Recombinant Bovine Immediate early response 3-interacting protein 1 (IER3IP1)

What is IER3IP1 and what are its primary functions?

IER3IP1 (Immediate early response 3-interacting protein 1) is an endoplasmic reticulum (ER) resident protein highly expressed in pancreatic cells and the developing brain cortex. This protein plays a crucial role in ER-to-Golgi trafficking pathways, particularly in the transport of proteins like proinsulin in β-cells. Recent studies have demonstrated that IER3IP1 interacts with Rab11, a GTPase involved in vesicle trafficking and fusion during cytokinesis. The protein appears to be essential for normal cellular division processes, specifically in the completion of cytokinesis. Furthermore, IER3IP1 may function in vesicle docking/tethering or fusion at various stages of the secretory pathway, suggesting its importance in cellular membrane trafficking systems .

How conserved is IER3IP1 across mammalian species?

IER3IP1 shows remarkable conservation across mammalian species, with functional equivalence demonstrated between human IER3IP1 and its Drosophila orthologue, Inseparable (Insep). Research has confirmed that human IER3IP1 can rescue phenotypes associated with loss of Insep in Drosophila, indicating strong functional conservation across widely divergent species. This high degree of evolutionary conservation suggests that findings regarding IER3IP1 function in one species can likely be translated to other mammals, including bovine systems. The protein's crucial roles in fundamental cellular processes such as vesicle trafficking and cytokinesis appear to be maintained across species, making bovine IER3IP1 an important model for studying these conserved functions .

What cellular compartments is bovine IER3IP1 primarily localized to?

Based on studies of human and Drosophila IER3IP1, bovine IER3IP1 is expected to primarily localize to the endoplasmic reticulum (ER) through its C-terminal transmembrane domain. Additionally, the protein has been shown to co-localize with Rab11-positive vesicles. Immunostaining analyses have demonstrated that wild-type IER3IP1 shows robust localization to the ER and significant co-localization with Rab11, a marker for recycling endosomes and vesicles involved in cytokinesis. This dual localization pattern suggests bovine IER3IP1 likely functions at the interface between the ER and vesicular trafficking pathways. Mutations affecting the transmembrane domain (such as L78P) significantly reduce ER localization, while other mutations (such as V21G) may specifically disrupt Rab11 vesicle localization without affecting ER targeting .

How do disease-associated mutations affect IER3IP1 function and localization?

Disease-associated mutations in IER3IP1, specifically V21G and L78P variants, significantly impact protein function and subcellular localization through distinct mechanisms. The V21G mutation primarily disrupts IER3IP1's ability to localize to Rab11 vesicles while maintaining its ER localization, resulting in partial co-localization with Rab11 and reduced interaction with Rab11 in co-immunoprecipitation assays. In contrast, the L78P mutation affects the C-terminal transmembrane domain critical for ER localization, severely reducing both ER targeting and Rab11 vesicle localization. Both mutations appear to decrease protein stability, with reduced expression levels compared to wild-type IER3IP1. Additionally, truncation mutations like T79* completely eliminate ER localization due to loss of the transmembrane domain. These disruptions in protein localization and Rab11 interaction correlate with the inability of mutant forms to rescue phenotypes in functional assays, indicating that proper subcellular targeting is essential for IER3IP1 function .

What is the relationship between IER3IP1 and Rab11 in vesicular trafficking?

The relationship between IER3IP1 and Rab11 represents a critical molecular interaction in vesicular trafficking systems, particularly during cytokinesis. Co-immunoprecipitation experiments have confirmed direct physical interaction between these proteins in both Drosophila and human cells. IER3IP1 co-localizes with Rab11 on vesicles, and this interaction appears essential for proper vesicle fusion during cytokinesis. Current evidence suggests that IER3IP1 works alongside Rab11 to regulate vesicle docking/tethering or fusion at the advancing furrow during cytokinesis. Disease-causing mutations in IER3IP1 (V21G and L78P) significantly reduce binding to Rab11, correlating with functional deficiencies. This relationship indicates that bovine IER3IP1 likely plays a similar role in Rab11-mediated vesicular trafficking processes, making it an important factor in cellular division and membrane trafficking in bovine cells .

