Recombinant V-type proton ATPase catalytic subunit A, partial

What is the V-type proton ATPase catalytic subunit A and what is its role in the V-ATPase complex?

The V-type proton ATPase catalytic subunit A is a critical component of the V1 domain of V-ATPase complexes, responsible for ATP hydrolysis. V-ATPases are rotary motor enzymes that consist of two major sectors: a cytosolic V1-ATPase that catalyzes ATP hydrolysis and a membrane-embedded V0 proton channel that translocates protons across membranes, resulting in acidification of its resident organelle .

Subunit A contains the catalytic site involved in ATP binding and hydrolysis. It is present in three copies per complex (A3), forming alternating interfaces with three B subunits (B3) that together constitute the catalytic core of the enzyme . These AB pairs adopt three different conformations during the catalytic cycle, described as the ATP-bound, ADP-bound (post-hydrolysis), and empty states, corresponding to different affinities for nucleotides . The energy released from ATP hydrolysis drives conformational changes that rotate the central rotor, which ultimately powers proton translocation through the V0 domain.

How does the catalytic subunit A interact with other V-ATPase components?

The catalytic subunit A of V-ATPase participates in multiple critical interactions within the complex. Most prominently, it forms heterodimers with the B subunits, creating the three catalytic sites at the A:B interfaces where ATP hydrolysis occurs . Each of the three AB pairs in the V1 domain adopts a distinct conformation during the catalytic cycle .

The A subunits also interact with other V1 components, including subunits E and G which form peripheral stalks that connect the A3B3 catalytic core to the V0 membrane domain. Specifically, the N-terminus of subunit B2 and C-terminal residues of subunits E1 and G2 form important connections, including a structure where β-strands from both subunit B2 and E1 form a single β-sheet .

Additionally, the A subunit can be targeted by regulatory proteins. For example, SidK, a protein from the bacterial pathogen Legionella, binds to subunit A and affects V-ATPase function, although the structural consequences of this binding in mammalian V-ATPases are not fully elucidated .

What structural features enable catalytic subunit A to perform ATP hydrolysis?

The catalytic subunit A contains specialized domains optimized for ATP binding and hydrolysis. Based on structural analyses, each A subunit in the V1 sector participates in forming one of three catalytic sites at the interface with a B subunit. These catalytic AB pairs cycle through three conformations - ATP-binding, ATP-hydrolyzing, and product-release states .

The nucleotide-binding pocket within subunit A contains highly conserved residues that coordinate with the phosphate groups of ATP and position the molecule for hydrolysis. These sites alternate between an open conformation with high affinity for ATP, an occluded conformation suggesting low nucleotide affinity, and a post-hydrolysis ADP-bound state .

The conformational changes in subunit A during ATP hydrolysis are coupled to the rotation of the central rotor subunits (D and F), which transmits the energy to the membrane-embedded V0 sector for proton translocation. These structural changes are coordinated across all three A subunits in the complex to ensure efficient energy coupling .

What expression systems are most suitable for producing recombinant V-ATPase catalytic subunit A?

The choice of expression system for recombinant V-ATPase catalytic subunit A depends on several factors including the need for post-translational modifications, protein solubility, and downstream applications. Based on research practices, several expression systems have proven effective:

Yeast expression systems, particularly Saccharomyces cerevisiae, offer significant advantages because they naturally express V-ATPase components and possess the appropriate chaperones and post-translational machinery for proper folding . The endogenous VMA1 gene (encoding subunit A) can be deleted and replaced with recombinant versions containing affinity tags or mutations.

For higher yields of isolated subunit A, insect cell expression systems (Sf9 or Hi5 cells with baculovirus vectors) provide advantages over prokaryotic systems, especially for structural studies where proper folding is critical . Bacterial expression using E. coli can be employed for partial constructs or specific domains of subunit A that express well in soluble form, particularly for biochemical or interaction studies.

