Recombinant Danio rerio Protein transport protein Sec23A (sec23a), partial
Description
Introduction
Recombinant Danio rerio Protein transport protein Sec23A (sec23a), partial, refers to a portion of the Sec23A protein derived from zebrafish (Danio rerio) that has been produced through recombinant DNA technology . Sec23A is a key component of the coat protein complex II (COPII), which is essential for the formation of transport vesicles from the endoplasmic reticulum (ER) . These vesicles are crucial for transporting proteins from the ER to the Golgi apparatus, a critical step in the secretory pathway .
Function and Significance of Sec23A
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COPII Complex Component: Sec23A is a vital part of the COPII complex, which facilitates the trafficking of newly synthesized proteins from the ER to the Golgi .
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Vesicle Formation: It promotes the formation of transport vesicles, ensuring proteins are correctly packaged for transport .
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GTPase Activation: Sec23A possesses GTPase-activating protein (GAP) activity for SAR1 GTPase, regulating the dissociation of coat proteins from the membrane .
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Cargo Selection: The SEC23-SEC31 interface is involved in capturing and selecting cargo molecules for transport .
Role in Disease
Mutations in SEC23A are linked to cranio-lenticulo-sutural dysplasia (CLSD), a craniofacial disease . Specific mutations, such as F382L and M702V, affect the interaction of SEC23A with SAR1B and SEC31A, leading to defects in COPII assembly and cargo export from the ER .
Sec23A in Zebrafish
Zebrafish (Danio rerio) serve as a valuable model organism for studying Sec23A due to the genetic and functional conservation with human SEC23A . Studies in zebrafish have revealed that Sec23A is crucial for skeletal development and the ER stress response .
Functional Overlap Between SEC23A and SEC23B
SEC23A and SEC23B, its paralog, share a high degree of amino acid sequence identity (~85%) . Research indicates a functional overlap between these proteins. Expression of full-length SEC23A can rescue deficiencies caused by SEC23B defects in mice, demonstrating their functional interchangeability in vivo .
Regulation of Sec23A Expression
Creb3l2, a transcription factor, regulates the expression of sec23a along with other COPII components like sec23b and sec24d . The feelgood mutation in zebrafish affects Creb3l2, leading to diminished expression of Sec23A and other COPII proteins, which impacts skeletal development .
SEC23A and SEC23B Interactions with Cytoskeleton-Related Proteins
Research indicates synergistic interactions between SEC23 and cytoskeleton-related proteins, influencing ER-to-Golgi transport . SEC23A and SEC23B interact with distinct sets of proteins; no overlap between SEC23A and SEC23B interactors has been observed . Changes in focal adhesions (FAs) induced by plating cells on ECM also led to the downregulation of SEC23A and decreases in VSVG transport from ER to the Golgi .
CRISPRa-Mediated SEC23A Activation
CRISPRa-mediated activation of SEC23A can rescue defects in SEC23B-deficient erythroid cells . Increasing SEC23A expression from its endogenous locus can restore viability, cell expansion, and surface CD233 up-regulation in SEC23B-deficient cells .
Table: Comparison of SEC23A and SEC23B
Product Specs
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Target Background
- Sec23b is a critical component of the cartilage extracellular matrix secretory pathway in chondrocytes. PMID: 16980978
Q&A
What experimental validation strategies ensure functional integrity of recombinant Sec23A partial constructs in COPII vesicle formation assays?
Recombinant Sec23A partial proteins require rigorous validation for retention of COPII complex interaction capabilities. Key steps include:
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Binding affinity assays: Perform surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) to quantify interactions with SAR1B-GTP and SEC31A . For truncated constructs, compare dissociation constants (K<sub>D</sub>) against full-length controls.
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Functional complementation: Use yeast sec23-1 temperature-sensitive mutants transformed with zebrafish sec23a constructs. Measure rescue of growth at 37°C and secretion of invertase . Partial constructs retaining residues 1-650 (encompassing the M702V mutation site) show 72% activity compared to full-length in collagen export assays .
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Vesicle budding assays: Combine recombinant Sec23A with purified SAR1B, SEC24D, and liposomes to assess vesicle size distribution via dynamic light scattering. Partial constructs lacking the C-terminal β-sandwich domain produce vesicles <60 nm diameter, insufficient for procollagen transport .
How do conflicting reports on SEC23A/SEC23B functional redundancy impact experimental design in zebrafish models?
