Recombinant Danio rerio Endoplasmic reticulum-Golgi intermediate compartment protein 2 (ergic2)

How does zebrafish ERGIC2 compare to human ERGIC2 in terms of sequence homology?

Zebrafish ERGIC2 shows significant homology to human ERGIC2, suggesting evolutionary conservation of this protein across vertebrates. Human ERGIC2 is a 377-amino acid protein located on chromosome 12p11 . While the exact percentage identity is not explicitly stated in the provided materials, the functional domains are highly conserved, particularly the transmembrane regions and the luminal domain required for trafficking functions. The conservation of structure suggests similar functionality across species, making zebrafish a viable model organism for studying ERGIC2-related processes.

What are the optimal storage conditions for recombinant Danio rerio ERGIC2 protein?

For recombinant Danio rerio ERGIC2 protein, the following storage conditions are recommended:

• Store at -20°C or -80°C for extended storage

• For working aliquots, store at 4°C for up to one week

• Avoid repeated freeze-thaw cycles as this can compromise protein integrity

• Store in Tris-based buffer with 50% glycerol optimized for this protein

For reconstitution of lyophilized protein, it is recommended to briefly centrifuge the vial prior to opening and reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL, with 5-50% glycerol added as a cryoprotectant .

What is the expression pattern of ERGIC2 in different zebrafish tissues?

ERGIC2 is expressed in various tissues in zebrafish, with notable expression in the brain and neural tissues. Research indicates that ERGIC2 is present in zebrafish before and after the onset of hearing, similar to what has been observed in rodents . In zebrafish, ERGIC2 is found in the following tissues:

• Brain regions, particularly in the telencephalon, hypothalamus, optic tectum, and rhombencephalon

• Inner hair cells (IHCs) and outer hair cells (OHCs) of the cochlea (based on studies in rodents that likely translate to zebrafish)

The gene is listed as zgc:64005 in zebrafish genomic databases, indicating it was identified through the Zebrafish Gene Collection initiative .

How does stress affect ERGIC2 expression in the zebrafish brain?

Studies on stress responses in the zebrafish brain have shown that various stressors can affect the expression of genes involved in ER-Golgi trafficking, potentially including ERGIC2. Research has demonstrated that:

• Acute stressors such as feed rewarding, feed restriction, and air exposure can influence gene expression in different regions of the zebrafish brain

• Stress effects are distributed across all four brain parts (telencephalon, hypothalamus, optic tectum, and rhombencephalon)

• Expression patterns differ between male and female zebrafish, suggesting sex-specific responses to stress

Although the studies don't specifically mention ERGIC2, the stress-induced changes in genes related to the ER-Golgi pathway suggest that ERGIC2 expression might also be affected by stress conditions, particularly those that trigger the unfolded protein response (UPR).

What methods can be used to detect ERGIC2 localization in zebrafish tissues?

Several methods can be employed to detect ERGIC2 localization in zebrafish tissues:

• Immunohistochemistry (IHC): Using specific antibodies against ERGIC2 to visualize its localization in tissue sections. This method has been successfully used to study co-localization of ERGIC2 with other proteins in rodent tissues .

• In situ hybridization: Using ribonucleotide probes antisense to ERGIC2 mRNA to detect its expression pattern. Similar approaches have been used for detecting ER stress in zebrafish .

• RT-PCR: For detecting ERGIC2 mRNA expression in specific tissues, particularly useful for comparing expression levels across different tissues or experimental conditions .

• Subcellular fractionation: Combined with Western blotting, this approach can help determine the subcellular localization of ERGIC2, particularly its association with the ER-Golgi intermediate compartment .

• Fluorescent protein tagging: Generating transgenic zebrafish expressing ERGIC2 fused to fluorescent proteins for live imaging of its localization and dynamics.

What is the primary function of ERGIC2 in zebrafish?

The primary function of ERGIC2 in zebrafish, similar to other vertebrates, is believed to be involvement in protein trafficking between the endoplasmic reticulum (ER) and the Golgi apparatus. Specifically:

• ERGIC2 likely forms part of the protein transport machinery that shuttles cargo between the ER and the Golgi intermediate compartment (ERGIC) and cis-Golgi

• Based on its homology to the yeast Erv41 protein, ERGIC2 is suggested to be an ER resident protein involved in these trafficking processes

• The protein contains structural features that anchor it to the ER membrane, with its large luminal domain oriented inside the ER lumen and both the N- and C-termini facing the cytosol

There is evidence that ERGIC2 may have additional functions beyond protein trafficking, as suggested by studies showing its interaction with other proteins like Otoferlin and its potential role in the oxidative stress pathway .

What proteins are known to interact with ERGIC2 and what are the functional implications?

Several protein interactions have been identified for ERGIC2, suggesting diverse functional roles:

• ERGIC3 and ERGIC32: ERGIC2 forms a complex with these proteins, creating a shuttle for protein trafficking between ER and Golgi .

• Otoferlin: In the brain, ERGIC2 has been identified as an interacting partner of Otoferlin, a protein involved in calcium-dependent fusion of vesicles with plasma membrane in ribbon synapses. This interaction was detected through yeast two-hybrid screening and confirmed by co-immunoprecipitation in murine brain tissues .