Mammalian expression systems might be preferred when studying mammalian V-ATPase subunit A, especially when interactions with regulatory proteins or other mammalian-specific components are of interest .

What purification strategies yield the highest activity for recombinant catalytic subunit A?

Purification of active recombinant V-ATPase catalytic subunit A requires strategies that maintain its native conformation and enzymatic capabilities. The following approach has proven effective in research settings:

• Affinity Chromatography: Initial capture using affinity tags (His6, FLAG, or Strep) positioned at either terminus of subunit A, with careful consideration of tag position to avoid interference with catalytic activity or complex assembly.

• Size Exclusion Chromatography: Critical for separating properly folded monomeric or oligomeric forms from aggregates, which is essential for activity studies.

• Ion Exchange Chromatography: Often employed as an intermediate step to remove contaminants based on charge differences.

For studies requiring highest activity, purification of the entire V1 complex rather than isolated subunit A is often preferred, as the catalytic activity depends on proper assembly of the A3B3 hexamer . When purifying from yeast, detergent-solubilized membranes can be fractionated using density gradient centrifugation before affinity purification steps.

The activity of purified preparations can be assessed through ATP hydrolysis assays, with successful preparations showing specific activities in the range of ~1-2.5 μmol × (min × mg)−1 . Reconstitution into lipid nanodiscs or proteoliposomes may be necessary for studies requiring membrane association.

How can I optimize expression of partial constructs of the catalytic subunit A for structural studies?

Optimizing the expression of partial constructs of V-ATPase catalytic subunit A for structural studies requires careful consideration of domain boundaries, solubility, and stability. Implementation of the following strategies can significantly improve success:

• Domain Boundary Selection: Bioinformatic analysis of sequence conservation and secondary structure predictions should guide the selection of construct boundaries. Avoid cutting within structured domains or conserved motifs. For V-ATPase subunit A, the non-homologous region (NHR) and catalytic domains can often be expressed separately .

• Fusion Partners: N-terminal fusion with solubility-enhancing tags such as MBP (maltose-binding protein), SUMO, or Thioredoxin can dramatically improve expression and solubility of partial constructs. Include a precision protease cleavage site for tag removal.

• Codon Optimization: Adjust codon usage to match the expression host, particularly for regions with rare codons that may cause translational pausing and misfolding.

• Expression Conditions: Lower temperatures (16-20°C) after induction, along with reduced inducer concentrations, often favor proper folding over rapid expression, resulting in higher yields of soluble protein.

For crystallographic studies, surface entropy reduction (replacing clusters of high-entropy residues like lysine and glutamate with alanines) can promote crystal formation. For cryoEM applications, constructs should retain interfaces with neighboring subunits if structural studies of subcomplexes are planned .

Successful partial constructs have been used to determine high-resolution structures of V-ATPase components, contributing significantly to our understanding of conformational states in the ATP hydrolysis cycle .

What assays accurately measure the ATP hydrolysis activity of recombinant catalytic subunit A?

Several complementary approaches are used to accurately measure ATP hydrolysis activity of recombinant V-ATPase catalytic subunit A:

• Colorimetric Phosphate Release Assays: The most common approach quantifies inorganic phosphate released during ATP hydrolysis using malachite green or similar reagents. For V-ATPase studies, these assays typically measure activities in the range of ~1-2.5 μmol × (min × mg)−1 for properly assembled complexes . This method allows for high-throughput screening but may be sensitive to phosphate contamination.

• Coupled Enzyme Assays: NADH-linked assays (pyruvate kinase and lactate dehydrogenase) monitor ATP hydrolysis in real-time by coupling ADP production to NADH oxidation, which decreases absorbance at 340 nm. This provides superior kinetic data but requires careful controls for enzyme coupling efficiency.

• Radioactive Assays: [γ-32P]ATP hydrolysis assays offer exceptional sensitivity for detecting low levels of activity, particularly useful when working with partial constructs or mutant proteins with compromised function.

