Recombinant Saccharomyces cerevisiae V-type proton ATPase subunit e (VMA9)
Description
Molecular Structure and Classification
Recombinant Saccharomyces cerevisiae V-type proton ATPase subunit e (VMA9) is a 73-amino acid hydrophobic protein encoded by the VMA9 gene (GenBank ID: NM_001184518.1, UniProt ID: Q3E7B6) . It belongs to the V0 subcomplex of the vacuolar-type ATPase (V-ATPase), a proton-translocating enzyme critical for acidifying intracellular compartments like vacuoles and endosomes .
Functional Role in Yeast V-ATPase
VMA9 is an integral membrane subunit of the V0 sector, essential for:
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V0 assembly: Disruption of VMA9 prevents proper localization of V1 and V0 subunits to vacuolar membranes .
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Proton translocation: While in vitro studies suggest VMA9 is dispensable for proton pumping after detergent solubilization , its absence in vivo impairs vacuolar acidification .
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Interaction with assembly factors: Binds to Vma21p (an assembly chaperone) in the ER, stabilizing V0 subunits like Vph1p and Vma6p .
Recombinant Production and Applications
Recombinant VMA9 is commercially produced as a His-tagged protein (N-terminal) for structural and functional studies .
Physiological Relevance
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Vacuolar acidification: vma9Δ mutants exhibit defective vacuole acidification, leading to sensitivity to high pH, calcium, and oxidants .
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Hop iso-α-acid resistance: VMA9-dependent V-ATPase activity is required for vacuolar sequestration of hop acids in brewing yeast .
In Vitro Activity
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Proton pumping: Purified V-ATPase lacking VMA9 retains full activity in vitro, suggesting its role may be primarily structural during assembly .
Interactome and Functional Partners
VMA9 interacts with core V-ATPase subunits and assembly factors:
Challenges and Future Directions
Product Specs
Note: We prioritize shipping the format currently in stock. However, if you have specific format requirements, please specify them in your order. We will strive to fulfill your request.
Note: All protein shipments are standardly packed with blue ice packs. If you require dry ice packaging, please inform us in advance, as additional charges may apply.
Generally, the shelf life of liquid formulations is 6 months at -20°C/-80°C. Lyophilized formulations typically have a shelf life of 12 months at -20°C/-80°C.
Target Background
- Vma9p is an integral membrane protein, synthesized and inserted into the endoplasmic reticulum (ER). It subsequently localizes to the limiting membrane of the vacuole. PMID: 16569636
KEGG: sce:YCL005W-A
STRING: 4932.YCL005W-A
Q&A
What is the functional role of VMA9 in the V-ATPase complex, and how is this validated experimentally?
VMA9 encodes subunit e of the V-ATPase V0 sector, essential for proton translocation across membranes. Key functional validation methods include:
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Gene deletion studies: Δvma9 yeast strains show defective vacuolar acidification, confirmed via pH-sensitive fluorescent dyes like quinacrine .
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Co-immunoprecipitation assays: Demonstrates physical interaction with other V0 subunits (e.g., Vph1p) .
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Subcellular localization tracking: GFP-tagged Vma9p localizes to vacuolar membranes, validated by confocal microscopy .
Table 1: Core Functional Attributes of VMA9
| Property | Value/Observation | Source |
|---|---|---|
| Gene ID | 2732686 | |
| Protein mass | ~9 kDa (predicted) | |
| Localization | Vacuolar membrane | |
| Interaction partners | Vph1p, Vma21p |
What experimental designs are optimal for expressing recombinant VMA9 in heterologous systems?
Critical factors for recombinant expression:
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Vector selection: Use yeast-compatible vectors (e.g., pYES2/CT) with inducible promoters (GAL1) to avoid toxicity .
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Tag placement: N-terminal tags (e.g., 6xHis) minimize interference with transmembrane domains .
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Host strain optimization: Use Δvma9 Saccharomyces cerevisiae to prevent endogenous interference .
Methodological Note:
Co-expression with chaperones (e.g., Kar2p) improves solubility in Pichia pastoris systems . Quantify proton transport activity using inverted membrane vesicles and ATP hydrolysis assays .
How do structural variations in VMA9 isoforms affect V-ATPase assembly across species?
Comparative structural analysis reveals:
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Conserved motifs: HEAT/armadillo repeats in VMA9 homologs (e.g., AGOS_AAL005W in Eremothecium gossypii) mediate regulatory interactions .
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Species-specific adaptations: Mammalian isoforms exhibit tissue-specific N-terminal extensions absent in yeast, altering regulatory binding sites .
Data Contradiction Analysis:
While early studies proposed VMA9 as a passive structural component , cryo-EM data shows conformational flexibility during proton translocation, suggesting dynamic regulatory roles . Resolve discrepancies by combining mutagenesis (e.g., truncating the N-terminal autoinhibitory domain) with single-molecule ATPase activity assays .
What strategies mitigate genomic instability in VMA9-knockout strains during long-term studies?
Δvma9 strains exhibit elevated recombination rates (5× baseline) due to defective pH homeostasis . Mitigation approaches:
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Checkpoint activation: RAD9-dependent G2/M arrest reduces nonreciprocal translocations by 90% .
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Medium supplementation: Buffering at pH 6.5 restores near-wild-type growth rates .
Table 2: Genomic Stability Parameters in Δvma9 Strains
| Condition | Recombination Rate (His+ events/10^6 cells) | Karyotypic Abnormalities |
|---|---|---|
| Unbuffered | 12.7 ± 1.4 | 68% |
| pH 6.5 buffered | 2.1 ± 0.3 | 12% |
| RAD9+ background | 1.8 ± 0.2 | <5% |
| Data synthesized from |
How can conflicting data on VMA9’s role in V-ATPase disassembly be reconciled?
Two dominant models exist:
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Static subunit model: VMA9 remains V0-integrated during glucose starvation-induced disassembly .
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Dynamic release model: Partial VMA9 dissociation alters proton pore conformation .
Resolution strategy:
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Pulse-chase SILAC labeling: Track subunit turnover kinetics under disassembly conditions.
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Hydrogen-deuterium exchange mass spectrometry: Maps conformational changes in V0 during ATPase inactivation .
What proteomic techniques resolve VMA9’s low-abundance membrane protein interactions?
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Crosslinking MS: Use membrane-permeable DSSO crosslinkers to stabilize transient interactions .
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Nanodisc reconstitution: Embed V0 complexes in lipid nanodiscs for native-state surface plasmon resonance analysis .
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Limited proteolysis: Identifies solvent-exposed regions via differential trypsin susceptibility .
How are in silico models of VMA9 validated against experimental data?
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Molecular dynamics simulations: Compare predicted vs. observed B-factors from X-ray crystallography (PDB 1HJO) .
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Free energy calculations: Validate proton translocation pathways using experimental ΔpH measurements .
Can engineered VMA9 variants enhance recombinant vaccine platforms?
Phase I trials of yeast-based vaccines (e.g., GI-4000 series) demonstrate:
