Recombinant Saccharomyces cerevisiae Vacuolar ATPase assembly integral membrane protein VMA21 (VMA21)

What is the basic function of VMA21 in yeast cells?

VMA21 serves as an essential assembly factor for the V-ATPase V₀ region in the endoplasmic reticulum (ER) membrane. It is an integral membrane protein that coordinates the assembly of V₀ subunits and escorts the assembled V₀ complex into ER-derived transport vesicles . VMA21 contains a C-terminal ER-retrieval motif that allows it to be transported back to the ER to participate in additional rounds of V₀ assembly after escorting assembled V₀ to the Golgi . This recycling mechanism ensures efficient V-ATPase assembly while preventing premature acidification of the ER, as V₁ only binds V₀ after it reaches the Golgi membrane .

What is the structural organization of VMA21?

VMA21 is a relatively small integral membrane protein. While the complete high-resolution structure of VMA21 alone has not been fully characterized in the search results, cryo-electron microscopy has revealed that VMA21 interacts with the rotor ring (c-ring) of the V₀ complex . Its binding appears to be somewhat variable in position, which has made it challenging to obtain coherent density for VMA21 in some 3D reconstructions . The protein contains transmembrane domains that anchor it to the ER membrane and a critical C-terminal ER-retrieval motif that facilitates its recycling from the Golgi back to the ER .

How does VMA21 differ from other V-ATPase assembly factors?

VMA21 differs from the other assembly factors (Vma12p and Vma22p) in several key aspects. While Vma12p and Vma22p form a complex (Vma12-22p) that remains in the ER, VMA21 actually accompanies the fully assembled V₀ complex out of the ER to the Golgi . After the V₁ sector assembles with V₀, VMA21 dissociates and is transported back to the ER via its ER-retrieval motif . This makes VMA21 unique among the assembly factors as it plays a dual role in both assembly and quality control of the V-ATPase complex . Additionally, experimental evidence suggests that VMA21 interacts with the proteolipid subunits of V₀, particularly through Vma11p, while the Vma12-22p complex is involved in recruiting subunits a, e, and f to the rotor ring of V₀ .

What is the sequential process of V-ATPase assembly involving VMA21?

The assembly of the V-ATPase complex follows a defined sequence in which VMA21 plays a central role:

• VMA21 first interacts with the proteolipid subunits (particularly Vma11p) in the ER membrane

• This initial binding helps coordinate the formation of the proteolipid ring (c-ring)

• The Vma12-22p complex then aids in recruiting additional V₀ subunits (a, e, and f) to the c-ring

• VMA21 remains associated with this complex during assembly

• The fully assembled V₀:Vma12-22p:Vma21p complex is then packaged into COPII-coated transport vesicles for export from the ER to the Golgi

• In the Golgi, the V₁ sector assembles with V₀, which coincides with the dissociation of VMA21 from V₀

• VMA21 is then retrieved back to the ER via its ER-retrieval motif to participate in additional rounds of assembly

This sequential process ensures that only properly assembled V₀ complexes leave the ER and that proton pumping is activated only after V₁ association in the Golgi .

What experimental evidence demonstrates VMA21's role in preventing premature V₁-V₀ association?

Pulse-chase experiments with wild-type VMA21 showed that its interaction with the V₀ sector is transient, with very little 100-kDa V₀ subunit coimmunoprecipitating with VMA21 after a 60-minute chase period . This suggests that VMA21 normally dissociates from V₀ after assembly is complete. Interestingly, a mutant form of VMA21 lacking the ER-retrieval motif (VMA21(QQ)p) remained associated with V₀ even after the chase period, indicating a long-lived interaction .

How does the interaction between VMA21 and other assembly factors contribute to quality control?

