Recombinant Pongo abelii Protein RER1 (RER1)

What is the RER1 protein and what are its key functions?

RER1 (Retention in Endoplasmic Reticulum 1) is an evolutionarily conserved protein localized to the endoplasmic reticulum (ER) and Golgi apparatus. This protein plays a critical role in maintaining protein homeostasis (proteostasis) within cells, particularly through its function in protein trafficking and quality control mechanisms . RER1 is essential for proper transport of proteins between the ER and Golgi compartments, acting as a retrieval receptor for certain ER-resident proteins . Additionally, RER1 facilitates the assembly of multisubunit protein complexes, including the tetrameric γ-secretase complex, iron transporters, and nicotinic acetylcholine receptors . These functions collectively contribute to cellular health by preventing proteotoxic stress and ensuring proper protein folding and assembly.

Why is RER1 from Pongo abelii useful for comparative research?

Studying RER1 from Pongo abelii provides valuable insights into evolutionary conservation of essential cellular mechanisms. Since RER1 is conserved from yeast to mammals , the orangutan variant offers an important comparative model for understanding primate-specific adaptations in protein quality control systems. Research comparing RER1 function across species illuminates how fundamental cellular processes have been preserved throughout evolution while potentially uncovering species-specific modifications. The high degree of conservation indicates that findings from the Pongo abelii RER1 protein can often be extrapolated to human systems, making it valuable for biomedical research while avoiding some ethical constraints associated with human samples. Additionally, comparative studies across primates can highlight critical structural and functional domains that have remained unchanged due to selective pressure.

How does RER1 deficiency impact cellular stress response pathways?

RER1 deficiency triggers profound cellular stress responses, particularly through activation of the unfolded protein response (UPR). Research in Drosophila has demonstrated that loss of RER1 leads to proteotoxic stress and subsequent activation of the PERK-mediated phosphorylation of eukaryotic initiation factor 2α (eIF2α) . This phosphorylation event attenuates general protein translation while selectively enhancing translation of stress-response factors. In developing tissues, RER1-deficient cells exhibit hallmarks of cellular stress, including elevated levels of cleaved Death caspase-1 (Dcp-1) and Acridine Orange positivity, indicating apoptotic cell death .

What is the relationship between RER1 expression and Myc-driven cellular processes?

Research has revealed a fascinating relationship between RER1 expression and the oncogenic transcription factor Myc. Studies in Drosophila wing epithelium have shown that RER1 levels are upregulated in cells overexpressing Myc . This upregulation appears to be a cytoprotective adaptation that helps cells manage the increased proteotoxic stress associated with Myc-driven enhancement of protein synthesis. Experimental evidence indicates that this elevation of RER1 is functionally significant, as loss of RER1 is sufficient to suppress Myc-induced overgrowth .

The mechanism underlying this relationship likely involves RER1's role in protein quality control and ER homeostasis. Myc-overexpressing cells have heightened protein synthesis rates, which increases the burden on cellular protein folding machinery. The concomitant upregulation of RER1 helps manage this burden by facilitating proper protein trafficking and assembly, thereby alleviating proteotoxic stress. This relationship highlights RER1's importance in supporting cellular adaptation to conditions of high protein synthesis demand and suggests potential implications for understanding Myc-driven tumorigenesis. Researchers investigating Myc-dependent cancers might consider RER1 as a potential modulator of cellular fitness under oncogenic stress.

How does RER1 contribute to multiprotein complex assembly?

RER1 plays a sophisticated role in the assembly of multisubunit protein complexes through several coordinated mechanisms. Studies across multiple model systems have demonstrated that RER1 facilitates proper assembly of complexes including the tetrameric γ-secretase complex, yeast iron transporters, and nicotinic acetylcholine receptors (nAChRs) . The precise mechanisms involve:

• Quality control during early assembly steps by retrieving unassembled subunits from the Golgi back to the ER where assembly can proceed

• Recognition of specific ER-retention motifs on unassembled subunits

• Temporal coordination of subunit availability during complex assembly

• Prevention of premature trafficking of incomplete complexes

These functions are critical for ensuring that only properly assembled complexes reach their final cellular destinations. Proper complex assembly is vital for cellular function, as misassembled complexes can contribute to proteotoxicity and cellular dysfunction. The involvement of RER1 in assembly of diverse protein complexes suggests it may have a broader role in cellular proteostasis beyond its initially characterized functions in ER-Golgi trafficking.

The table below summarizes key multiprotein complexes known to depend on RER1 for proper assembly:

What are the optimal storage and handling conditions for recombinant Pongo abelii RER1 protein?

Proper storage and handling of recombinant Pongo abelii RER1 protein is critical for maintaining its stability and functional integrity in experimental applications. The recommended storage conditions are as follows:

For long-term storage: The protein should be maintained at -20°C or preferably -80°C for extended periods . The commercial preparation is typically supplied in a Tris-based buffer containing 50% glycerol, which has been optimized to preserve protein structure during freeze-thaw cycles .

