Recombinant Schizosaccharomyces pombe V-type proton ATPase subunit e (vma9)

What is the function of V-type proton ATPase subunit e (vma9) in Schizosaccharomyces pombe?

The V-type proton ATPase subunit e (vma9) is an essential component of the vacuolar H+-ATPase (V-ATPase) complex in S. pombe. This complex functions as an ATP-dependent proton pump responsible for acidification of intracellular compartments in eukaryotic cells. The vma9 subunit contributes to the structural integrity and functional assembly of the V0 domain of the V-ATPase complex, which is embedded in the membrane and forms the proton-conducting channel. Research with other V-ATPase subunits in S. pombe has demonstrated that the complex is critical for endocytosis, ion and pH homeostasis, and intracellular targeting of vacuolar proteins and vacuolar biogenesis .

How does vma9 deletion affect cellular phenotypes in S. pombe?

While specific vma9 deletion data is not directly presented in the search results, studies of other V-ATPase subunits provide insight into likely phenotypes. Deletion of V-ATPase subunits in S. pombe, such as vma1 (encoding subunit A) or vma3 (encoding subunit c), results in multiple phenotypic defects:

• Loss of vacuolar acidification capacity in vivo

• Increased sensitivity to neutral pH environments

• Hypersensitivity to high concentrations of divalent cations, including Ca²⁺

• Impaired endocytosis (inhibited delivery of FM4-64 to vacuolar membrane and reduced accumulation of Lucifer Yellow CH)

• Missorting of vacuolar carboxypeptidase Y

• Abnormal vacuole morphology

• Mating defects

As vma9 is part of the same complex, similar phenotypes would be expected upon its deletion, though potentially with varying severity depending on its specific role in the complex assembly and function.

What expression systems are optimal for producing recombinant S. pombe vma9?

Based on established protocols for similar S. pombe proteins, E. coli expression systems have proven effective for recombinant production. The methodology typically involves:

• Cloning the vma9 gene into an expression vector containing an N-terminal or C-terminal His-tag for purification purposes

• Transforming the construct into an appropriate E. coli strain (BL21(DE3) or similar)

• Inducing protein expression with IPTG under optimized temperature conditions (typically 16-25°C to enhance solubility)

• Lysing cells and purifying the protein via immobilized metal affinity chromatography (IMAC)

This approach has been successfully employed for other S. pombe proteins, such as ATP synthase subunit 9 (atp9), which was expressed in E. coli with an N-terminal His-tag . The recombinant protein can be produced in sufficient quantities for biochemical and structural studies while maintaining proper folding.

What protocols are recommended for the purification and storage of recombinant vma9?

The following purification and storage protocol is recommended based on established methods for similar S. pombe proteins:

Purification Protocol:

• Harvest E. coli cells expressing His-tagged vma9 by centrifugation (5,000 × g, 15 min, 4°C)

• Resuspend cell pellet in lysis buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, 1 mM PMSF, protease inhibitor cocktail)

• Lyse cells by sonication or pressure-based methods

• Clarify lysate by centrifugation (20,000 × g, 30 min, 4°C)

• Apply supernatant to Ni-NTA resin pre-equilibrated with lysis buffer

• Wash with increasing imidazole concentrations (20-50 mM)

• Elute protein with elution buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 250 mM imidazole)

• Perform size exclusion chromatography for further purification if needed

Storage Conditions:

• Formulate in Tris/PBS-based buffer containing 6% trehalose, pH 8.0

• Store at -20°C/-80°C after adding glycerol to a final concentration of 50%

• Avoid repeated freeze-thaw cycles

• For short-term storage, keep working aliquots at 4°C for up to one week

What analytical techniques are most effective for characterizing recombinant vma9?

Several analytical techniques have proven effective for characterizing recombinant V-ATPase subunits:

Protein Purity and Identity Assessment:

• SDS-PAGE: To verify protein purity (>90% purity is typically achievable)

• Western blot: For specific detection using anti-His antibodies or custom antibodies against vma9

• Mass spectrometry: For precise molecular weight determination and peptide mapping

Structural Characterization:

• Circular dichroism (CD): To assess secondary structure elements

• Dynamic light scattering (DLS): To evaluate protein homogeneity and detect aggregation

• X-ray crystallography or cryo-EM: For high-resolution structural studies of the V-ATPase complex

Functional Analysis:

• ATPase activity assays: To measure ATP hydrolysis capacity when assembled with other V-ATPase subunits

• Proton translocation assays: Using fluorescent pH indicators in reconstituted liposomes

• Protein-protein interaction studies: Using yeast two-hybrid, pull-down assays, or surface plasmon resonance to identify binding partners

How can recombinant vma9 be used to study V-ATPase assembly in vitro?