For accurate assessment of catalytic subunit A activity, it's critical to distinguish between the activity of individual subunit A (which is typically minimal) and the activity in the context of assembled A3B3 hexamers or complete V1 complexes . Concanamycin A sensitivity can be used to confirm that measured activity is specifically from V-type ATPases rather than contaminating P-type or F-type ATPases .

How do mutations in the catalytic subunit A affect V-ATPase assembly and function?

Mutations in V-ATPase catalytic subunit A can have diverse effects on assembly and function, depending on the location and nature of the mutation:

Disease-linked mutations in subunit A have been mapped onto recent structures, providing molecular insights into their effects . For example, in yeast, mutations affecting Glu44 in subunit E (which interacts with subunit A) alter the assembly/disassembly dynamics of the V-ATPase complex in response to glucose availability .

Experimentally, the effects of mutations can be assessed through complementation studies in yeast strains lacking the endogenous VMA1 gene, where growth on alkaline media or media containing elevated calcium concentrations depends on functional V-ATPase .

What is the role of the catalytic subunit A in V-ATPase regulation by reversible disassembly?

The catalytic subunit A plays a central role in the regulation of V-ATPase through reversible disassembly, a process that allows cells to rapidly modulate V-ATPase activity in response to environmental conditions:

In yeast, glucose deprivation triggers rapid disassembly of the V-ATPase complex, with the V1 sector (including the A3B3 catalytic hexamer) dissociating from the membrane-bound V0 sector. This regulatory mechanism conserves ATP under energy-limited conditions . Recent studies have identified proteins that facilitate this process, including Oxr1p, which interacts directly with V-ATPase components during disassembly .

The catalytic subunit A participates in this regulation through:

• Conformational Changes: The nucleotide-binding state of subunit A influences the stability of V1-V0 association. ATP binding and hydrolysis drive conformational changes that can either stabilize or destabilize this association.

• Interaction Surfaces: The non-homologous region (NHR) of subunit A contains residues that affect disassembly rates without altering catalytic activity . Mutations in these regions can create disassembly-resistant V-ATPases.

• Regulator Binding: Regulatory proteins like Oxr1p interact with specific surfaces on the V1 sector, including regions on subunit A and other peripheral components like subunit E (with Glu44 in subunit E being part of the Oxr1p binding site) .

Experimental evidence shows that the disassembly process is accelerated by ATP, with complete disassembly occurring within minutes when both Oxr1p and ATP are present . After disassembly, the V1 sector adopts an auto-inhibited conformation where subunit H prevents wasteful ATP hydrolysis in the detached complex .

How can I design experiments to study the conformational changes in catalytic subunit A during the ATP hydrolysis cycle?

Designing experiments to study conformational changes in V-ATPase catalytic subunit A during ATP hydrolysis requires sophisticated approaches that capture dynamic states of this complex molecular machine:

• Cryo-electron Microscopy (cryo-EM): This has become the method of choice for visualizing different conformational states of V-ATPase. By using either ATP analogs (AMP-PNP for pre-hydrolysis state) or ADP (for post-hydrolysis state), or by trapping transition states using vanadate or beryllium fluoride, researchers can capture catalytic subunit A in different conformations . Modern processing methods allow classification of particles into distinct conformational states, revealing how the three copies of subunit A adopt different conformations simultaneously.

• FRET-based Approaches: Strategic placement of fluorophore pairs within or between subunit A and its interaction partners allows real-time monitoring of conformational changes during ATP hydrolysis. This approach requires careful introduction of labeling sites that don't disrupt function.

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): This technique provides information about solvent accessibility and conformational flexibility of different regions of subunit A under various nucleotide-bound states, revealing how specific domains respond during the catalytic cycle.

• Disulfide Crosslinking: Introducing cysteine pairs at strategic locations allows trapping of specific conformational states through formation of disulfide bonds, which can then be analyzed by structural methods or functional assays.