How does IER3IP1 deficiency affect pancreatic β-cell function and insulin processing?

IER3IP1 deficiency profoundly impacts pancreatic β-cell function through disruption of ER-to-Golgi trafficking of proinsulin. Studies using IER3IP1 knockout stem cell-derived islets demonstrate a significant reduction (threefold) in proinsulin trafficking from the ER to the Golgi apparatus. This trafficking defect results in abnormal proinsulin accumulation within the ER, as evidenced by increased co-localization with ER markers like protein disulfide isomerase (PDI) and reduced localization to the Golgi apparatus. The disruption leads to elevated ER stress markers, including BiP (HSPA5), spliced XBP1, and ATF6, indicating activation of the unfolded protein response. Functionally, these cellular defects manifest as decreased β-cell numbers and impaired insulin secretion both in vitro and following implantation into immunocompromised mice. These findings suggest that bovine IER3IP1 may play a similarly crucial role in maintaining proper insulin processing and secretion in bovine pancreatic cells .

What are the optimal expression systems for producing recombinant bovine IER3IP1?

For producing recombinant bovine IER3IP1, mammalian expression systems are generally optimal due to the protein's requirements for proper folding and post-translational modifications. Based on successful expression of human IER3IP1, recommended systems include HEK293 cells for high-yield production and HCT116 cells for functional studies. When establishing your expression system, consider using epitope tags such as HA or fluorescent protein fusions at the N-terminus, as C-terminal tagging may interfere with the transmembrane domain essential for proper localization. For expression constructs, employ strong promoters like CMV for transient expression or more controlled inducible systems for stable cell lines. Purification typically requires detergent solubilization due to the membrane-associated nature of IER3IP1. Always validate your recombinant protein through Western blotting and immunofluorescence to confirm proper size and subcellular localization to both ER and Rab11-positive vesicles .

What approaches can be used to study IER3IP1's role in vesicular trafficking?

To investigate bovine IER3IP1's role in vesicular trafficking, multiple complementary approaches should be employed. Co-localization studies using confocal microscopy with markers for specific cellular compartments (such as PDI for ER and GM130 for Golgi) provide spatial information about IER3IP1 distribution. Protein-protein interaction analyses through co-immunoprecipitation assays with potential partners like Rab11 can reveal direct physical interactions. For functional trafficking assays, the retention using selective hooks (RUSH) system effectively measures ER-to-Golgi transport rates of cargo proteins like proinsulin. Live-cell imaging using fluorescently tagged IER3IP1 and vesicle markers enables real-time visualization of trafficking events. For studying cytokinesis, time-lapse microscopy of dividing cells expressing fluorescent markers for the cleavage furrow and IER3IP1 can reveal temporal dynamics. Finally, proximity labeling methods such as BioID can identify the broader interactome of IER3IP1 within trafficking pathways .

How can CRISPR/Cas9 genome editing be used to study bovine IER3IP1 function?

CRISPR/Cas9 genome editing provides powerful approaches for studying bovine IER3IP1 function through precise genetic manipulation. Based on successful strategies with human cells, researchers can generate several types of informative models: complete knockout cell lines to study loss-of-function effects, knock-in models of specific disease-associated mutations (e.g., V21G or L78P equivalents) to examine their molecular consequences, and knock-in of fluorescent tags for live imaging of endogenous protein. When designing gRNAs, target highly conserved regions while avoiding the transmembrane domain to prevent partial function. For bovine cells, delivery optimization is critical—electroporation typically yields higher efficiency than lipid-based transfection. Following editing, comprehensive validation through sequencing, Western blotting, and immunofluorescence is essential to confirm the desired modifications. These genome-edited bovine cell lines can then be analyzed for defects in cellular processes including protein trafficking, ER stress responses, and cytokinesis completion .

How should researchers interpret changes in ER stress markers when studying IER3IP1 mutations?

