Recombinant Saccharomyces cerevisiae Plasma membrane-associated coenzyme Q6 reductase PGA3 (PGA3)
Product Specs
Note: While we prioritize shipping the format currently in stock, please specify your format preference in order notes for customized preparation.
Note: Standard shipping includes blue ice packs. Dry ice shipping requires prior arrangement and incurs additional charges.
Generally, liquid formulations have a 6-month shelf life at -20°C/-80°C, while lyophilized forms have a 12-month shelf life at -20°C/-80°C.
The tag type is finalized during production. If you require a specific tag, please inform us, and we will prioritize its development.
Target Background
KEGG: sce:YML125C
STRING: 4932.YML125C
Q&A
What is the primary biochemical role of PGA3 in Saccharomyces cerevisiae plasma membranes?
Methodological Answer:
PGA3 functions as a critical component of the plasma membrane electron transport system, primarily mediating NADH-driven reduction of coenzyme Q6 (CoQ6) to maintain extracellular ascorbate stabilization. This activity was confirmed through comparative studies using coq3Δ mutants (defective in CoQ6 biosynthesis) and wild-type strains. Researchers employ NADH-ascorbate free radical reductase assays with inhibitors like chloroquine/dicumarol to isolate PGA3-specific activity . For validation:
-
Measure CoQ6-dependent electron transfer using ferricyanide/cytochrome c as alternate acceptors
-
Compare activity in atp2Δ (respiration-deficient but Q6-competent) vs. coq3Δ mutants
-
Use detergent solubilization (Zwittergent 3-14) to confirm membrane association
How do researchers confirm CoQ6 dependence in PGA3-mediated redox systems?
Methodological Answer:
Genetic complementation and exogenous Q6 supplementation are key strategies:
-
Transform coq3Δ mutants with plasmid-borne COQ3 to rescue Q6 biosynthesis
-
Cultivate mutants in Q6-supplemented media and quantify membrane Q6 via HPLC-ECD
-
Compare redox activity recovery in mitochondria vs. plasma membrane fractions
Data Contradiction Analysis:
While Q6 restoration rescues ascorbate reductase activity in plasma membranes, mitochondrial Q6 uptake in EG103 strains remains impaired due to endocytic trafficking defects . This highlights compartment-specific Q6 trafficking mechanisms.
What experimental approaches resolve conflicting data on PGA3’s role in superoxide-dependent vs. independent pathways?
Methodological Answer:
Studies show PGA3 operates via two distinct mechanisms:
-
CoQ6-dependent pathway: Blocked by quinone antagonists (dicumarol) and absent in coq3Δ mutants
-
Iron-regulated pathway: Activated in low-iron conditions, independent of Q6 but requiring ferric reductase
Key experimental designs:
-
Use SOD (superoxide dismutase) to test superoxide involvement
-
Culture strains in iron-depleted media (+ bathophenanthroline) to induce ferric reductase activity
-
Quantify FM4-64 uptake defects in erg2/coq3Δ double mutants to link endocytosis to Q6 trafficking
How do researchers address challenges in detecting PGA3 activity in recombinant systems?
Methodological Answer:
Common pitfalls include improper membrane fractionation and interference from mitochondrial homologs. Solutions involve:
-
Differential centrifugation with sucrose gradients to isolate pure plasma membranes
-
Use atp2Δ or cor1Δ controls to exclude respiratory chain artifacts
-
Fluorescence quenching assays with resorufin-NADH to quantify redox coupling
Critical Data Table:
| Strain | CoQ6 (nmol/mg) | NADH-AFR Activity (Units) | SOD Sensitivity |
|---|---|---|---|
| Wild-type | 12.3 ± 0.8 | 45.2 ± 3.1 | No |
| coq3Δ | 0.0 | 4.7 ± 0.9 | No |
| coq3Δ + Q6 | 9.1 ± 1.2 | 38.6 ± 2.7 | No |
| atp2Δ | 11.8 ± 0.7 | 42.1 ± 2.9 | Yes |
| Data synthesized from |
What genetic tools enable functional analysis of PGA3 in engineered yeast strains?
Methodological Answer:
Advanced CRISPR-Cas9 systems (e.g., pCEC-red) allow marker-free editing:
-
Design gRNAs targeting PGA3 loci using Golden Gate Assembly for high-efficiency cloning
-
Use homology-directed repair with Q6 biosynthetic genes (COQ3, COQ7) for pathway analysis
-
Validate edits via colony PCR and LC-MS quantification of Q6 species
Example Workflow:
-
Transform with plasmid overexpressing COQ3 under inducible promoter
-
Profile Q6 distribution in Golgi/vacuole fractions via subcellular fractionation
How do researchers reconcile discrepancies in PGU1 gene presence vs. PGA3 activity?
Methodological Answer:
Some S. cerevisiae strains retain PGU1 (polygalacturonase) homologs but lack PGA3 activity due to:
-
Post-translational modifications (assess via zymogram assays)
-
Epigenetic silencing (use chromatin immunoprecipitation for histone marks)
Contradiction Resolution:
Blanco et al. reported conserved PGU1 in all strains, but recent data show 28% of wine yeasts lack active PGA3 despite gene presence . This suggests strain-specific regulatory elements upstream of PGA3.
Technical Notes for Experimental Design
-
Critical Controls: Always include coq3Δ + vector control and atp2Δ (respiratory-deficient) strains to isolate plasma membrane-specific effects
-
Activity Assays: Use 100 μM NADH + 50 μM ascorbate free radical (pH 6.0) for optimal PGA3 activity measurement
-
Inhibitor Concentrations: 10 μM chloroquine (Q6 antagonist); 50 U/mL SOD (superoxide scavenger)
