Recombinant Schizosaccharomyces pombe V-type proton ATPase subunit d (vma6), partial

What is the V-type proton ATPase (V-ATPase) and what role does subunit d (vma6) play in its function?

V-type proton ATPases (V-ATPases) are ATP-dependent proton pumps responsible for acidification of intracellular compartments in eukaryotic cells. These multi-subunit enzyme complexes consist of two primary domains: the cytoplasmic V₁ domain, which hydrolyzes ATP, and the membrane-embedded V₀ domain, which translocates protons across the membrane .

Subunit d, encoded by the vma6 gene in S. pombe, is a critical component of the V₀ domain. It serves as a connecting element between the V₁ and V₀ domains, helping to couple ATP hydrolysis to proton transport. Studies with deletion mutants (vma6Δ) have demonstrated that without subunit d, the V-ATPase fails to properly assemble, and cells exhibit various phenotypes associated with defective vacuolar acidification . The structural importance of subunit d is further evidenced in mammalian systems, where it forms critical interactions with accessory proteins near the cytoplasmic surface of the V₀ region .

Without functional vma6, the proton pumping capability of V-ATPases is compromised, affecting numerous cellular processes including endocytosis, protein sorting, and ion homeostasis. This highlights the essential structural and functional role of vma6 in maintaining proper V-ATPase activity.

What phenotypes are observed in S. pombe cells with vma6 gene disruption?

Disruption of the vma6 gene in S. pombe results in several observable phenotypes consistent with the loss of vacuolar acidification. These mutant phenotypes provide valuable insights into the physiological roles of V-ATPases in fission yeast.

The following table summarizes the key phenotypes observed in vma6-disrupted S. pombe cells:

These phenotypes collectively indicate that V-ATPase activity, which requires the vma6 gene product, is essential for endocytosis, ion and pH homeostasis, intracellular protein targeting, and vacuolar biogenesis in S. pombe . The pleiotropic nature of these defects underscores the fundamental importance of V-ATPase function in numerous cellular processes.

How does the V-ATPase complex structure differ between yeast and mammals?

The V-ATPase complex exhibits both significant similarities and notable differences between yeast (such as S. pombe) and mammals, reflecting evolutionary adaptations to different cellular requirements.

The V-ATPase complex shows these key similarities between yeast and mammals:

• Mammalian V₀ regions are composed of ac₍ₓ₎c''de along with additional accessory proteins ATP6AP1/Ac45 and ATP6AP2/PRR that are absent in yeast V-ATPases

• Mammals express multiple isoforms of several subunits in both V₁ and V₀ domains in a tissue-dependent and cellular compartment-dependent manner, including two isoforms of subunit B, two of C, two of E, three of G, four of a, two of d, and two of e

• Specific interactions occur in mammalian V-ATPases that are not present in yeast, such as the interaction between subunit d1 and the C-terminal tails of ATP6AP1/Ac45 and ATP6AP2/PRR

These structural differences likely reflect adaptations to more complex cellular functions and tissue-specific requirements in mammals. The presence of additional regulatory components in mammalian V-ATPases suggests more sophisticated control mechanisms that may be necessary for specialized functions in different cell types and physiological contexts.

What methods are commonly used to express and purify recombinant vma6 from S. pombe?

Expression and purification of recombinant S. pombe vma6 typically involves several methodological approaches that must address the challenges of protein solubility, folding, and yield. The following outlines established protocols and considerations for successful production of functional vma6 protein.