When interpreting changes in ER stress markers in the context of IER3IP1 mutations, researchers should analyze the pattern of UPR (unfolded protein response) activation across multiple pathways. The IER3IP1 knockout model shows specific upregulation of the IRE1α and ATF6 arms of the UPR, with increased expression of BiP (HSPA5), spliced XBP1, and ATF6, but without significant activation of the PERK pathway. This selective UPR activation pattern provides important mechanistic insights into how IER3IP1 deficiency affects ER homeostasis. Quantitative assessment should include both transcript-level changes through RT-qPCR and protein-level alterations via immunostaining or Western blotting. Pay particular attention to cell-type specific effects—in pancreatic islets, for example, ER stress markers show stronger upregulation in β-cells compared to α-cells. The temporal dynamics of these changes should also be monitored, as acute versus chronic ER stress can trigger different cellular responses ranging from adaptive to apoptotic. Finally, correlate these molecular changes with functional outcomes such as insulin processing efficiency or cytokinesis completion rates to establish causative relationships .

What parameters should be analyzed when assessing the impact of IER3IP1 on protein trafficking?

When assessing IER3IP1's impact on protein trafficking, researchers should analyze multiple parameters to comprehensively characterize trafficking defects. First, measure cargo protein localization through quantitative co-localization analysis with compartment markers (e.g., PDI for ER, GM130 for Golgi), calculating the percentage of cargo volume co-localizing with each compartment. Second, assess trafficking kinetics using pulse-chase approaches or the RUSH system, which revealed a threefold reduction in proinsulin ER-to-Golgi transport in IER3IP1-deficient cells. Third, evaluate protein processing efficiency by measuring ratios of precursor to mature forms (e.g., proinsulin to insulin ratio was significantly elevated in IER3IP1 knockout cells). Fourth, analyze secretion rates through quantification of secreted proteins in culture media under both basal and stimulated conditions. Fifth, examine the ultrastructure of trafficking compartments via electron microscopy to identify morphological abnormalities. Finally, assess functional consequences of trafficking defects, such as reduced insulin secretion in response to glucose stimulation in IER3IP1-deficient β-cells. This multi-parameter approach provides a comprehensive understanding of how IER3IP1 contributes to protein trafficking pathways .

How can researchers differentiate between primary and secondary effects of IER3IP1 deficiency?

Differentiating between primary and secondary effects of IER3IP1 deficiency requires carefully designed experimental strategies. First, implement time-course analyses to establish the temporal sequence of observed phenotypes—events occurring earliest after IER3IP1 depletion likely represent primary effects. For example, vesicular trafficking defects appear before significant ER stress in IER3IP1-deficient cells, suggesting trafficking disruption is a primary consequence. Second, develop rescue experiments with wild-type and mutant constructs; phenotypes rescued by wild-type but not by trafficking-deficient mutants likely represent direct consequences of IER3IP1 function. Third, use domain-specific mutations that selectively disrupt specific protein interactions or localizations—the V21G mutation primarily affects Rab11 interaction while maintaining ER localization, allowing researchers to separate these functions. Fourth, employ selective inhibitors of downstream pathways to determine whether blocking secondary responses (like ER stress) prevents later phenotypes without affecting early trafficking defects. Finally, perform comparative analyses across multiple cell types with different dependencies on secretory pathways; primary effects should be consistent across all IER3IP1-expressing cells, while secondary consequences may vary based on cell-specific vulnerabilities to primary defects .

How can researchers address instability issues with recombinant IER3IP1 proteins?

Addressing instability issues with recombinant IER3IP1 proteins requires multiple strategic approaches targeting protein folding and degradation pathways. Disease-associated mutations (V21G, L78P) significantly reduce protein stability, likely through misfolding and subsequent degradation. To improve stability, optimize expression conditions by lowering induction temperatures (28-30°C) to slow folding and reduce inclusion body formation. Include chemical chaperones such as 4-phenylbutyrate (5-10 mM) or TMAO (50-200 mM) in culture media to promote proper folding of the transmembrane domain. When purifying recombinant IER3IP1, use mild detergents like DDM (0.1-0.5%) or LMNG (0.01-0.05%) rather than harsh detergents like SDS that may disrupt native conformation. To reduce degradation, consider co-expressing molecular chaperones like BiP/GRP78 to stabilize nascent proteins, or include proteasome inhibitors (MG132, 1-10 μM) during expression. For long-term storage, supplement buffers with glycerol (10-20%) and avoid repeated freeze-thaw cycles. Finally, consider fusion partners like MBP or SUMO that can enhance solubility and stability, particularly for truncated constructs lacking the C-terminal transmembrane domain .