For working stocks: Aliquots can be stored at 4°C for up to one week . It is crucial to note that repeated freezing and thawing should be avoided as this can lead to protein denaturation and loss of activity . Therefore, it is recommended to prepare small working aliquots when dividing the stock.

When handling the protein, researchers should minimize exposure to room temperature and maintain sterile conditions to prevent microbial contamination. For optimal results in functional assays, it is advisable to include appropriate protease inhibitors in working buffers and to validate protein integrity by SDS-PAGE or other analytical methods before critical experiments. The specific buffer composition may need to be adjusted depending on the downstream application, but care should be taken to maintain conditions that preserve the native structure of the protein.

What experimental approaches can effectively assess RER1 function in protein trafficking?

Investigating RER1 function in protein trafficking requires sophisticated experimental strategies that can capture the dynamic nature of ER-Golgi transport. Several complementary approaches are recommended:

• Live-cell fluorescence imaging: Utilizing GFP-tagged RER1 constructs enables visualization of its subcellular localization and trafficking dynamics . This approach can be enhanced with photoactivatable or photoconvertible fluorescent proteins to track specific protein populations over time.

• Cargo trafficking assays: Monitoring the movement of known RER1-dependent cargoes using pulse-chase experiments with metabolic labeling. This typically involves:

  • Pulse-labeling newly synthesized proteins with radioactive amino acids

  • Chasing with non-radioactive media for varying time periods

  • Immunoprecipitating the cargo protein of interest

  • Analyzing by autoradiography to determine maturation and trafficking rates

• Proximity labeling methods: Techniques such as BioID or APEX2 can identify proteins in close proximity to RER1 at different cellular locations, revealing transient trafficking interactions.

• Subcellular fractionation: Isolating ER and Golgi fractions followed by immunoblotting for RER1 and its cargo proteins can provide quantitative assessment of protein distribution between compartments.

• RUSH system (Retention Using Selective Hooks): This allows synchronized release of cargo from the ER, enabling precise temporal analysis of trafficking kinetics in the presence or absence of functional RER1.

For knockdown or knockout studies, researchers should consider genetic approaches such as CRISPR-Cas9 editing or RNAi, while being mindful that complete loss of RER1 may be lethal, as demonstrated in Drosophila studies . Conditional or tissue-specific approaches may be necessary to study RER1 function in complex organisms.

How can researchers effectively study the interaction between RER1 and cell competition mechanisms?

Investigating the relationship between RER1 and cell competition requires specialized experimental systems that can create and monitor cellular mosaics. Based on findings in Drosophila , the following methodological approaches are recommended:

• Mosaic analysis: Generate tissue mosaicism using techniques like the FLP/FRT system (in Drosophila) or mosaic analysis with double markers (MADM) in mammals . These systems create genetically distinct cell populations within the same tissue, allowing direct comparison between RER1-deficient and wild-type cells.

• Live imaging of competitive interactions: Implement long-term live imaging with fluorescently labeled cell populations (e.g., RFP for one population, GFP for another) to track cell behaviors at the boundary between populations. Time-lapse microscopy can capture:

  • Changes in cell morphology

  • Cell elimination events

  • Cellular protrusions

  • Migratory behaviors

• Cell competition markers assessment: Analyze established molecular markers of cell competition including:

  • Phosphorylated JNK signaling components

  • Cleaved caspases (e.g., Dcp-1 in Drosophila)

  • Engulfment markers

  • Expression of fitness comparison factors like Flower or Azot

• Quantitative analysis approaches:

  • Clone size measurements over time

  • Quantification of apoptotic cells specifically at clone boundaries

  • Spatial distribution analysis of death events relative to genotype boundaries

  • Statistical comparison of clone survival rates

• Mechanical force analysis: Implement techniques like laser ablation or force inference methods to measure mechanical tensions at the interface between RER1-deficient and normal cells.

When designing these experiments, researchers should carefully consider the developmental timing, as the strength of competitive interactions may vary at different developmental stages. Additionally, manipulating Myc levels in conjunction with RER1 status can provide insight into how cellular fitness is modulated through these interacting pathways .

What analytical methods are appropriate for assessing RER1-dependent proteostasis?