Recent structural studies have revealed that the three AB pairs in V-ATPase adopt distinct conformations corresponding to different stages of the catalytic cycle, with one site having high affinity for ATP, another in a post-hydrolysis state, and the third in an occluded conformation with low nucleotide affinity . These approaches provide mechanistic insights into how ATP hydrolysis drives rotary motion.

What strategies can identify interaction partners specific to the catalytic subunit A?

Identifying interaction partners specific to V-ATPase catalytic subunit A requires combinatorial approaches that distinguish direct interactions from indirect associations within the complex:

• Proximity Labeling Methods: BioID or APEX2 fused to subunit A can biotinylate proteins in close proximity in living cells, allowing identification of both stable and transient interaction partners. This approach successfully identified regulatory proteins like Oxr1p that interact with the V-ATPase complex and influence its disassembly .

• Crosslinking Mass Spectrometry (XL-MS): Chemical crosslinkers with various spacer lengths can capture direct interactions between subunit A and partner proteins. Combined with mass spectrometry, this approach maps the interaction interfaces at amino acid resolution. This method has helped elucidate how subunit A interacts with other V1 components, including the peripheral stalks formed by subunits E and G .

• Yeast Two-Hybrid Screens with Domain-Specific Baits: Using specific domains of subunit A rather than the full-length protein can reveal domain-specific interactions that might be masked in global approaches. This is particularly useful for identifying regulatory proteins that interact with the non-homologous region of subunit A.

• Co-Immunoprecipitation with Structure-Guided Mutations: Introducing mutations at specific surface-exposed regions of subunit A and assessing how they affect interaction partner binding can map binding interfaces with high precision.

These approaches have revealed that subunit A interacts not only with structural components of the V-ATPase complex but also with regulatory proteins like SidK (a bacterial effector protein) and components of the RAVE complex that facilitate reassembly of V1 and V0 sectors . The interaction with peripheral stalks formed by subunits E and G has been structurally characterized, showing how β-strands from subunits B2 and E1 form a shared β-sheet .

How do post-translational modifications regulate the function of catalytic subunit A?

Post-translational modifications (PTMs) of V-ATPase catalytic subunit A represent an important regulatory mechanism that fine-tunes enzyme activity, complex assembly, and cellular localization:

Experimental approaches to study these PTMs include site-directed mutagenesis of modified residues, phosphomimetic mutations (replacing phosphorylated residues with aspartate or glutamate), and the use of phosphatase or deubiquitinase inhibitors to preserve modifications during purification. Mass spectrometry-based strategies can provide a comprehensive map of PTMs and their dynamics under different cellular conditions.

Why might recombinant catalytic subunit A lack ATPase activity despite successful expression?

Recombinant V-ATPase catalytic subunit A may lack ATPase activity despite successful expression for several reasons, each requiring specific troubleshooting approaches:

• Improper Folding: The complex tertiary structure of subunit A may not form correctly in heterologous expression systems. This is particularly common when expressing eukaryotic proteins in bacterial systems. Solutions include:

  • Switching to eukaryotic expression systems like yeast or insect cells

  • Co-expressing molecular chaperones

  • Lowering expression temperature to slow folding

  • Adding folding enhancers like arginine or trehalose to lysis buffers

• Missing Required Partner Subunits: Isolated subunit A typically shows minimal ATPase activity, as the catalytic function requires assembly with subunit B to form catalytic sites at their interface . For activity studies, co-expression or co-purification with subunit B or reconstitution of the A3B3 hexamer is often necessary.

• Inhibitory Tags or Fusion Partners: Affinity tags placed near the catalytic site or at interfaces with other subunits can interfere with activity. Consider:

  • Moving tags to the opposite terminus

  • Removing tags after purification using precision proteases

  • Using smaller tags (e.g., His6 instead of larger fusion proteins)

• Inactive Conformation: Upon V-ATPase disassembly, the V1 sector adopts an auto-inhibited conformation where subunit H prevents wasteful ATP hydrolysis . Recombinant preparations may inadvertently capture this inactive state. Reconstitution with appropriate V0 components or manipulation of the subunit H position may restore activity.