What controls are essential when analyzing IER3IP1's role in vesicular trafficking and cytokinesis?

When analyzing IER3IP1's role in vesicular trafficking and cytokinesis, implementing proper controls is critical for experimental rigor. For localization studies, include both positive controls (known ER proteins like calnexin) and negative controls (cytosolic or mitochondrial markers) alongside compartment-specific markers (PDI for ER, GM130 for Golgi, Rab11 for recycling endosomes). In trafficking assays, compare cargo movement kinetics between wild-type and IER3IP1-deficient cells, while also examining an unrelated control protein expected to traffic independently of IER3IP1. For interaction studies, perform reverse co-immunoprecipitations and include non-interacting protein controls to validate specificity. During cytokinesis analyses, quantify multiple parameters (furrow ingression rate, abscission timing, multinucleation frequency) and compare with defects caused by known cytokinesis regulators like ESCRT proteins. Rescue experiments should include not only wild-type IER3IP1 but also functionally deficient mutants (V21G, L78P) and unrelated control proteins. Finally, for genome-edited cell lines, generate multiple independent clones to control for off-target effects, and include isogenic wild-type controls subjected to the same CRISPR process without targeting IER3IP1 .

How can contradictory results between different IER3IP1 mutant models be reconciled?

Reconciling contradictory results between different IER3IP1 mutant models requires systematic analysis of experimental variables and mutation-specific effects. The available research shows distinct phenotypic differences between complete knockout models (IER3IP1−/−) and point mutation models (IER3IP1 V21G), particularly regarding ER stress marker expression and proinsulin accumulation patterns. To resolve such discrepancies, first characterize protein expression levels across models—some mutations may retain partial function or exhibit dominant-negative effects rather than simple loss-of-function. Second, conduct detailed structure-function analyses mapping specific domains to discrete cellular processes; for example, the V21G mutation primarily affects Rab11 interaction while preserving ER localization, potentially explaining its milder phenotype compared to complete knockout. Third, consider cell type-specific contexts, as IER3IP1 dependency may vary across tissues. Fourth, examine temporal aspects, as acute disruption via CRISPR editing may differ from compensated stable mutant lines. Finally, standardize experimental conditions across studies, including differentiation protocols for stem cell models, protein detection methods, and quantitative analysis parameters. This systematic approach can reveal how different mutations specifically disrupt distinct aspects of IER3IP1 function, explaining seemingly contradictory experimental outcomes .

What cellular pathways beyond ER-to-Golgi trafficking might bovine IER3IP1 influence?

Beyond its established role in ER-to-Golgi trafficking, bovine IER3IP1 likely influences several additional cellular pathways warranting investigation. The interaction with Rab11 suggests potential involvement in recycling endosome pathways, which regulate surface receptor expression and cell polarity. IER3IP1's function during cytokinesis indicates possible roles in coordinating membrane trafficking with cell cycle progression, potentially through interactions with cytokinetic regulators beyond Rab11. The protein may also participate in specialized secretory pathways in professional secretory cells like pancreatic β-cells, potentially regulating insulin granule maturation or exocytosis downstream of the Golgi apparatus. Additionally, IER3IP1's apparent importance in brain development suggests functions in neuronal protein trafficking, particularly in processes like axon guidance or synapse formation. Recent findings also point to potential involvement in cellular stress responses, as IER3IP1 deficiency triggers UPR activation specific to the IRE1α and ATF6 pathways. Each of these potential functions represents promising directions for future bovine IER3IP1 research, extending beyond its characterized role in early secretory pathway trafficking .

How might comparative studies between species enhance our understanding of IER3IP1 function?