Recombinant vma9 provides a valuable tool for investigating V-ATPase assembly mechanisms through several experimental approaches:

Reconstitution Experiments:

• Express and purify multiple recombinant V-ATPase subunits individually

• Combine purified subunits in controlled ratios under various buffer conditions

• Monitor complex assembly using native PAGE, analytical ultracentrifugation, or cryo-EM

• Test the functional activity of reconstituted complexes through ATPase activity assays

Structure-Function Analysis:

• Generate site-directed mutants of vma9 targeting conserved residues

• Assess the ability of mutant proteins to assemble with other V-ATPase subunits

• Evaluate the functional consequences of mutations on proton pumping activity

• Correlate structural alterations with functional defects

Cross-linking Studies:

• Use chemical cross-linkers to capture protein-protein interactions during assembly

• Identify interaction interfaces by mass spectrometry analysis of cross-linked products

• Map the temporal sequence of assembly events by performing time-course experiments

What genetic approaches can be used to study vma9 function in S. pombe?

Several genetic approaches have been established for studying V-ATPase subunits in S. pombe:

Gene Disruption and Replacement:

• Generate vma9 deletion strains using homologous recombination techniques

• Create point mutations using site-directed mutagenesis

• Reintroduce wild-type or mutant vma9 alleles to assess complementation

• Analyze resulting phenotypes related to vacuolar function, stress resistance, and cell growth

Fluorescent Tagging for Localization:

• Generate C-terminal or N-terminal GFP fusions of vma9

• Observe subcellular localization under various conditions

• Track dynamic changes in localization during cell cycle or in response to stress

• Perform co-localization studies with other V-ATPase subunits

Genetic Interaction Studies:

• Perform synthetic genetic array (SGA) analysis with vma9 mutants

• Identify genetic interactions by creating double mutants with other genes of interest

• Map functional relationships within the V-ATPase complex and related pathways

• Quantify genetic interaction strengths to build functional interaction networks

How does vma9 function relate to drug sensitivity and resistance in S. pombe?

V-ATPase function has been implicated in drug sensitivity patterns in S. pombe, suggesting vma9 may play an important role in this context:

Mechanisms of Drug Sensitivity:

• Disruption of vacuolar acidification through V-ATPase mutations can alter intracellular pH gradients

• Changes in pH homeostasis may affect drug accumulation and distribution within cells

• Mutations in genes regulating V-ATPase function (e.g., rav1) result in sensitivity to structurally unrelated compounds including clinical drugs like doxorubicin

• Drug accumulation studies show increased intracellular concentration of fluorescent compounds like doxorubicin in V-ATPase regulatory mutants

Research Applications:

• Use vma9 mutants to screen for compounds that selectively target cells with altered vacuolar function

• Employ fluorescent drug analogs to track intracellular distribution in wild-type versus vma9 mutant cells

• Investigate the mechanism by which V-ATPase dysfunction leads to multidrug sensitivity

• Explore potential synergies between V-ATPase inhibitors and other therapeutic agents

How does S. pombe vma9 compare to orthologs in other model organisms?

Comparative analysis of V-ATPase subunit e across species reveals both conserved and divergent features:

The V-ATPase subunit e is generally less conserved at the sequence level compared to catalytic subunits like subunit A (vma1), but maintains functional conservation across species. The protein typically features a single transmembrane domain and plays crucial roles in V-ATPase assembly and stability rather than directly participating in catalysis.

What structural and functional differences exist between vma9 and other V-ATPase subunits?