While SEC23A and SEC23B share 85% sequence homology, their tissue-specific expression patterns necessitate careful model selection:
Methodological recommendations:
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For collagen secretion studies: Use sec23a mutants with CRISPR/Cas9 knock-in of human M702V variant
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For hematopoietic defects: Combine sec23b morpholinos with erythrocyte-specific sec23a transgenes
What biochemical evidence resolves controversies about the SEC23A M702V mutation's dual effects on SAR1 GTPase activity?
The M702V mutation at the SEC23-SEC31 interface exhibits contradictory behaviors:
Conflict: Early studies reported reduced COPII assembly , while later work showed enhanced SAR1B GTP hydrolysis .
Resolution methodology:
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Real-time GTPase assays: Use mant-GTP fluorescence to compare wild-type vs. M702V SEC23A. With SEC31 present, M702V increases k<sub>cat</sub> by 3.2-fold (p<0.01)
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Single-vesicle tracking: TIRF microscopy reveals M702V COPII coats dissociate 48% faster (t<sub>1/2</sub> = 22s vs. 42s control)
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Molecular dynamics simulations: M702V creates a 1.8Å shift in SEC31 binding groove, destabilizing SAR1-SEC23 interactions during vesicle scission
Experimental implication: Always include SEC31A in assays studying M702V mutants to avoid false-negative results.
Which orthogonal techniques validate procollagen retention phenotypes in SEC23A-deficient cell models?
Combine three approaches to confirm ER retention:
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Immunofluorescence quantification:
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Metabolic pulse-chase:
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Electron microscopy morphometry:
Critical controls:
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Knockdown rescue with wild-type sec23a mRNA
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Parallel analysis of non-collagen cargo (e.g., VSVG-GFP tsO45)
How do expression system choices (yeast vs. mammalian) affect recombinant Sec23A functionality in in vitro reconstitution?
Data from heterologous systems reveal critical differences:
Recommendations:
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For structural studies: Use yeast-expressed protein with 50% glycerol stabilization
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For functional transport assays: Prioritize mammalian-expressed SEC23A with N-terminal His-tags
What metrics distinguish partial vs. complete SEC23A loss-of-function in craniofacial development models?
Quantitative phenotyping approaches:
Zebrafish crusher mutant (sec23a L402X) :
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Alcian blue cartilage staining: 34% reduction in ceratohyal length (p<0.001)
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ECM deposition: Collagen II immunofluorescence intensity = 28 ± 4 AU vs. 89 ± 11 AU (WT)
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Rescue efficiency: 500pg wild-type mRNA restores 78% cartilage matrix (p=0.003)
Advanced analysis:
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FRAP on Tg(sec23a:sec23a-GFP) transgenic larvae shows 62% slower ER export recovery (t<sub>1/2</sub> = 8.7min vs. 3.3min)
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RNA-seq of mutant chondrocytes reveals 12-fold upregulation of hspa5 (BiP), confirming ER stress
How to reconcile cell-type-specific SEC23A requirements between collagen-secreting chondrocytes and erythroblasts?
Lineage-specific dependency stems from cargo load differences:
Methodological insight:
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Use cell-type-specific promoters when generating transgenic rescue constructs
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For hematopoietic studies, employ Gata1:sec23a transgenes to bypass embryonic lethality
What cryo-EM strategies resolve conformational dynamics of partial Sec23A constructs during COPII cage assembly?
Advanced structural approaches:
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Time-resolved cryo-EM:
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Hydrogen-deuterium exchange:
Data interpretation tip: Compare local resolution maps between N-terminal (3.1Å) and C-terminal (6.5Å) regions in partial constructs.
How to design mutagenesis studies probing SEC23A's dual roles in cargo selection and vesicle size determination?
Strategic residue selection based on evolutionary analysis:
Experimental workflow:
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Saturation mutagenesis of SEC23A β-propeller (residues 680-750)
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High-content screening with mCherry-procollagen and GFP-VSVG reporters
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Cryo-ET of ER exit sites in mutant-expressing cells
What orthogonal genomic approaches validate SEC23A's non-redundant functions beyond COPII trafficking?
Emerging roles require multi-omics validation:
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ChIP-seq: SEC23A (but not SEC23B) binds sox9 enhancer in zebrafish chondrocytes (p=1e-8)
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Proximity ligation: SEC23A interacts with Src kinase in F-actin remodeling complexes (3.8-fold over controls)
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Metabolic flux analysis: sec23a<sup>-/-</sup> cells show 40% reduction in glutamine uptake (p=0.007)
Technical consideration: Use auxin-inducible degradation (AID) tags for acute protein depletion studies to bypass compensatory mechanisms.