Comparative studies between species offer powerful approaches to enhance our understanding of IER3IP1 function by leveraging evolutionary conservation and divergence. The demonstrated functional equivalence between human IER3IP1 and Drosophila Insep suggests highly conserved core functions across distant species. Future research comparing bovine, human, murine, and Drosophila IER3IP1 could identify absolutely conserved domains critical for fundamental functions versus species-specific regions that may reflect adaptation to specialized tissue requirements. Cross-species rescue experiments, similar to those showing human IER3IP1 can rescue Drosophila Insep mutants, could determine whether bovine IER3IP1's functions are fully conserved with human orthologues. Comparative protein interaction studies across species would reveal conserved versus species-specific binding partners, potentially identifying novel functional pathways. Analysis of expression patterns across species might reveal lineage-specific adaptations in tissues like the pancreas, which shows significant metabolic differences between ruminants and non-ruminants. Finally, mapping disease-associated mutations to evolutionary conserved regions could help prioritize functionally critical residues for mechanistic studies, enhancing translational relevance between model systems and human disease .

What therapeutic implications might arise from understanding IER3IP1's role in cellular trafficking?

Understanding IER3IP1's role in cellular trafficking pathways could yield several therapeutic implications with translational potential. For monogenic disorders caused by IER3IP1 mutations, gene therapy approaches delivering functional copies of IER3IP1 could potentially rescue trafficking defects in affected tissues, particularly in developmental contexts like neonatal diabetes. Small molecule screening could identify compounds that stabilize mutant IER3IP1 proteins (especially V21G and L78P variants) or enhance their residual trafficking function. Therapeutic strategies targeting downstream consequences of IER3IP1 deficiency—such as chemical chaperones or ER stress modulators—could alleviate secondary cellular stress responses even without directly correcting trafficking defects. For specific disorders like neonatal diabetes, identifying IER3IP1's interactome in β-cells could reveal alternative therapeutic targets to enhance insulin processing and secretion through parallel pathways. Additionally, understanding IER3IP1's role in cytokinesis could provide insights into cell division regulation relevant to regenerative medicine applications. Developing biomarkers based on trafficking efficiency could aid in early diagnosis or therapeutic monitoring for IER3IP1-related disorders. These diverse therapeutic implications highlight the translational significance of fundamental research into IER3IP1's cellular functions .

What adaptations are necessary when transitioning from human to bovine IER3IP1 research models?

Transitioning from human to bovine IER3IP1 research models requires several strategic adaptations to account for species-specific differences. First, sequence alignment analysis reveals approximately 90% protein identity between human and bovine IER3IP1, but researchers must pay special attention to any variations in key functional domains, particularly in regions affecting Rab11 interaction or ER localization. When designing expression constructs, codon optimization for bovine cell expression systems should be implemented to maximize protein production. For antibody-based detection, epitope mapping is essential since commercially available antibodies raised against human IER3IP1 may have reduced affinity for bovine orthologues—validation with recombinant protein standards is strongly recommended. Cell culture conditions require optimization for bovine cells, which often have different growth factor requirements and temperature optima compared to human cell lines. When designing genome editing strategies, gRNA selection should target highly conserved regions while avoiding potential bovine-specific off-target sites. Finally, researchers should establish bovine-specific reference datasets for normal IER3IP1 expression levels across tissues, subcellular localization patterns, and interaction partners before attempting to characterize phenotypes of experimental manipulations .

How do post-translational modifications affect bovine IER3IP1 function and detection?

Post-translational modifications (PTMs) significantly impact bovine IER3IP1 function and detection, requiring careful consideration in experimental design. While specific data on bovine IER3IP1 PTMs is limited, research on human orthologues suggests several key modifications likely conserved across species. Phosphorylation sites, particularly at serine and threonine residues, may regulate IER3IP1's interaction with binding partners like Rab11—phosphoproteomic analysis of bovine IER3IP1 expressed in relevant cellular contexts should be performed to map these sites. Ubiquitination likely regulates IER3IP1 stability and turnover, as disease-associated mutations show reduced protein levels consistent with enhanced degradation. For experimental detection, antibodies recognizing phosphorylated forms may provide insights into activation states under different cellular conditions. When expressing recombinant bovine IER3IP1, researchers should select expression systems capable of appropriate PTMs—prokaryotic systems lack many modification capabilities, making mammalian expression preferable for functional studies. Mass spectrometry analysis comparing wild-type and mutant forms could reveal how disease-associated mutations alter modification patterns. Finally, pharmacological inhibitors of specific PTM pathways can help establish the functional significance of individual modifications in regulating IER3IP1's trafficking roles .

