Recombinant Arabidopsis thaliana Derlin-2.1 (DER2.1)
Description
Introduction to Derlin-2.1 in Arabidopsis thaliana
Derlin-2.1 (DER2.1) is a member of the Derlin family of proteins found in Arabidopsis thaliana, a model organism widely used in plant molecular biology. The gene encoding DER2.1 is identified by several synonyms including At4g21810, F17L22.270, T8O5.20, and AtDerlin2-1, with the protein identified by UniProt ID Q8VZ96 . Derlins are evolutionarily conserved proteins that play critical roles in the endoplasmic reticulum-associated degradation (ERAD) pathway, which is responsible for identifying, retrotranslocating, and ultimately degrading misfolded proteins that accumulate in the ER.
The ERAD pathway is a crucial component of cellular protein quality control mechanisms. In Arabidopsis and other organisms, accumulation of misfolded proteins in the ER causes ER stress and activates a signaling pathway called the unfolded protein response (UPR) . This response aims to restore normal ER function by increasing the production of molecular chaperones, enhancing protein folding capacity, and upregulating components of the ERAD machinery to remove terminally misfolded proteins.
Role in ER-Associated Degradation (ERAD)
Based on comparative studies with Derlin family proteins in other organisms, DER2.1 in Arabidopsis likely plays a central role in the ERAD pathway. In mammals, Derlin-2 interacts with multiple proteins including Derlin-1, Sel1L, VIMP, p97, HRD-1, OS-9, and Ubc6e to form a dislocation complex involved in removing misfolded substrates from the ER .
In this complex, Derlin proteins are thought to form a channel through which misfolded proteins are dislocated from the ER lumen to the cytosol. Once in the cytosol, these proteins are typically ubiquitinated and subsequently degraded by the 26S proteasome. This process is essential for maintaining ER homeostasis and preventing the accumulation of potentially toxic misfolded proteins.
The ERAD pathway in Arabidopsis involves similar components to those in other eukaryotes, suggesting that DER2.1 functions in an analogous manner. The protein likely interacts with HRD1, a ubiquitin ligase that marks misfolded proteins for degradation, and other components of the Arabidopsis ERAD machinery .
Interaction with the Unfolded Protein Response (UPR)
The function of DER2.1 is closely linked to the unfolded protein response (UPR), a cellular stress response activated by the accumulation of misfolded proteins in the ER. Studies in mice have shown that Derlin-2 deficiency leads to constitutive activation of the UPR, particularly the IRE-1-mediated branch .
In Arabidopsis, the UPR involves three main sensors: PERK, ATF6, and IRE-1, which detect ER stress and activate downstream signaling pathways. These pathways lead to increased production of molecular chaperones, attenuation of protein synthesis, and upregulation of ERAD components, including potentially DER2.1 .
The relationship between DER2.1 and the UPR in Arabidopsis is likely bidirectional. On one hand, DER2.1 helps alleviate ER stress by facilitating the removal of misfolded proteins through ERAD. On the other hand, the expression of DER2.1 itself may be upregulated during the UPR to enhance ERAD capacity when needed.
Comparative Analysis with Derlin Family Proteins in Other Species
The Derlin family of proteins is evolutionarily conserved across eukaryotes, suggesting fundamental importance in cellular function. Studies on Derlin-2 in mice have provided valuable insights that may be relevant to understanding DER2.1 function in Arabidopsis.
In mice, Derlin-2 deficiency results in constitutive upregulation of ER chaperones and IRE-1-mediated induction of the unfolded protein response in most tissues . Whole-body deletion of Derlin-2 in mice is largely lethal, with the few surviving animals developing skeletal dysplasia, likely due to defects in collagen matrix protein secretion . This severe phenotype underscores the critical importance of Derlin-2 in protein quality control.
Applications and Research Significance
Recombinant DER2.1 protein serves as a valuable tool for investigating protein quality control mechanisms in plants. Applications include:
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Structural studies: The purified recombinant protein can be used to determine the three-dimensional structure of DER2.1, providing insights into how it forms dislocation pores in the ER membrane.
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Protein-protein interaction analyses: The recombinant protein can be used to identify and characterize interactions with other components of the ERAD machinery in Arabidopsis.