V-ATPase is a multi-subunit complex composed of two main domains (V₁ and V₀) with distinct functions. Subunit e (vma9) has specific characteristics compared to other subunits:

Structural Position:

• vma9 (subunit e) is a component of the V₀ domain, which is embedded in the membrane

• Unlike larger subunits (A, B) of the V₁ domain that participate in ATP hydrolysis, vma9 is not directly involved in catalysis

• vma9 is one of the smaller subunits, primarily serving structural and regulatory roles

Functional Roles:

What are common challenges in working with recombinant vma9 and how can they be addressed?

Researchers often encounter several challenges when working with small membrane proteins like vma9:

Low Expression Yields:

• Problem: Small membrane proteins often express poorly in heterologous systems

• Solution: Optimize codon usage for E. coli, use specialized strains (C41/C43), lower induction temperature (16°C), or try fusion partners (MBP, SUMO)

Protein Solubility Issues:

• Problem: Tendency to form aggregates or inclusion bodies

• Solution: Express with solubility-enhancing tags, use mild detergents (DDM, LMNG) during extraction, optimize buffer conditions (add glycerol, adjust salt concentration)

Protein Instability:

• Problem: Rapid degradation during purification or storage

• Solution: Include protease inhibitors throughout purification, minimize handling time, store in optimized buffer with stabilizing agents (e.g., 6% trehalose)

Functional Assay Limitations:

• Problem: Challenging to assess functionality of isolated subunit

• Solution: Develop reconstitution assays with other V-ATPase components, measure specific protein-protein interactions, or use complementation in yeast vma9 mutants

How can researchers troubleshoot experiments involving vma9 mutants in S. pombe?

When working with vma9 mutants in S. pombe, several experimental challenges may arise:

Growth Defect Severity:

• Problem: Extreme growth defects making strain maintenance difficult

• Solution: Use conditional promoters or temperature-sensitive alleles to maintain strains under permissive conditions

Phenotype Verification:

• Problem: Confirming that observed phenotypes are directly due to vma9 disruption

• Solution: Perform genetic complementation with wild-type vma9, create point mutants rather than complete deletions, use multiple independent mutant isolates

Vacuole Visualization:

• Problem: Difficulties in assessing vacuolar morphology changes

• Solution: Use multiple vacuolar stains (FM4-64, CMAC), optimize staining protocols for mutants with altered endocytic capacity

pH Measurement Challenges:

• Problem: Quantifying changes in vacuolar pH in vma9 mutants

• Solution: Use ratiometric pH-sensitive fluorescent proteins targeted to vacuoles, calibrate with ionophores, employ plate reader assays for population measurements

What emerging technologies might enhance our understanding of vma9 function?

Several cutting-edge technologies offer promising avenues for deeper insights into vma9 function:

Cryo-Electron Microscopy:

• High-resolution structural analysis of the entire V-ATPase complex

• Visualization of conformational changes during the catalytic cycle

• Mapping vma9 interactions within the complex architecture

CRISPR-Cas9 Genome Editing:

• Precise introduction of mutations to study structure-function relationships

• Creation of conditional alleles for essential functions

• Generation of tagged versions at the endogenous locus for more physiological studies

Single-Molecule Biophysics:

• Direct observation of V-ATPase assembly process

• Measurement of proton pumping at the single-molecule level

• Analysis of rotational dynamics of the V₀ and V₁ domains

Integrative Multi-Omics Approaches:

• Combining proteomics, metabolomics, and transcriptomics to understand broader impacts of vma9 dysfunction

• Systems-level analysis of cellular responses to V-ATPase perturbation

• Network modeling of V-ATPase regulation in different cellular contexts

How might recombinant vma9 contribute to understanding broader aspects of cellular physiology?

Research using recombinant vma9 has implications for understanding several important biological processes:

Membrane Trafficking and Organelle Homeostasis:

• Investigation of how V-ATPase activity coordinates with membrane fusion/fission machinery

• Understanding organelle identity maintenance through pH regulation

• Exploring the role of compartment acidification in protein sorting and quality control

Cellular Stress Responses:

• Examination of how V-ATPase function interfaces with stress response pathways

• Investigation of vma9's role in cellular adaptation to environmental challenges

• Understanding how V-ATPase activity is regulated during different metabolic states

Drug Resistance Mechanisms:

• Exploration of connections between vacuolar pH and multidrug resistance

• Development of strategies to overcome drug resistance by targeting V-ATPase function

• Investigation of V-ATPase as a potential therapeutic target