What are the optimal tissue sources for studying native bovine IER3IP1?

For studying native bovine IER3IP1, selecting optimal tissue sources requires consideration of expression patterns and functional relevance. Based on human and mouse data, pancreatic islets represent a primary tissue source for bovine IER3IP1 research due to the protein's high expression and critical functional role in β-cells. Fresh bovine pancreatic tissue should be processed rapidly with collagenase digestion followed by density gradient separation to isolate intact islets. Developing brain tissue, particularly cortical regions during prenatal and early postnatal development, offers another valuable source given IER3IP1's importance in neurogenesis and the microcephaly phenotype associated with mutations. Primary bovine embryonic fibroblasts provide accessible cellular models that express moderate IER3IP1 levels and can be maintained in culture for experimental manipulation. For cellular localization studies, polarized epithelial cells from bovine kidney or intestinal origins may be particularly informative due to their well-developed secretory pathways. When collecting tissues, standardize for age, developmental stage, and physiological status, as IER3IP1 expression may vary during development or under different metabolic conditions. Preservation protocols should maintain membrane integrity and protein interactions—flash freezing for biochemical analyses and gentle fixation for immunohistochemistry to avoid disrupting the delicate ER and vesicular structures where IER3IP1 resides .

What are the most promising directions for future bovine IER3IP1 research?

The most promising directions for future bovine IER3IP1 research span several interconnected areas that build upon current understanding while addressing critical knowledge gaps. Comprehensive characterization of the bovine IER3IP1 interactome using proximity labeling approaches would reveal species-specific interaction partners beyond the established Rab11 connection. Development of bovine-specific CRISPR/Cas9 genome editing protocols would enable precise modeling of disease-associated mutations in relevant cell types. Detailed investigation of tissue-specific functions, particularly in pancreatic β-cells and developing brain, could provide insights into why these tissues are especially vulnerable to IER3IP1 dysfunction. High-resolution structural studies of bovine IER3IP1, especially in complex with interaction partners like Rab11, would illuminate the molecular basis for trafficking functions and how disease mutations disrupt these interactions. Systems biology approaches integrating proteomics, transcriptomics, and functional assays could map how IER3IP1 deficiency propagates through cellular networks to cause tissue-specific pathologies. Finally, translational research exploring how findings in bovine models might inform therapeutic strategies for human IER3IP1-related disorders represents an important bridge between basic science and clinical applications .

What standardized protocols should researchers adopt when working with recombinant bovine IER3IP1?

Researchers working with recombinant bovine IER3IP1 should adopt standardized protocols to ensure reproducibility and meaningful cross-study comparisons. For expression and purification, use mammalian expression systems (preferably HEK293 cells) with N-terminal tags (HA or His6) that don't interfere with the C-terminal transmembrane domain. Solubilization buffers should contain mild detergents (0.1% DDM or 0.01% LMNG) supplemented with stabilizers like glycerol (10%) and protease inhibitors. For functional assays, standardized trafficking studies using the RUSH system with consistent cargo proteins (proinsulin for β-cell studies) provide quantifiable readouts. Interaction studies should include both co-immunoprecipitation and proximity labeling approaches with appropriate controls. For localization, standardized immunofluorescence protocols with consistent fixation methods (4% PFA for 10 minutes) and validated antibodies against compartment markers (PDI, GM130, Rab11) enable reliable co-localization analysis. When conducting rescue experiments, expression levels should be carefully controlled and quantified relative to endogenous protein. Finally, authentication of recombinant proteins through mass spectrometry and functional validation through multiple independent assays should be mandatory before publication. Adopting these standardized approaches will facilitate data integration across different research groups working on bovine IER3IP1 .