Experimental verification of proper folding can be assessed through circular dichroism spectroscopy, limited proteolysis, and thermal shift assays before concluding that activity issues stem from other causes.

How can I overcome stability issues when working with partial constructs of catalytic subunit A?

Stability issues with partial constructs of V-ATPase catalytic subunit A are common challenges in research and can be addressed through several strategic approaches:

• Rational Construct Design:

  • Use structural information and domain prediction tools to avoid cutting within folded domains

  • Consider constructing from domain boundaries observed in limited proteolysis experiments

  • Include a few additional residues at domain boundaries to ensure complete secondary structure elements

  • Target natural proteolytic sites identified in mass spectrometry studies of V-ATPase

• Buffer Optimization:

  • Screen buffers with varying pH values (typically 6.5-8.0 for V-ATPase components)

  • Test different salt concentrations (150-300 mM NaCl often improves stability)

  • Add stabilizing additives such as glycerol (5-10%), specific nucleotides (ATP/ADP at 0.1-1 mM), or osmolytes like trehalose

  • Include reducing agents (DTT or TCEP) to prevent cysteine oxidation and aggregation

• Protein Engineering Approaches:

  • Introduce surface mutations to reduce hydrophobic patches exposed by truncation

  • Remove flexible regions identified by hydrogen-deuterium exchange mass spectrometry

  • Consider adding stabilizing inter-domain disulfide bonds based on structural analysis

  • Use computational tools to identify and mutate aggregation-prone sequences

• Co-expression Strategies:

  • Co-express with natural binding partners that stabilize the chosen fragment

  • Consider artificial binding partners like nanobodies or designed ankyrin repeat proteins (DARPins) that can stabilize specific conformations

Successful applications of these strategies have enabled structural studies of V-ATPase components, including studies where partial constructs of subunit A were co-crystallized with regulatory proteins .

What controls should be included when studying interactions between catalytic subunit A and potential regulatory proteins?

When investigating interactions between V-ATPase catalytic subunit A and potential regulatory proteins, comprehensive controls are essential to establish specificity and functional relevance:

• Protein-Protein Interaction Controls:

  • Negative Controls: Include unrelated proteins of similar size/charge to rule out non-specific binding. For V-ATPase studies, F-type ATPase subunits make appropriate negative controls.

  • Binding Site Mutants: Generate mutations in predicted interface residues of subunit A to confirm the specific binding site. For instance, mutations in the non-homologous region of subunit A or in Glu44 of subunit E have been shown to affect interactions with regulatory proteins like Oxr1p .

  • Competition Assays: Demonstrate that unlabeled protein can compete with labeled protein for binding, confirming specificity.

• Functional Validation Controls:

  • Activity Assays: Measure V-ATPase activity with and without the regulatory protein to demonstrate functional consequences. For example, Oxr1p causes a decrease in ATPase activity when added to purified V-ATPase .

  • Disassembly Assays: For proteins involved in assembly/disassembly regulation, quantify the V1-V0 association using techniques like biolayer interferometry (BLI) or fluorescence-based assays .

  • In vivo Phenotypic Assays: In yeast, growth on alkaline or high calcium media depends on functional V-ATPase and can reveal physiological relevance of interactions .

• Technical Controls:

  • Tag-Only Controls: Ensure that affinity tags are not mediating the observed interactions.

  • Reciprocal Co-IPs: Perform immunoprecipitation in both directions (i.e., pull down with antibodies against both subunit A and the potential regulator).

  • Concentration Series: Test interactions across a range of concentrations to establish dose-dependence and saturation.