Expression Systems:

• E. coli expression:

  • Vectors: pET series, pGEX (for GST fusion), pMAL (for MBP fusion)

  • Strains: BL21(DE3), Rosetta, Arctic Express (for difficult-to-express proteins)

  • Induction conditions: IPTG concentration (0.1-1.0 mM), temperature (15-37°C), duration (3-24 hours)

  • Considerations: May require solubility tags for proper folding

• Yeast expression:

  • S. cerevisiae or native S. pombe systems for proper post-translational modifications

  • Vectors: pYES2, pRS series with appropriate promoters

  • Advantages: Native-like environment, proper folding machinery

• Baculovirus-insect cell systems:

  • Suitable for complex eukaryotic proteins requiring specific modifications

  • Higher yield than mammalian systems while maintaining eukaryotic processing

Purification Strategy:

• Initial extraction:

  • PCR amplification of vma6 gene from S. pombe genomic DNA

  • Cell lysis optimization (sonication, French press, enzymatic methods)

  • Buffer composition optimization to maintain protein stability

• Affinity chromatography:

  • His-tag purification using Ni-NTA or TALON resins

  • GST-tag purification using glutathione-sepharose

  • FLAG or HA-tag immunoaffinity approaches

• Additional purification steps:

  • Ion exchange chromatography (based on theoretical pI of vma6)

  • Size exclusion chromatography for final polishing and buffer exchange

  • Tag removal using specific proteases if necessary

Similar approaches have been used successfully for V-ATPase components, as evidenced by protocols employed for epitope tagging and purification of V-ATPase assembly factors and subunits in related research . The choice of expression system and purification strategy should be guided by the intended application, with structural studies generally requiring higher purity achieved through multiple purification steps.

What experimental approaches can be used to study the assembly process of V-ATPase complexes containing the vma6 subunit?

Studying the assembly of V-ATPase complexes containing vma6 requires sophisticated experimental approaches that can capture both spatial and temporal aspects of this multi-step process. Several complementary methodologies can provide comprehensive insights into the assembly mechanisms.

Genetic Approaches:

• Generation of conditional mutants or deletion strains (vma6Δ) to study assembly defects

• Creation of epitope-tagged versions (HA, c-myc) of vma6 and other V-ATPase components for tracking assembly intermediates

• Development of temperature-sensitive mutants to synchronize assembly processes

• Epistasis analysis with assembly factors like Voa1p, an endoplasmic reticulum (ER)-localized integral membrane glycoprotein that functions in V₀ assembly

Biochemical Methods:

• Blue Native PAGE to analyze intact complexes and sub-complexes

• Co-immunoprecipitation with tagged subunits to identify interaction partners during assembly

• Pulse-chase experiments with metabolic labeling to track temporal aspects of assembly

• Glycerol gradient centrifugation to separate assembly intermediates

• Crosslinking followed by mass spectrometry to identify spatial relationships between subunits

Imaging Techniques:

• Fluorescence microscopy with tagged subunits to track localization during assembly

• FRET (Förster Resonance Energy Transfer) to study proximity of subunits during assembly

• Super-resolution microscopy for detailed spatial analysis of assembly intermediates

Structural Approaches:

• Cryo-electron microscopy of partially assembled complexes at different stages

• Hydrogen-deuterium exchange mass spectrometry to probe conformational changes during assembly

Research has successfully employed methods such as epitope tagging of assembly factors and V-ATPase subunits and creation of deletion mutants (e.g., vma6Δ::Kanr) to study assembly. For example, the discovery of Voa1p as a V₀ assembly factor demonstrated how targeted genetic approaches can identify key components of the assembly machinery . Combining multiple approaches provides the most comprehensive understanding of this complex process.

How can researchers analyze the impact of vma6 mutations on vacuolar acidification and endocytosis in S. pombe?

Analysis of vma6 mutations on vacuolar acidification and endocytosis requires specialized techniques to quantitatively assess these processes. The following methodological approaches provide rigorous frameworks for such investigations.

Vacuolar Acidification Assays:

• pH-sensitive fluorescent probes:

  • Quinacrine accumulation in acidic compartments (fluorescence microscopy or flow cytometry)

  • BCECF-AM or pHluorin for ratiometric pH measurements

  • Protocol: Load cells with the probe, measure fluorescence before and after treatment with ionophores/inhibitors

  • Quantification: Compare fluorescence intensity or ratios between wild-type and mutant strains

• Biochemical assays:

  • ATP-dependent proton pumping in isolated vacuolar vesicles

  • Acridine orange quenching assays to measure proton gradient formation

  • Measurement parameters: Initial rate, maximum pH gradient, ATP:H⁺ coupling ratio