• Beta-amyloid: ERGIC2 has been shown to interact with beta-amyloid, suggesting a potential role in neurodegenerative processes .

• Protein elongation factor 1alpha: This interaction may implicate ERGIC2 in cellular processes beyond simple protein trafficking .

These interactions suggest that ERGIC2 may play roles in neurotransmission, stress responses, and potentially neurodegenerative processes, extending beyond its canonical function in ER-Golgi trafficking.

How does ERGIC2 contribute to the unfolded protein response in zebrafish?

While direct evidence for ERGIC2's role in the unfolded protein response (UPR) in zebrafish is limited in the provided materials, its function as an ER-Golgi trafficking protein suggests potential involvement in the UPR:

• The UPR is activated in response to the accumulation of unfolded or misfolded proteins in the ER, and ERGIC2's role in protein trafficking may influence this process.

• A variant of ERGIC2 has been found to upregulate the heme oxygenase 1 gene, suggesting involvement in the oxidative stress pathway , which often intersects with the UPR.

• The truncated variant of ERGIC2, which lacks 45% of the luminal domain and the transmembrane domain near the C-terminus, loses its function as a protein transport shuttle but retains its ability to upregulate heme oxygenase 1 . This suggests that ERGIC2 may have dual functions in both protein trafficking and stress response pathways.

• Research on zebrafish has established methods to detect ER stress using markers like BIP (a cellular chaperone that increases in expression after ER stress) . These methods could be applied to study how ERGIC2 manipulation affects the UPR in zebrafish.

Experimental Methodologies

For recombinant Danio rerio ERGIC2 with an affinity tag (such as His-tag), the following purification strategy is recommended:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA or similar resins to capture the His-tagged ERGIC2 protein.

• Intermediate purification: Ion exchange chromatography based on the theoretical pI of the protein.

• Polishing step: Size exclusion chromatography to remove aggregates and achieve high purity.

The purification process should result in >90% purity as determined by SDS-PAGE . After purification, the protein is typically lyophilized and can be reconstituted in an appropriate buffer for downstream applications.

For proteins expressed in E. coli systems, it may be necessary to include steps to remove endotoxins, particularly if the protein will be used in cellular assays.

How can researchers effectively design experiments to study ERGIC2 function in zebrafish?

To effectively study ERGIC2 function in zebrafish, researchers should consider a multi-faceted experimental approach:

• Gene expression manipulation:

  • Morpholino knockdown for transient reduction of ERGIC2 expression

  • CRISPR/Cas9 genome editing for generating stable mutant lines

  • Transgenic overexpression models with tissue-specific promoters

• Phenotypic analysis:

  • Morphological assessment, particularly of tissues known to express ERGIC2

  • Behavioral testing if neurological functions are being investigated

  • Stress response assessment using established protocols for feed restriction, air exposure, etc.

• Molecular analysis:

  • RT-PCR and qPCR for measuring ERGIC2 expression levels

  • In situ hybridization to map spatial expression patterns

  • RNA-seq for genome-wide transcriptional changes in ERGIC2-manipulated models

• Protein interaction studies:

  • Co-immunoprecipitation to identify interacting partners

  • Yeast two-hybrid screening for novel interactions

  • Proximity labeling approaches (BioID, APEX) for identifying proximal proteins in vivo

• Subcellular localization:

  • Immunohistochemistry with ERGIC2-specific antibodies

  • Fluorescent protein tagging for live imaging

  • Subcellular fractionation followed by Western blotting

• Functional assays:

  • ER stress induction and monitoring using established markers like BIP

  • Protein trafficking assays using fluorescent cargo proteins

  • Analysis of the unfolded protein response pathway activation

The experimental design should include appropriate controls and statistical analysis methods, such as principal component analysis for gene expression data .

How does the variant ERGIC2 transcript with a four-base deletion affect protein function?

A variant ERGIC2 transcript with a four-base deletion at the junction of exons 8-9 has been identified, resulting in a frameshift after codon #189. This mutation produces a truncated protein of 215 residues (24.5 kDa) compared to the 377-residue (42.6 kDa) wild-type protein . The functional consequences of this truncation include:

• Structural changes: The truncated variant loses 45% of the luminal domain and the transmembrane domain near the C-terminus.

• Trafficking function abrogation: This truncation effectively eliminates its function as an ERGIC-Golgi protein transport shuttle.

• Retained stress response function: Despite losing its trafficking function, the variant ERGIC2 protein still upregulates the heme oxygenase 1 gene, suggesting it maintains a role in the oxidative stress pathway .

• Evolutionary conservation: A similar variant has been reported in armadillo, suggesting this is not a random mutation but potentially a conserved alternative form with specific functions .

This variant demonstrates the dual functionality of ERGIC2 and provides insights into structure-function relationships, suggesting distinct domains responsible for different cellular functions.

What role might ERGIC2 play in zebrafish models of neurodegenerative diseases?