VMA21 works in concert with Vma12p and Vma22p to create a sophisticated quality control system for V-ATPase assembly:

• The Vma12-22p complex helps recruit subunits a, e, and f to the rotor ring while simultaneously blocking premature binding of V₁

• VMA21 binds the V₀:Vma12-22p complex and also interacts with assembly intermediates containing the rotor ring

• Structural analysis has revealed that VMA21 also binds a complex containing YAR027W and YAR028W proteins, suggesting its involvement in monitoring additional assembly intermediates

• The ER-retrieval motif of VMA21 ensures that it can be recycled back to the ER after successful assembly, creating an efficient system for continued production of properly assembled V₀ complexes

This multi-step quality control system ensures that only properly assembled V₀ complexes exit the ER, and that proton pumping is activated only after V₁ assembly with V₀ in the Golgi membrane .

What are the optimal expression systems for producing recombinant VMA21?

For recombinant expression of VMA21, yeast-based expression systems are often preferred due to the protein's native context in Saccharomyces cerevisiae. Several approaches have proven successful:

• Expression in S. cerevisiae using endogenous promoters with epitope tags (such as HA, FLAG, or other affinity tags) for detection and purification

• Complementation systems using vma21Δ yeast strains transformed with plasmids encoding tagged versions of VMA21

• Commercial expression systems like the one offered by GenScript that provide cDNA ORF clones of VMA21 in expression vectors such as pcDNA3.1

For structural studies, expression systems that yield sufficient quantities of protein while maintaining proper folding and membrane insertion are critical. The successful cryo-EM studies described in the search results utilized epitope-tagged versions of VMA21 (such as VMA21p-3×FLAG) expressed in yeast to isolate complexes containing VMA21 and its binding partners .

What purification strategies are most effective for isolating VMA21-containing complexes?

Effective purification of VMA21-containing complexes requires careful consideration of their membrane-associated nature and transient interactions. Based on the successful approaches described in the search results:

• Affinity purification using epitope tags (such as 3×FLAG or HA tags) is effective for isolating VMA21 and associated proteins

• Non-denaturing conditions using mild detergents (such as 1% C₁₂E₉) are critical for maintaining protein-protein interactions during solubilization of membrane proteins

• Immunoprecipitation under non-denaturing conditions using antibodies against the epitope tags has been successful for analyzing VMA21's interactions with V₀ subunits

• For structural studies, additional purification steps such as size exclusion chromatography may be necessary to obtain homogeneous samples suitable for cryo-EM analysis

When tracking the dynamic interactions of VMA21 with V₀ complexes, pulse-chase experiments combined with immunoprecipitation have proven valuable for determining the half-life of these interactions and how they change during trafficking through the secretory pathway .

What imaging techniques are most informative for studying VMA21's role in V-ATPase assembly?

Several advanced imaging techniques have proven valuable for understanding VMA21's role in V-ATPase assembly:

• Cryo-electron microscopy (cryo-EM) has been particularly powerful, allowing visualization of VMA21's interactions with V₀ complexes and assembly intermediates at high resolution (up to 4.4 Å)

• 3D classification of cryo-EM datasets has been crucial for identifying different populations of VMA21-containing complexes, including the V₀:Vma12-22p complex and a complex containing YAR027W and YAR028W proteins

• Immunofluorescence microscopy can be useful for tracking the cellular localization of tagged VMA21 and its co-localization with other V-ATPase components

• For dynamic studies, live-cell imaging with fluorescently tagged proteins can provide insights into the trafficking of VMA21 and V₀ complexes through the secretory pathway

These imaging approaches, particularly high-resolution cryo-EM, have been instrumental in revealing how VMA21 and other assembly factors coordinate V-ATPase assembly and quality control .

How does the structural conformation of VMA21 change during different stages of V-ATPase assembly?