Comprehensive investigation of RER1-dependent proteostasis requires a multi-modal analytical approach that addresses protein folding, trafficking, and quality control. The following methodological framework is recommended:

• ER stress sensor analysis: Quantify activation of canonical unfolded protein response (UPR) pathways through:

  • Phosphorylation status of PERK, IRE1α, and eIF2α by immunoblotting

  • XBP1 splicing assays using RT-PCR to detect the spliced active form

  • ATF6 cleavage and nuclear translocation via cellular fractionation and immunoblotting

  • Luciferase reporters driven by UPR response elements

• Protein aggregation assessment:

  • Detergent solubility fractionation to separate soluble from aggregated proteins

  • Filter trap assays to capture high-molecular-weight aggregates

  • Thioflavin T or Congo Red binding to detect amyloid-like structures

  • Microscopic visualization of protein aggregates using fluorescent proteins

• Protein degradation pathway analysis:

  • Cycloheximide chase experiments to measure protein half-lives

  • Proteasome activity assays using fluorogenic substrates

  • Autophagy flux measurements using LC3-II/I ratios with and without lysosomal inhibitors

  • Ubiquitination profiling of cellular proteins

• Global proteomics approaches:

  • Stable isotope labeling with amino acids in cell culture (SILAC) to compare protein levels and turnover rates

  • Pulse-SILAC to measure newly synthesized vs. pre-existing protein pools

  • Quantitative mass spectrometry to identify RER1-dependent changes in the proteome

  • Thermal proteome profiling to assess protein stability changes

• Imaging-based techniques:

  • Fluorescence recovery after photobleaching (FRAP) to measure protein mobility

  • Split fluorescent protein complementation to visualize protein-protein interactions in vivo

  • Förster resonance energy transfer (FRET) to detect conformational changes in reporter proteins

Given that loss of RER1 activates proteotoxic stress responses in Drosophila , researchers should design experiments that can distinguish primary effects of RER1 deficiency from secondary adaptations to cellular stress. This may involve time-course experiments or the use of stress pathway inhibitors to dissect the sequence of events following RER1 disruption.

How can recombinant RER1 protein be incorporated into in vitro binding studies?

Incorporating recombinant Pongo abelii RER1 protein into in vitro binding studies requires careful experimental design to preserve the protein's native structure and function. The following methodological approach is recommended:

• Preparation of functional RER1 protein: Since RER1 is a membrane protein with multiple transmembrane domains, it requires appropriate reconstitution systems to maintain functionality. Options include:

  • Nanodiscs or lipid bilayer systems to mimic the native membrane environment

  • Detergent micelles using mild non-ionic detergents (e.g., DDM, LMNG) that preserve protein structure

  • Cell-free expression systems combined with immediate reconstitution into liposomes

• Binding assay formats:

  • Solid-phase binding assays where purified RER1 is immobilized on a surface (considering its orientation)

  • Solution-based assays using techniques like microscale thermophoresis or isothermal titration calorimetry

  • Pull-down assays using tagged RER1 protein and candidate binding partners

  • Surface plasmon resonance to measure real-time binding kinetics

• Candidate binding partner preparation: Potential RER1 interacting proteins should be expressed and purified with appropriate tags that don't interfere with the interaction domains. For membrane proteins that interact with RER1, similar reconstitution approaches should be applied.

• Controls and validation:

  • Use of binding-deficient RER1 mutants as negative controls

  • Competition assays with known RER1 ligands

  • Concentration-dependent binding curves to determine affinity constants

  • Cross-validation with orthogonal binding techniques

When designing these experiments, researchers should be mindful that the storage buffer for commercial recombinant RER1 (containing 50% glycerol) may interfere with some binding assays and may require buffer exchange procedures. Additionally, the binding conditions should reflect the physiological environment of the ER-Golgi interface, including appropriate pH (typically 7.2-7.4 for ER, 6.7-7.0 for Golgi) and calcium concentrations.

What considerations should guide the design of RER1 genetic rescue experiments?

Designing effective RER1 genetic rescue experiments requires careful attention to both construct design and experimental implementation. Based on successful rescue experiments in Drosophila , the following considerations are critical:

• Expression system selection:

  • Endogenous promoter: Using the native RER1 promoter and regulatory elements ensures physiologically relevant expression patterns and levels

  • Inducible systems: For temporal control of rescue, systems like Gal4/UAS, Tet-On/Off, or heat-shock inducible promoters offer flexibility

  • Tissue-specific promoters: For targeted rescue in specific tissues without affecting others

• Construct design considerations:

  • Tagging strategy: If fluorescent or epitope tags are needed for visualization or purification, careful placement is essential to avoid interfering with function. N-terminal GFP tagging has been successfully used in Drosophila rescue experiments

  • Codon optimization: Adapting codons to the host organism can improve expression efficiency

  • Inclusion of introns: For some expression systems, including native introns can enhance expression

  • Cloning vector selection: Vectors with appropriate selection markers for the model system

• Delivery methods:

  • For cell culture: Transfection or viral transduction with appropriate controls for transfection efficiency

  • For Drosophila: Germline transformation via P-element, ΦC31 integrase, or CRISPR-mediated knock-in

  • For mammals: Viral vectors, electroporation, or generation of transgenic animals

• Validation strategies:

