SEC31A, several complementary techniques prove most effective:

• In vitro reconstitution systems:

  • Parallel reconstitution of COPII vesicle formation using purified components from both zebrafish and mammals

  • Quantitative assessment of cargo incorporation efficiency for specific proteins

  • Cross-species mixing experiments (e.g., zebrafish SEC31A with mammalian SEC23/SEC24) to identify species-specific constraints

• Quantitative proteomics:

  • COPII vesicle isolation followed by mass spectrometry to identify and quantify cargo repertoires

  • Stable isotope labeling (SILAC) to directly compare vesicle content between species

  • Comparison of SEC31A interactomes across species to identify conserved and divergent binding partners

• Cargo trafficking assays:

  • Fluorescently tagged model cargo proteins expressed in both zebrafish and mammalian cells

  • Live imaging to compare trafficking kinetics and efficiency

  • RUSH (Retention Using Selective Hooks) system to synchronize cargo release and measure export rates

• Mutational analyses:

  • Introduction of equivalent mutations at the SEC23-SEC31A interface in both species

  • Assessment of cargo-specific effects (e.g., collagen vs. smaller cargo)

  • Structure-function mapping of domains required for specific cargo selection

• Cross-species rescue experiments:

  • Expression of zebrafish SEC31A in mammalian SEC31A-knockout cells and vice versa

  • Quantitative assessment of rescue efficiency for different cargo types

  • Domain-swapping experiments to identify regions responsible for species-specific functions

These approaches provide complementary insights into the conservation and divergence of SEC31A-mediated cargo selection mechanisms, establishing the extent to which zebrafish studies can inform our understanding of mammalian SEC31A function .

How do SEC31A mutations in zebrafish compare with human disease-causing mutations in terms of molecular mechanisms?

The comparison between SEC31A mutations in zebrafish and human disease-causing mutations reveals important insights about molecular mechanisms:

Understanding these similarities and differences is crucial for accurately modeling human SEC31A-related diseases in zebrafish. While the core molecular mechanisms are generally conserved, species-specific factors must be considered when extrapolating findings from zebrafish to human pathophysiology .

By synthesizing these insights, researchers can leverage the strengths of zebrafish models while accounting for their limitations, ultimately advancing our understanding of SEC31A function and related disorders.

What are the most promising future directions for research on recombinant Danio rerio SEC31A?

The most promising future directions for research on recombinant Danio rerio SEC31A include:

• Structure-function studies: Determining the high-resolution structure of zebrafish SEC31A, particularly in complex with other COPII components, would provide crucial insights into species-specific aspects of COPII trafficking and cargo selection mechanisms .

• Cargo specificity mechanisms: Investigating how different domains of zebrafish SEC31A contribute to cargo-specific export efficiency could reveal fundamental principles of size-selective transport that are applicable across species .

• Therapeutic development: Using recombinant zebrafish SEC31A in high-throughput screens to identify small molecules that can modulate COPII function, potentially leading to therapeutics for SEC31A-related human diseases .

• Comparative systems biology: Integrating proteomics, transcriptomics, and functional genomics to develop comprehensive models of how SEC31A functions within the broader secretory network across evolutionary distance .

• Post-translational regulation: Exploring how post-translational modifications regulate zebrafish SEC31A function could reveal novel regulatory mechanisms conserved in vertebrate evolution .