Recent studies investigating the role of Oxr1p in V-ATPase disassembly employed several of these controls, including BLI experiments with combinations of Oxr1p and nucleotides, in vivo fluorescence microscopy in wild-type and deletion strains, and activity assays with purified components .

What emerging technologies could advance our understanding of catalytic subunit A function?

Several cutting-edge technologies are poised to revolutionize our understanding of V-ATPase catalytic subunit A function, providing unprecedented insights into its dynamics and regulation:

• Time-resolved Cryo-EM: This emerging technique captures multiple conformational states during rapid mixing experiments, potentially visualizing the complete conformational trajectory of subunit A during the ATP hydrolysis cycle at near-atomic resolution. Recent advances in V-ATPase structural biology have already utilized cryo-EM to resolve different conformational states of the catalytic AB pairs , but time-resolved approaches could reveal short-lived transition states.

• In-cell NMR and EPR Spectroscopy: These techniques can monitor structural changes of specifically labeled residues in subunit A within living cells, bridging the gap between in vitro mechanistic studies and cellular physiology. This could be particularly valuable for understanding how V-ATPase regulation via reversible disassembly occurs in different cellular compartments.

• AlphaFold2 and Related AI Approaches: Deep learning methods are dramatically improving protein structure prediction and could be applied to model conformational dynamics, protein-protein interaction surfaces, and the effects of disease-associated mutations in subunit A. These computational approaches could guide experimental design and interpretation.

• Single-Molecule FRET and Optical Tweezers: These techniques can track conformational changes and measure force generation in individual V-ATPase complexes, providing insights into how ATP hydrolysis by subunit A drives mechanical rotation and proton pumping. Single-molecule approaches are particularly suited for understanding the mechanochemical coupling in this molecular motor.

• Proximity Proteomics in Specific Cellular Compartments: TurboID or APEX2 fusions to subunit A variants targeted to specific organelles could reveal compartment-specific interaction partners and regulatory mechanisms, providing a comprehensive view of how V-ATPase function is fine-tuned throughout the cell.

These technologies, especially when combined in integrative approaches, promise to resolve long-standing questions about the coupling between ATP hydrolysis in subunit A and proton translocation by the V0 domain, as well as the regulatory mechanisms that control V-ATPase activity in different cellular contexts.

How might understanding catalytic subunit A inform therapeutic approaches for V-ATPase-related diseases?

Understanding the structure, function, and regulation of V-ATPase catalytic subunit A has significant implications for developing therapeutic strategies for V-ATPase-related diseases:

• Precision Targeting of Disease-Associated Mutations: Numerous mutations in V-ATPase subunits, including subunit A, are linked to human diseases . Detailed structural information about these mutations enables rational design of small molecules that could stabilize mutant proteins or compensate for functional deficits. For instance, mutations that affect V-ATPase assembly could be targeted with molecular chaperones or stabilizers that promote proper complex formation.

• Isoform-Specific Inhibitors: Different tissues express specific isoforms or variants of V-ATPase subunits. Understanding the unique structural features of catalytic subunit A variants could enable development of tissue-specific V-ATPase inhibitors, reducing off-target effects. This approach could be particularly valuable for targeting cancer cells that often upregulate specific V-ATPase isoforms.

• Allosteric Modulators of V-ATPase Assembly/Disassembly: The reversible disassembly of V-ATPase represents a natural regulatory mechanism . Small molecules that mimic the action of proteins like Oxr1p could provide novel ways to modulate V-ATPase activity without directly targeting the catalytic site. These modulators could potentially adjust V-ATPase activity without complete inhibition.

• Targeting Protein-Protein Interactions: The interactions between subunit A and other V-ATPase components or regulatory proteins represent potential therapeutic targets. Disrupting specific interactions could modulate V-ATPase function in a more nuanced way than direct inhibition of catalytic activity.

• Gene Therapy Approaches: For genetic diseases caused by mutations in V-ATPase subunits, detailed understanding of subunit A structure and function guides the development of gene therapy strategies, including which mutations might be amenable to correction and what level of protein function needs to be restored for therapeutic benefit.