Endocytosis Assessment:

• Membrane trafficking:

  • FM4-64 uptake and trafficking assays to track membrane internalization

  • Time-course imaging to distinguish between defects in internalization versus later trafficking steps

  • Quantification: Measure kinetics of dye internalization and delivery to the vacuole

• Fluid-phase endocytosis:

  • Lucifer Yellow CH accumulation assays

  • Flow cytometry or fluorescence microscopy-based quantification

  • Parameters: Rate of uptake, total accumulation, compartmentalization

• Protein trafficking:

  • Tracking of specific cargo proteins like carboxypeptidase Y using fluorescent tags or immunodetection

  • Pulse-chase analysis to follow protein transport kinetics

  • Subcellular fractionation to determine protein localization

Experimental Design Considerations:

• Include appropriate controls: wild-type cells, complemented mutants, other V-ATPase subunit mutants

• Perform time-course experiments to distinguish between kinetic and steady-state defects

• Combine multiple assays to assess different aspects of the same process

• Consider temperature sensitivity by performing assays at both permissive and restrictive temperatures

• Use quantitative image analysis for objective measurement of phenotypes

Research has demonstrated that vma6 disruption strongly inhibits both the delivery of FM4-64 to the vacuolar membrane and the accumulation of Lucifer Yellow CH, indicating that V-ATPase activity is essential for these endocytic processes in S. pombe . These established protocols provide a foundation for analyzing the specific effects of different vma6 mutations on these cellular processes.

What are the structure-function relationships of the vma6 subunit within the V-ATPase complex and how can they be investigated?

The structure-function relationships of vma6 (subunit d) within the V-ATPase complex involve its crucial positioning at the interface between the V₁ and V₀ domains. Investigating these relationships requires integrated structural and functional approaches.

Key Structural Aspects of vma6:

• Forms a critical connection between the V₁ and V₀ domains

• Interacts with both the central rotor components and stationary parts of the complex

• In mammals, the C-terminal tails of accessory proteins (ATP6AP1/Ac45 and ATP6AP2/PRR) interact with subunit d

• These interactions occur near the cytoplasmic surface of the V₀ region, where the C-terminal tail of ATP6AP1/Ac45 and the short C-terminal α-helix of ATP6AP2/PRR are sandwiched between subunits of the c-ring and subunit d1

Investigation Approaches:

• Mutational Analysis:

  • Targeted mutagenesis of conserved residues based on sequence alignments

  • Alanine-scanning mutagenesis of surface-exposed regions

  • Creation of chimeric proteins between species to map functional domains

  • Truncation analysis to identify essential regions for interaction and function

• Structural Studies:

  • Cryo-electron microscopy of full V-ATPase complexes at different functional states

  • X-ray crystallography of isolated subunit d or d-containing subcomplexes

  • Computational modeling and molecular dynamics simulations to predict dynamic behaviors

  • Structure-guided crosslinking to validate predicted interactions

• Functional Correlation:

  • Proton pumping assays with reconstituted systems containing mutant forms

  • ATP hydrolysis measurements to assess coupling efficiency

  • Growth complementation studies in deletion strains with mutated versions

  • pH homeostasis assays to link structure to physiological function

• Interaction Mapping:

  • Co-immunoprecipitation with tagged vma6 variants

  • Yeast two-hybrid or split-ubiquitin assays to map interaction domains

  • Surface plasmon resonance to measure binding affinities with partner proteins

  • Hydrogen-deuterium exchange mass spectrometry to identify interaction surfaces

Recent structural studies of V-ATPases have revealed important details about subunit organization and interactions . For example, the interaction between mammalian subunit d1 and the accessory proteins ATP6AP1/Ac45 and ATP6AP2/PRR suggests additional regulatory roles beyond the basic structural function observed in simpler eukaryotes like yeast . These findings provide a foundation for targeted structure-function studies of vma6 and its homologs.

How can researchers evaluate the impact of vma6 mutations on ion homeostasis and pH regulation in S. pombe?