  • Expression level assessment: Western blotting, qRT-PCR, or fluorescence quantification

  • Subcellular localization confirmation: Microscopy to verify proper ER/Golgi localization

  • Functional readouts: Assessment of phenotypic rescue through multiple parameters

• Experimental design:

  • Positive controls: Wild-type RER1 expression

  • Negative controls: Empty vector or expression of unrelated proteins

  • Structure-function studies: Rescue with mutant versions of RER1 to identify critical functional domains

  • Dose-dependency: Titration of expression levels to determine minimum requirements for rescue

A particularly effective approach demonstrated in Drosophila involved creating a genomic rescue construct expressing GFP-tagged Rer1 via the endogenous promoter . This approach successfully rescued the lethality of homozygous rer1 mutant flies, confirming both the specificity of the mutation and the functionality of the GFP-tagged rescue construct . Similar strategies can be adapted for other model systems while accounting for species-specific considerations.

How might RER1 function in neurodegenerative disease processes?

The implication of RER1 in neurodegenerative disease processes represents a promising frontier for research, given its established roles in proteostasis and protein complex assembly. Several mechanistic connections warrant investigation:

• Protein aggregation modulation: Given RER1's role in proteostasis and protein quality control , it may influence the aggregation propensity of disease-associated proteins like amyloid-β, tau, α-synuclein, or huntingtin. Research should explore whether:

  • RER1 expression levels correlate with disease progression in neurodegenerative conditions

  • RER1 directly interacts with disease-associated proteins during their biosynthesis

  • Modulation of RER1 function affects the accumulation of protein aggregates in cellular and animal models

• Neuron-specific protein complex assembly: RER1's role in assembling multisubunit complexes has particular relevance for neurons, which depend on numerous complex protein assemblies for function. Investigations should address:

  • Whether RER1 regulates assembly of neuron-specific receptor complexes beyond nAChRs

  • If RER1 dysfunction contributes to synaptic protein complex imbalances

  • How neuronal activity influences RER1-dependent trafficking and complex assembly

• ER stress in neurodegeneration: Since loss of RER1 activates ER stress pathways , and chronic ER stress is implicated in neurodegenerative diseases, researchers should examine:

  • The status of RER1 expression and function in brain regions affected by neurodegeneration

  • Whether restoring or enhancing RER1 function can alleviate ER stress in disease models

  • Potential neuroprotective effects of RER1 upregulation under conditions of proteotoxic stress

• Trafficking of disease-relevant proteins: As RER1 regulates protein trafficking between ER and Golgi , studies should investigate its role in:

  • Trafficking of APP and secretases involved in amyloid-β generation

  • Movement of proteins involved in lysosomal function and autophagy

  • Maintaining polarized trafficking in neurons, which is often disrupted in neurodegenerative states

Methodologically, researchers should employ both cell-based systems and animal models with neuron-specific manipulation of RER1 expression. Post-mortem brain tissue from patients with neurodegenerative diseases should be examined for alterations in RER1 levels or localization. Additionally, genetic studies could analyze whether RER1 variants associate with disease risk or progression in human populations.

What evolutionary insights can be gained from comparing RER1 across primate species?

Comparative analysis of RER1 across primate species offers valuable insights into the evolution of fundamental cellular processes and potentially species-specific adaptations. Research in this area should pursue:

• Sequence conservation analysis: Detailed comparison of RER1 sequences across primate species, including Pongo abelii, can reveal:

  • Ultra-conserved domains that likely perform essential functions

  • Variable regions that might confer species-specific properties

  • Selective pressure signatures indicating functional constraints or adaptations

  • Correlation between RER1 sequence variation and primate evolutionary relationships

• Structure-function comparative studies: Using recombinant RER1 proteins from different primate species to:

  • Compare binding affinities for known interaction partners

  • Assess interchangeability in rescue experiments across species

  • Identify species-specific interaction networks

  • Evaluate differential responses to cellular stressors

• Expression pattern comparisons: Examination of:

  • Tissue-specific expression profiles across primate species

  • Developmental regulation of RER1 expression

  • Response to stress conditions in different primate cell types

  • Alternative splicing patterns and their conservation

• Regulatory evolution: Analysis of:

  • Promoter and enhancer element conservation across primates

  • Transcription factor binding sites and their functional significance

  • microRNA regulation of RER1 expression

  • Epigenetic modifications and their conservation

This research approach would benefit from integrating computational phylogenetic analysis with experimental functional validation. Advanced techniques such as ancestral sequence reconstruction could provide insights into the evolutionary trajectory of RER1 function. The availability of recombinant Pongo abelii RER1 protein provides an excellent starting point for comparative biochemical studies with human and other primate RER1 variants. Since RER1 is involved in essential cellular processes, understanding its evolution could illuminate how fundamental mechanisms of protein quality control have been maintained or adapted throughout primate evolution.