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Functional assays: In vitro assays using the recombinant protein can help elucidate the mechanism by which DER2.1 facilitates the dislocation of misfolded proteins from the ER.
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Antibody production: The recombinant protein can be used to generate antibodies for detecting and studying endogenous DER2.1 in plant tissues.
The study of DER2.1 and other components of the ERAD pathway in Arabidopsis contributes to our understanding of how plants maintain protein homeostasis under normal and stress conditions. This knowledge has broader implications for plant stress responses and potential applications in improving crop resilience.
Product Specs
Note: While we prioritize shipping the format currently in stock, we are happy to accommodate specific format requests. Please indicate your preference in the order notes, and we will fulfill your needs to the best of our ability.
Note: All proteins are shipped with standard blue ice packs. If dry ice shipping is required, please communicate with us beforehand. Additional fees will apply.
Generally, liquid forms have a shelf life of 6 months at -20°C/-80°C. Lyophilized forms typically have a shelf life of 12 months at -20°C/-80°C.
Target Background
Q&A
What is Derlin-2.1 and what is its role in Arabidopsis thaliana?
Derlin-2.1 (DER2.1) is a transmembrane protein in Arabidopsis thaliana with the UniProt accession number Q8VZ96. It is also known as AtDerlin2-1. The protein is encoded by the gene DER2.1 (At4g21810), with alternative locus names F17L22.270 and T8O5.20 .
Derlins generally function in the endoplasmic reticulum-associated degradation (ERAD) pathway, which is responsible for the retrograde transport of misfolded proteins from the ER to the cytosol for degradation. While specific research on Arabidopsis DER2.1 is still developing, studies in other organisms suggest it plays a crucial role in protein quality control and cellular stress responses.
How is the Derlin-2.1 gene organized in the Arabidopsis genome?
The DER2.1 gene is located on chromosome 4 of the Arabidopsis thaliana genome (At4g21810). It encodes the full-length protein of 244 amino acids. For researchers planning gene manipulation experiments, it's important to note that the gene has been annotated with alternative locus names including F17L22.270 and T8O5.20, which may be relevant when designing primers or CRISPR guide RNAs for targeted genetic modifications .
When working with the Arabidopsis 1001 Genomes database or AraPheno repository, researchers can access detailed genomic information about DER2.1 across different natural accessions, which is valuable for studying natural genetic variation and its effects on DER2.1 function .
What are the optimal storage conditions for recombinant Arabidopsis Derlin-2.1?
For optimal stability and activity of recombinant Arabidopsis thaliana Derlin-2.1, follow these research-validated storage protocols:
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Store at -20°C for regular use
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For extended storage, conserve at -20°C or -80°C
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Avoid repeated freezing and thawing cycles, as this significantly reduces protein stability
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For working experiments, prepare aliquots and store at 4°C for up to one week
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The protein is typically provided in a storage buffer containing Tris-based buffer with 50% glycerol, optimized for stability
The shelf life depends on several factors including buffer ingredients, storage temperature, and the inherent stability of the protein itself. Generally, liquid preparations have a shelf life of approximately 6 months at -20°C/-80°C, while lyophilized forms can maintain stability for up to 12 months at -20°C/-80°C .
How is recombinant Derlin-2.1 produced for research applications?
Recombinant Arabidopsis thaliana Derlin-2.1 is primarily produced using in vitro E. coli expression systems. The recombinant protein typically includes an N-terminal 10xHis-tag to facilitate purification and detection in experimental applications .
The expression region encompasses amino acids 1-244, representing the full-length protein. For researchers planning to express this protein themselves, it's important to note that as a transmembrane protein, DER2.1 may require specialized expression and purification protocols to maintain its native conformation and functionality.
What experimental approaches can be used to study DER2.1 function in Arabidopsis?
When investigating DER2.1 function in Arabidopsis thaliana, researchers can employ several methodological approaches:
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Gene expression analysis: Using RNA-Seq data from various Arabidopsis studies to analyze DER2.1 expression patterns across different tissues, developmental stages, and stress conditions. The Arabidopsis gene expression predictor can be particularly useful, as it allows inference of gene expression relationships with an average correlation coefficient (r) of 0.9861±0.0177 .