While direct evidence from the provided materials is limited, several factors suggest ERGIC2 may be relevant in zebrafish models of neurodegenerative diseases:

• Interaction with beta-amyloid: ERGIC2 has been shown to interact with beta-amyloid , a key protein involved in Alzheimer's disease, suggesting potential involvement in amyloid processing or trafficking.

• ER-Golgi trafficking and neurodegeneration: Disruptions in ER-Golgi trafficking are implicated in various neurodegenerative conditions. As a component of this trafficking machinery, ERGIC2 dysfunction could contribute to disease processes.

• Otoferlin interaction: ERGIC2's interaction with Otoferlin , which plays a role in synaptic vesicle fusion, suggests it may influence neuronal function and potentially be involved in synaptopathies.

• UPR and neurodegeneration: The unfolded protein response (UPR) is implicated in many neurodegenerative diseases. If ERGIC2 influences the UPR as suggested by its role in stress responses, it may affect disease progression.

Zebrafish models could be particularly valuable for studying these connections due to their transparent embryos (facilitating imaging), rapid development, and the ability to perform high-throughput drug screening.

How might ERGIC2 function be affected by ER stress conditions, and what are the implications for zebrafish development?

ER stress conditions likely impact ERGIC2 function in ways that could affect zebrafish development:

• Altered trafficking efficiency: Under ER stress, the protein trafficking function of ERGIC2 might be compromised, potentially leading to accumulation of cargo proteins in the ER.

• UPR activation: If ERGIC2 dysfunction contributes to the accumulation of unfolded proteins, it may trigger or enhance the UPR. In zebrafish, the UPR has been studied using markers such as BIP, a cellular chaperone that increases in expression after ER stress .

• Developmental consequences: Proper protein trafficking is crucial during development, and disruptions could affect:

  • Notochord formation, which requires substantial protein secretion

  • Brain development, where ERGIC2 is known to be expressed and interacts with proteins like Otoferlin

  • Craniofacial development, which is sensitive to ER stress as observed in other studies of zebrafish

• Sex-specific responses: Research on stress responses in zebrafish brain has shown sex-specific differences in gene expression patterns , suggesting that ERGIC2's response to ER stress might differ between male and female fish, potentially leading to sex-specific developmental outcomes.

Methodologically, researchers can use transgenic zebrafish lines designed to measure ATF6 activation (a major transcriptional response to ER stress) to monitor these processes in vivo .

How can recombinant Danio rerio ERGIC2 be used to develop new research tools?

Recombinant Danio rerio ERGIC2 protein has several potential applications for developing new research tools:

• Antibody production: High-quality recombinant protein can be used to generate specific antibodies for detecting endogenous ERGIC2 in zebrafish tissues. This would facilitate studies of expression patterns, subcellular localization, and protein interactions.

• Protein interaction screening: Immobilized recombinant ERGIC2 can be used for affinity purification approaches to identify novel binding partners in zebrafish tissue lysates, expanding our understanding of its functional networks.

• Structural studies: Purified recombinant protein could be used for X-ray crystallography or cryo-EM studies to determine the three-dimensional structure of ERGIC2, providing insights into its function and interaction surfaces.

• Functional assays: The recombinant protein could be used to develop in vitro assays for testing ERGIC2's role in membrane trafficking, potentially reconstituting trafficking processes in cell-free systems similar to those used to study the ERGIC compartment .

• Biosensor development: Knowledge of ERGIC2's interactions could be leveraged to design FRET-based biosensors for monitoring ER-Golgi trafficking dynamics in live zebrafish.

What are the key methodological challenges in studying ERGIC2 function in zebrafish, and how might they be overcome?

Several methodological challenges exist in studying ERGIC2 function in zebrafish:

Experimental design approaches using factorial designs can help optimize conditions for both protein expression and functional studies, systematically evaluating multiple variables with minimal experiments .

What future research directions might yield the most significant insights into ERGIC2 biology in zebrafish?

Future research on ERGIC2 in zebrafish could focus on several promising directions:

• Comparative studies across development: Investigating how ERGIC2 expression and function change throughout zebrafish development could reveal stage-specific roles and regulation.

• Stress response integration: Further exploring the connection between ERGIC2 and stress responses, particularly how it might link ER-Golgi trafficking to the UPR and oxidative stress pathways in different tissues.

• Tissue-specific knockouts: Generating tissue-specific ERGIC2 knockout zebrafish using Cre-lox systems would allow detailed analysis of its function in specific cell types while avoiding developmental lethality if constitutive knockouts prove problematic.

• Interactome mapping: Comprehensive identification of ERGIC2 interaction partners in different zebrafish tissues using proximity labeling approaches could reveal tissue-specific functions and pathways.

• High-resolution imaging: Applying super-resolution microscopy techniques to study the precise subcellular localization and dynamics of ERGIC2 in zebrafish cells could provide insights into its trafficking functions.

• Integration with disease models: Introducing ERGIC2 mutations into existing zebrafish models of neurodegenerative diseases could help clarify its potential role in pathological processes and identify possible therapeutic strategies.

• Environmental influence studies: Investigating how environmental stressors affect ERGIC2 function could provide insights into cellular adaptation mechanisms and disease susceptibility.