Recent structural studies have mapped disease-associated mutations in V-ATPase subunits including subunit A , providing a foundation for rational therapeutic design. Additionally, the identification of regulatory mechanisms involving proteins like ATP6AP1/Ac45 and ATP6AP2/PRR, which influence V-ATPase assembly and function , offers new potential targets for therapeutic intervention.

What are the challenges in studying catalytic subunit A across different species and organelles?

Investigating V-ATPase catalytic subunit A across different species and cellular compartments presents several significant challenges that researchers must address:

Addressing these challenges requires multidisciplinary approaches combining structural biology, cell biology, genetics, and biochemistry. Recent advances in cryo-EM have facilitated structural studies of intact V-ATPase complexes from different sources , while genetic tools enable in vivo functional studies across model organisms .

What is the comparative structure and function of catalytic subunit A across different biological systems?

V-ATPase catalytic subunit A exhibits both conserved and divergent features across various biological systems, reflecting both the fundamental importance of its catalytic function and the specialized regulatory mechanisms that have evolved in different organisms and cellular compartments.

Table 1: Comparison of V-ATPase Catalytic Subunit A Across Species

The catalytic mechanism involving ATP binding and hydrolysis is remarkably conserved across species, with each subunit A containing the Walker A and B motifs characteristic of P-loop NTPases . The three copies of subunit A in each V-ATPase complex adopt different conformational states during the catalytic cycle, a feature preserved from yeast to mammals .

Despite these conserved elements, important differences exist in the regulatory regions and interaction surfaces of subunit A. These differences influence how V-ATPase activity is regulated in different biological contexts, from glucose-responsive disassembly in yeast to tissue-specific regulation in complex multicellular organisms .

What mutations in catalytic subunit A are associated with disease phenotypes?

Mutations in the V-ATPase catalytic subunit A (ATP6V1A in humans) have been linked to several disease conditions, with the specific mutation locations providing insights into structure-function relationships and potential therapeutic approaches.

Table 2: Disease-Associated Mutations in V-ATPase Catalytic Subunit A

These disease-associated mutations cluster in several functionally important regions of subunit A:

The phenotypic consequences of these mutations reflect the importance of V-ATPase function in various tissues, particularly those requiring precise pH regulation such as neurons, osteoclasts, and renal cells . Understanding the molecular mechanisms of these mutations provides potential avenues for therapeutic intervention, including approaches to stabilize mutant proteins or compensate for reduced activity.

How do affinity and kinetic parameters of ATP hydrolysis by catalytic subunit A compare across experimental conditions?

The catalytic activity of V-ATPase subunit A exhibits significant variability depending on experimental conditions, assembly state, and the presence of regulatory factors. This table summarizes key kinetic and affinity parameters across different experimental contexts.

Table 3: Kinetic and Affinity Parameters for ATP Hydrolysis by V-ATPase Catalytic Subunit A

Key insights from this comparative analysis include:

• The ATP hydrolysis activity of subunit A is highly dependent on its assembly state, with minimal activity as an isolated subunit and increasing activity as it assembles into progressively more complete complexes .

• The coupling between ATP hydrolysis and proton pumping enhances catalytic efficiency, with the intact V-ATPase complex showing the highest turnover rates and lowest Km values.

• Regulatory mechanisms like reversible disassembly mediated by Oxr1p provide rapid means to modulate activity in response to cellular conditions . This regulation occurs much faster in vivo (minutes) than with purified components in vitro, suggesting additional cellular factors may be involved.

• The presence of ATP dramatically accelerates Oxr1p-mediated disassembly, with complete dissociation occurring within minutes when both are present .

These parameters highlight the complex regulation of V-ATPase activity and provide benchmarks for assessing the functional consequences of mutations or the effects of potential therapeutic modulators.