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Mutation analysis: Creating and phenotyping mutant lines using approaches similar to those used for studying other genes in Arabidopsis, such as TDP2 and WSS1A. This can involve analyzing growth phenotypes, root length measurements, and sensitivity to various stress conditions .
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Triple testcross (TTC) design: For studying genetic interactions, researchers can use approaches similar to those employed in the analysis of biomass-related traits in Arabidopsis. This involves crossing recombinant inbred lines (RILs) with parental lines and their F1 hybrids to investigate dominance and epistatic effects .
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Phenotypic characterization: Utilizing repositories like AraPheno (https://arapheno.1001genomes.org) to access and analyze population-scale phenotypes for Arabidopsis thaliana inbred lines, which can help in understanding the relationship between genetic variation in DER2.1 and observable phenotypes .
How can I analyze gene expression data related to DER2.1 in Arabidopsis?
To effectively analyze gene expression data for DER2.1 in Arabidopsis thaliana, researchers should consider the following methodological approach:
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Utilize RNA-Seq datasets: According to research methodologies, quality RNA-Seq data should have mapping rates ≥70% with at least 5000 genes having counts per million (cpm) ≥1. For DER2.1 analysis, focus on samples meeting these quality criteria .
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Apply appropriate normalization: Transform expression values using log2(cpm+1) to ensure data is appropriately scaled for statistical analysis.
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Implement predictive modeling: Consider using linear models similar to those described in gene expression predictor studies. These models can be represented as Y = XB + e, where X represents transcription factor (TF) gene expression, Y represents target gene expression, B is the coefficient matrix, and e represents random errors .
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Assess statistical significance: Apply t-tests following hypothesis test methods for multiple linear regression to determine the significance of individual regression coefficients, which helps identify significant interacting TF-target gene pairs .
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Visualize interaction networks: Use tools like Cytoscape (V3.4.0) to visualize gene networks and modules, which can help identify functional relationships between DER2.1 and other genes .
What approaches can be used to analyze phenotypic data in DER2.1 research?
When analyzing phenotypic data related to DER2.1 in Arabidopsis thaliana, researchers should employ the following methodological framework:
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Root meristem assay: For analyzing sensitivity to genotoxic agents, quantify the number of dead cells in the root meristem after propidium iodide (PI) staining. PI is a fluorescent dye that permeates dead cells, thus differentiating living and dead cells. This approach has been successfully used to analyze the effects of mutations in DNA repair genes in Arabidopsis .
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Statistical comparison: Compare the number of dead cells in untreated versus treated conditions across different genotypes (wild-type vs. mutant). This approach allows for the evaluation of statistical differences and can reveal the functional importance of specific genes in stress responses .
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AraPheno database utilization: Leverage the AraPheno repository, which contains population-scale phenotypes for Arabidopsis thaliana inbred lines. As of August, 2016, this database contained data from 6 studies, 260 phenotypes, 7,425 accessions, and 52,741 phenotype values, providing a rich resource for comparative analysis .
| Data Content | Data Statistics |
|---|---|
| Studies | 6 |
| Phenotypes | 260 |
| Accessions | 7,425 |
| Phenotyped accessions | 1,425 |
| Observational units | 4,064 |
| Phenotype values | 52,741 |
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Ontology-based analysis: Utilize trait ontology terms to categorize and analyze phenotypic data. The top trait-ontology terms in AraPheno include days to flowering, bacterial disease resistance, seed weight, and various mineral concentrations, which can provide context for interpreting DER2.1-related phenotypes .
How can DER2.1 research benefit from comparison with DNA repair pathways in Arabidopsis?
While DER2.1 and DNA repair proteins like TDP2 and WSS1A have different primary functions, the methodological approaches used to study DNA repair pathways provide valuable insights for DER2.1 research:
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Mutant analysis: Similar to the study of tdp2 mutants in topoisomerase 2 cleavage complex (TOP2cc) repair, researchers can generate and characterize DER2.1 mutants to understand its function. The analysis of growth phenotypes, including root length measurements and sensitivity to various stress conditions, can reveal the role of DER2.1 in cellular processes .
