Recombinant Saccharomyces cerevisiae Coatomer subunit gamma (SEC21), partial
Description
Molecular Identity and Functional Role of SEC21
SEC21 encodes the γ-subunit of the COPI coatomer, a heptameric complex (α, β, β', γ, δ, ε, ζ) crucial for vesicle formation and cargo sorting . The COPI coat, in conjunction with Arf1p, facilitates retrograde transport of proteins containing dilysine (KKXX) motifs from the Golgi to the ER .
Key domains:
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The γ-COP subunit (Sec21p) interacts with dilysine motifs via its N-terminal domain .
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Structural studies indicate that γ-COP forms part of the F-subcomplex (γ-COP, δ-COP, ζ-COP), which binds cargo, while the B-subcomplex (α-COP, β'-COP, ε-COP) mediates coatomer assembly .
Experimental Use of Recombinant SEC21
Recombinant SEC21 variants have been engineered to study COPI dynamics. Examples include:
Tagged Constructs
Temperature-Sensitive Mutants
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sec21-3: Exhibits cargo-specific anterograde transport defects at restrictive temperatures. Suppressed by overexpression of Mst27p, a COPI-binding protein .
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sec21-1 and ret1-1 (α-COP): Disrupted dilysine motif binding, impairing retrograde transport .
Methotrexate Resistance
A sec21 allele (S96L) conferred methotrexate resistance in yeast, suggesting a novel role for COPI in stress response .
Interactome and Regulatory Partners
SEC21 interacts with:
Notable pathways:
Applications in Biotechnology
Partial recombinant SEC21 constructs are used to:
Product Specs
Target Background
KEGG: sce:YNL287W
STRING: 4932.YNL287W
Q&A
What is the functional role of SEC21 in Saccharomyces cerevisiae intracellular trafficking?
SEC21 (encoded by the SEC21 gene) is the γ-subunit of the COPI coatomer complex, which mediates retrograde vesicular transport from the Golgi apparatus to the endoplasmic reticulum (ER) . Methodologically, its role can be validated through:
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Gene deletion analysis: Haploid sec21Δ mutants are inviable, necessitating conditional knockdown systems (e.g., galactose-inducible promoters) .
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Localization studies: Immunofluorescence microscopy using anti-Sec21p antibodies reveals punctate Golgi-associated staining .
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Biochemical assays: Co-immunoprecipitation with COPI subunits (Sec26p, Sec27p) confirms complex integrity .
How should researchers produce recombinant SEC21 with partial sequences in yeast?
Partial SEC21 constructs (e.g., truncations lacking the C-terminal WD40 domain) require:
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Vector design: Use high-copy plasmids (e.g., pYES2/CT) with inducible promoters (GAL1) to avoid cytotoxicity .
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Epitope tagging: Incorporate a C-terminal 6×His or HA tag for purification and detection .
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Expression optimization: Induce cultures at OD600 = 0.8 with 2% galactose for 6–8 hours at 30°C .
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Purification: Ni-NTA affinity chromatography under denaturing conditions (8 M urea, pH 8.0) due to the protein’s insolubility .
Table 1: Troubleshooting SEC21 Recombinant Expression
What standard assays validate SEC21’s structural integrity and activity?
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Circular dichroism (CD): Compare spectra of recombinant SEC21 against wild-type yeast lysates to confirm secondary structure retention .
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ATPase activity: Measure phosphate release using malachite green assays (baseline activity: 0.8–1.2 µmol/min/mg) .
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Binding assays: Surface plasmon resonance (SPR) quantifies interaction kinetics with COPI subunits (e.g., Kd = 120 nM for Sec26p) .
How does the sec21(S96L) mutation confer methotrexate resistance, and how is this studied?
The sec21(S96L) allele (serine → leucine at position 96) causes recessive, neomorphic methotrexate (MTX) resistance . Key methodologies include:
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Genetic complementation: Transform mutants with wild-type SEC21 on a MoBY plasmid . Monitor MTX sensitivity in SC-Ura + 2 mM MTX .
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Transcriptomic profiling: RNA-seq of sec21(S96L) strains reveals upregulation of folate biosynthesis genes (e.g., FOL1, FOL2) .
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Mitochondrial linkage: Assess ρ0 status via PCR for COX2 and growth on YP-glycerol .
Table 2: Phenotypic Comparison of sec21 Alleles
| Parameter | Wild-Type SEC21 | sec21(S96L) |
|---|---|---|
| MTX IC50 (mM) | 0.5 | 2.1 |
| Growth on YP-glycerol | + | - (ρ0 phenotype) |
| Sed5p binding affinity | High (Kd = 95 nM) | Reduced (Kd = 420 nM) |
How can researchers resolve contradictions in SEC21’s reported roles in anterograde vs. retrograde transport?
Discrepancies arise from context-dependent interactions:
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Epistatic analysis: Combine sec21 mutants with sed5ts (anterograde t-SNARE). Double mutants exhibit synthetic lethality, confirming parallel pathways .
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Vesicle budding assays: Isolate Golgi-enriched fractions and quantify COPII (anterograde) vs. COPI (retrograde) vesicles via anti-Sec13p/Sec21p immunoblotting .
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Live-cell imaging: Track GFP-tagged carboxypeptidase Y (CPY) in vti1Δ + sec21(S96L) strains. Delayed CPY trafficking indicates compensatory mechanisms .
What advanced strategies identify SEC21’s non-canonical binding partners?
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Cross-linking mass spectrometry: Treat yeast lysates with 1% formaldehyde, immunoprecipitate SEC21, and identify interactors via LC-MS/MS .
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Synthetic genetic array (SGA): Screen for synthetic sick/lethal interactions with sec21ts alleles. Overlap with COPI-unrelated pathways (e.g., YKT6) suggests moonlighting roles .
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Cryo-EM with nanodiscs: Reconstitute SEC21 into lipid nanodiscs for structural studies of membrane-proximal conformations .
How should researchers optimize SEC21 mutant studies given its essentiality?
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Conditional promoters: Use GAL1-driven SEC21 in a sec21Δ background. Repress with 2% glucose to deplete native protein .
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Diploid shuffle: Introduce mutant alleles on a URA3 plasmid in a heterozygous SEC21/sec21Δ strain. Counterselect with 5-FOA .
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CRISPR-dCas9 modulation: Titrate SEC21 expression using guide RNAs targeting the SEC21 promoter .
What statistical approaches address variability in SEC21 interaction studies?
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Error-weighted regression: Model SPR data with a modified Hill equation to account for COPI complex cooperativity .
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Bootstrapping: Resample co-IP/MS data 1,000× to calculate confidence intervals for low-abundance interactors .
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Bayesian networks: Integrate genetic (SGA) and proteomic (AP-MS) data to infer high-confidence interaction networks .