The structural dynamics of VMA21 during V-ATPase assembly are still being elucidated, but the available data suggests several conformational changes:

• Cryo-EM analysis indicates that VMA21 binds the c-ring of V₀, but with variable positions that lead to incoherent averaging in 3D reconstruction

• This variability suggests that VMA21 may adopt different conformations or binding positions depending on the assembly state of the V₀ complex

• The interaction between VMA21 and V₀ appears to be mediated primarily through the proteolipid subunit Vma11p, with this initial binding likely inducing conformational changes that facilitate further assembly

• The dissociation of VMA21 from V₀ upon V₁ binding suggests additional conformational changes that reduce VMA21's affinity for the fully assembled complex

Further high-resolution structural studies of VMA21 in different assembly intermediates will be needed to fully characterize these conformational dynamics.

What are the molecular mechanisms by which VMA21 prevents proton leakage during V-ATPase assembly?

VMA21 contributes to preventing premature proton pumping and potential proton leakage through several mechanisms:

• By coordinating the assembly of V₀ in the ER and escorting it to the Golgi before V₁ association, VMA21 ensures that proton pumping is activated only in post-ER compartments

• The Vma12-22p complex, which works with VMA21, plays a direct role in blocking premature binding of V₁ to V₀

• Structural data suggests that the binding of VMA21 and assembly factors to the c-ring may physically occlude conformations that would allow proton translocation

• The quality control function of VMA21 ensures that only properly assembled V₀ complexes exit the ER, preventing the formation of partially assembled complexes that might allow proton leakage

These mechanisms collectively ensure that the powerful proton-pumping activity of the V-ATPase is appropriately regulated during assembly, preventing unwanted acidification of the ER lumen .

What is the biochemical basis for VMA21's selective binding to V₀ assembly intermediates versus mature V-ATPase complexes?

The selective binding of VMA21 to V₀ assembly intermediates appears to be regulated through several biochemical mechanisms:

• Analysis of vmaΔ strains showed that VMA21's binding to V₀ subunits is mediated primarily by the proteolipid subunit Vma11p

• VMA21's interaction with Vph1p (the 100-kDa V₀ subunit) is dependent on all other V₀ subunits, indicating that assembly occurs in a defined sequence with specific binding affinities at each stage

• Pulse-chase experiments demonstrated that the interaction between VMA21 and V₀ is transient, with dissociation coinciding with V₁/V₀ assembly

• The conformational changes in V₀ that occur upon V₁ binding likely reduce the binding affinity for VMA21, explaining its dissociation from mature V-ATPase complexes

• The cryo-EM datasets showed few images of intact V-ATPase with bound VMA21, suggesting any interaction between VMA21 and intact V-ATPase is unstable

These findings indicate that VMA21's binding affinity is regulated by the conformational state of the V₀ complex, with highest affinity for assembly intermediates and reduced affinity for the mature V-ATPase.

How do mutations in VMA21 orthologues contribute to human disease?

The mammalian orthologue of VMA21 (known as VMA21) is essential for proper V-ATPase function, and its deficiency is associated with several human diseases:

• X-linked myopathy with excessive autophagy (XMEA) is caused by mutations in the human VMA21 gene

• VMA21 deficiency leads to dysfunction of lysosomes, which are acidified by V-ATPases

• The mammalian homologues of the yeast assembly factors Vma12p, Vma21p, and Vma22p (known as TMEM199, VMA21, and CCDC115, respectively) are all associated with congenital disorders of glycosylation when deficient

• Deficiency of CCDC115 (homologue of Vma22p) has been associated with follicular lymphoma

The structural data obtained from yeast VMA21 studies provides a framework for understanding how disease-causing mutations in human VMA21 might disrupt V-ATPase assembly, potentially opening avenues for therapeutic interventions .

What experimental approaches can be used to model VMA21-related diseases in laboratory settings?