Evaluating the impact of vma6 mutations on ion homeostasis and pH regulation requires a multi-faceted experimental approach that addresses both cellular phenotypes and underlying molecular mechanisms.

Methods for Assessing pH Homeostasis:

• Growth phenotyping:

  • pH sensitivity assays across a range of external pH values (pH 4.0-8.0)

  • Protocol: Serial dilution spot tests on media buffered to different pH values

  • Quantification: Growth rate determination, survival percentage, colony size

• Intracellular pH measurements:

  • Ratiometric measurement using pH-sensitive fluorescent probes

  • pH recovery kinetics following acid loading or alkalinization

  • Compartment-specific pH using targeted indicators

  • Microelectrode measurements for direct pH determination

• Gene expression analysis:

  • Transcriptional profiling of pH-responsive genes

  • qRT-PCR validation of selected pH-regulated transcripts

  • Reporter constructs for monitoring pH-dependent promoter activity

Methods for Assessing Ion Homeostasis:

• Cation sensitivity testing:

  • Growth assays with varying concentrations of divalent cations (Ca²⁺, Zn²⁺, Fe²⁺, Mn²⁺)

  • Generation of dose-response curves for different metal ions

  • Combinatorial testing with pH variation and ion stress

• Ion content measurement:

  • Direct measurement of cellular ion content using atomic absorption spectroscopy

  • Inductively coupled plasma mass spectrometry (ICP-MS) for multi-element analysis

  • Subcellular fractionation to determine ion distribution across compartments

• Ion flux and localization:

  • Ion-specific fluorescent probes for real-time monitoring

  • Example: Fura-2 for Ca²⁺ measurements, FluoZin-3 for Zn²⁺

  • Time-resolved imaging to capture dynamic responses to stimuli

Data Analysis Framework:

The following table outlines an analytical approach for integrating multiple experimental readouts:

Based on the search results, vma6 deletion mutants show sensitivity to neutral pH and high concentrations of divalent cations including Ca²⁺, indicating that V-ATPase activity is essential for ion and pH homeostasis in S. pombe . These established phenotypes provide valuable baseline data for comparative analysis of specific vma6 mutations.

What high-resolution structural techniques can be applied to study recombinant vma6 and its interactions within the V-ATPase complex?

High-resolution structural techniques are essential for understanding the molecular details of how vma6 functions within the V-ATPase complex. Multiple complementary approaches can provide comprehensive structural insights.

Cryo-electron Microscopy (cryo-EM):

• Single-particle analysis for structure determination at near-atomic resolution

• Advantages: Works with heterogeneous samples, no crystallization required, captures different conformational states

• Application to V-ATPase: Recent cryo-EM studies have successfully determined structures of V-ATPases from various sources, revealing critical details about subunit arrangement

• Processing strategy: Classification approaches to sort conformational heterogeneity

• Resolution enhancement: Use of direct electron detectors and motion correction software

• Sample preparation: Optimization of buffer conditions, use of detergents or nanodiscs for membrane proteins

X-ray Crystallography:

• Crystallization of isolated vma6 or subcomplexes containing vma6

• Advantages: Potentially atomic resolution, well-established phase determination methods

• Challenges: Obtaining well-diffracting crystals of membrane-associated proteins

• Strategies: Surface entropy reduction, use of crystallization chaperones, lipidic cubic phase crystallization

• Data collection optimization: Synchrotron radiation, microfocus beamlines for small crystals

Nuclear Magnetic Resonance (NMR):

• Solution NMR of isotopically labeled domains of vma6

• Solid-state NMR for membrane-associated regions

• Advantages: Dynamic information, solution-state measurements, binding interface mapping

• Applications: Structure determination of smaller domains, measurement of dynamics, interaction mapping

Integrative Structural Biology Approaches:

Technical Considerations and Recent Advances:

Recent structural studies using cryo-EM have been particularly successful for V-ATPases, revealing how subunit d1 (the mammalian homolog of vma6) interacts with other components of the complex, including accessory proteins at the interface between the V₁ and V₀ domains . These structures provide valuable templates for investigating vma6-specific interactions and conformational states.