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Double mutant studies: The generation of double mutants (e.g., tdp2 wss1A in DNA repair studies) can reveal functional relationships between DER2.1 and other proteins. By comparing the phenotypes of single and double mutants, researchers can determine whether proteins function in the same or parallel pathways .
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Quantitative phenotypic assays: Root meristem assays used to study DNA repair can be adapted to study DER2.1 function. These assays provide quantitative data on cellular responses to stress conditions and can reveal the functional importance of specific proteins .
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Cross-species comparison: Comparing the function of DER2.1 in Arabidopsis with its homologs in other organisms can provide insights into conserved mechanisms, similar to how studies of DNA repair proteins benefit from comparison with yeast and human homologs .
How can CRISPR-Cas9 technology be applied to study DER2.1 function?
CRISPR-Cas9 technology offers powerful approaches for investigating DER2.1 function in Arabidopsis thaliana:
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Gene knockout: Similar to the Cas9-mediated mutagenesis used to create tdp2 mutations in the wss1A-3 background, researchers can generate precise DER2.1 knockout lines. This approach can create mutations leading to premature stop codons, which can be confirmed at the mRNA level .
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Double mutant generation: CRISPR-Cas9 can be used to create mutations in DER2.1 in various genetic backgrounds, enabling the study of genetic interactions. For example, researchers could generate DER2.1 mutations in lines with alterations in other ERAD pathway components to study functional relationships .
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Domain-specific modifications: Rather than completely knocking out DER2.1, researchers can use CRISPR-Cas9 to make precise modifications to specific functional domains, allowing for a more nuanced understanding of protein function.
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Promoter modification: CRISPR-Cas9 can be used to modify the DER2.1 promoter region, enabling the study of gene regulation under various conditions or in different tissues.
How can Google's "People Also Ask" feature be leveraged to identify research gaps in DER2.1 studies?
Google's "People Also Ask" (PAA) feature can be a valuable tool for identifying research gaps and emerging questions in the field of DER2.1 research:
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Identification of common questions: The PAA feature shows frequently asked questions related to a search topic, which can help researchers identify areas of interest or confusion in the scientific community. As PAA appears in approximately 68% of desktop SERPs as of May 2023, it provides a good representation of common inquiries .
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Question clustering analysis: PAA questions are dynamically presented based on user interactions, creating clusters of related questions. By analyzing these clusters related to DER2.1 and Arabidopsis research, scientists can identify emerging research directions and unexplored connections .
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Methodological approach:
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Data collection: The PAA box is interactive - clicking on one question reveals additional related questions. This feature can be exploited to systematically collect a network of research questions surrounding DER2.1, creating a comprehensive map of current knowledge gaps .
What are the emerging trends in Arabidopsis protein research that might impact DER2.1 studies?
Research on Arabidopsis thaliana continues to evolve, with several emerging trends that will likely impact future studies of DER2.1:
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Integration of multi-omics data: The development of resources like AraPheno facilitates the integration of genotypic and phenotypic data, enabling a more comprehensive understanding of gene-phenotype relationships. This approach will be valuable for elucidating the function of DER2.1 in different genetic backgrounds and environmental conditions .
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Advanced genetic analysis: Techniques like the triple testcross design with recombinant inbred lines provide powerful tools for investigating genetic interactions, which could reveal how DER2.1 functions within broader cellular networks .
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Predictive modeling: The development of gene expression predictors for Arabidopsis, with high accuracy (average r of 0.9861±0.0177), enables researchers to make inferences about gene function and interactions based on expression data, which could provide insights into DER2.1 function without requiring extensive experimental work .
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Comparative analysis: As more data becomes available through resources like AraPheno, comparative analysis of different phenotypic data types will enhance our understanding of the genotype-phenotype map, including the role of DER2.1 in plant development and stress responses .
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Advanced DNA repair research methodologies: The sophisticated approaches used to study DNA repair pathways in Arabidopsis, such as the analysis of topoisomerase 2 cleavage complexes, provide methodological frameworks that can be adapted to study other cellular processes, including those involving DER2.1 .