Several experimental approaches can be employed to model VMA21-related diseases:

• Yeast models: Using vma21Δ strains complemented with mutant versions of VMA21 that mimic disease-causing mutations in humans can provide insights into functional consequences

• Mammalian cell culture: CRISPR/Cas9-mediated knockout or knockdown of VMA21 in relevant cell types can help study the cellular consequences of VMA21 deficiency

• Patient-derived cells: Fibroblasts or induced pluripotent stem cells (iPSCs) from patients with VMA21 mutations can be used to study disease mechanisms in human cells

• Animal models: While not described in the provided search results, conditional knockout mouse models of VMA21 could provide insights into tissue-specific effects of VMA21 deficiency

These models can be used to study the impact of VMA21 deficiency on V-ATPase assembly, lysosomal function, autophagy, and other cellular processes affected in diseases like XMEA.

How might the structural insights into VMA21 function inform therapeutic approaches for V-ATPase-related disorders?

The structural and functional insights into VMA21's role in V-ATPase assembly suggest several potential therapeutic approaches:

• Small molecule stabilizers: Compounds that stabilize the interaction between mutant VMA21 and V₀ components could potentially rescue assembly defects

• Gene therapy: Delivery of functional VMA21 genes could restore V-ATPase assembly in patients with VMA21 mutations

• Bypassing strategies: Approaches that stabilize partially assembled V-ATPase complexes or provide alternative acidification mechanisms could compensate for V-ATPase deficiency

• Targeted protein degradation: In cases where mutant VMA21 has dominant negative effects, selective degradation of the mutant protein could allow residual wild-type function

The high-resolution structural data showing how VMA21 and other assembly factors coordinate V-ATPase assembly provides rational targets for drug design efforts aimed at modulating these interactions .

What are the critical factors to consider when designing experiments to study VMA21-V₀ interactions?

When designing experiments to study VMA21-V₀ interactions, researchers should consider:

• Protein tagging strategy: Careful consideration of tag placement is essential as improper tagging can disrupt VMA21's function. C-terminal tags must be designed to preserve the ER-retrieval motif function

• Membrane protein solubilization: Use of appropriate detergents (such as 1% C₁₂E₉) that maintain protein-protein interactions while effectively solubilizing membrane proteins

• Time-resolved analysis: Given the transient nature of VMA21-V₀ interactions, pulse-chase experiments or other time-resolved approaches are crucial for capturing these dynamics

• Compartment-specific isolation: Methods that can distinguish between ER, Golgi, and vacuolar pools of VMA21 are important for tracking its movement through the secretory pathway

• Control strains: Utilization of vma deletion strains (vmaΔ) as controls to validate the specificity of observed interactions

These considerations help ensure that the experimental setup accurately captures the biological behavior of VMA21 and its interactions with V₀ components.

What common technical challenges arise when working with recombinant VMA21, and how can they be addressed?

Several technical challenges are commonly encountered when working with recombinant VMA21:

• Low expression levels: As a small integral membrane protein, VMA21 may express at low levels. This can be addressed by using strong promoters or optimizing codon usage for the expression system

• Protein stability: Membrane proteins can be unstable when removed from their native environment. Stabilizing additives in buffers and careful temperature control during purification can help maintain protein integrity

• Functional verification: Confirming that recombinant VMA21 is functional can be challenging. Complementation assays in vma21Δ yeast strains provide a robust approach to verify functionality

• Detecting protein-protein interactions: The transient nature of some VMA21 interactions makes them difficult to capture. Crosslinking approaches or rapid isolation techniques may help stabilize these interactions

• Structural heterogeneity: As seen in cryo-EM studies, VMA21 can bind to its partners with variable positions, leading to challenges in structural determination . Utilizing classification algorithms in image processing can help sort heterogeneous populations

Addressing these challenges requires careful experimental design and often the combination of multiple complementary approaches.

What are the optimal conditions for preserving VMA21 activity during recombinant protein production and purification?

To maximize VMA21 activity during recombinant production and purification:

• Expression system selection: Native or closely related host organisms (like S. cerevisiae for yeast VMA21) often provide the best environment for proper folding and post-translational modifications

• Temperature control: Expression at lower temperatures (20-25°C) can improve folding and reduce aggregation of membrane proteins

• Buffer composition:

  • pH: Maintain pH between 6.5-7.5 during purification

  • Salt concentration: 150-300 mM NaCl typically helps stabilize membrane proteins

  • Glycerol (10-15%): Can enhance stability during purification

  • Reducing agents: Include DTT or β-mercaptoethanol to prevent oxidation of cysteine residues

• Detergent selection: Mild non-ionic detergents like DDM, C₁₂E₉, or LMNG are often effective for solubilizing membrane proteins while preserving their native structure and interactions

• Protease inhibitors: Include a comprehensive protease inhibitor cocktail throughout the purification process to prevent degradation

• Rapid purification: Minimize time between cell lysis and final purification step to reduce exposure to potential denaturing conditions

Each of these factors contributes to maintaining the structural integrity and functional activity of VMA21 during the production and purification process.

How can mass spectrometry be optimized for studying VMA21 interactions and post-translational modifications?

Mass spectrometry (MS) offers powerful approaches for analyzing VMA21 interactions and modifications:

• Crosslinking Mass Spectrometry (XL-MS):

  • Chemical crosslinkers with various spacer lengths can capture transient interactions between VMA21 and its binding partners

  • MS/MS analysis of crosslinked peptides can identify specific interaction sites

  • This approach is particularly valuable for mapping the binding interface between VMA21 and proteolipid subunits

• Native MS for membrane protein complexes:

  • Specialized detergents compatible with electrospray ionization

  • Nanodiscs or amphipol-stabilized complexes can maintain native interactions

  • Can reveal stoichiometry and stability of VMA21-containing complexes

• Post-translational modification (PTM) analysis:

  • Enrichment strategies for phosphorylated, glycosylated, or ubiquitinated peptides

  • Multiple fragmentation techniques (CID, HCD, ETD) to maximize PTM identification

  • Quantitative approaches (SILAC, TMT) to compare PTM levels under different conditions

• Targeted proteomics:

  • Selected reaction monitoring (SRM) or parallel reaction monitoring (PRM) for sensitive quantification of VMA21 and interacting partners

  • Internal standard peptides for absolute quantification

These MS approaches can provide molecular-level insights into VMA21 function that complement structural and genetic studies.

What computational methods are most effective for predicting VMA21-substrate interactions and functional domains?

Several computational approaches can be valuable for studying VMA21:

• Homology modeling and threading:

  • Using known structures of related proteins to predict VMA21 structure

  • Integration of experimental constraints from crosslinking or mutagenesis studies

  • Refinement based on molecular dynamics simulations

• Molecular dynamics simulations:

  • Coarse-grained simulations to model membrane insertion and protein-lipid interactions

  • All-atom simulations to explore conformational dynamics

  • Steered molecular dynamics to investigate binding/unbinding events

• Protein-protein docking:

  • Prediction of interaction interfaces between VMA21 and V₀ components

  • Incorporation of experimental constraints to guide docking

  • Ensemble docking to account for conformational flexibility

• Evolutionary analysis:

  • Multiple sequence alignments to identify conserved residues across species

  • Coevolutionary analysis to predict residue pairs involved in interactions

  • Analysis of conservation patterns in disease-associated mutations

• Machine learning approaches:

  • Prediction of functional motifs and binding sites

  • Classification of variants based on potential functional impact

  • Integration of multiple data types for functional annotation

These computational methods can generate testable hypotheses about VMA21 function and guide experimental design.

How can single-molecule techniques be applied to study the dynamics of VMA21-mediated V-ATPase assembly?

Single-molecule techniques offer unique insights into the dynamic aspects of VMA21 function:

• Single-molecule FRET (smFRET):

  • Labeling of VMA21 and binding partners with donor-acceptor fluorophore pairs

  • Monitoring of conformational changes during assembly in real-time

  • Detection of transient intermediates not observable in bulk measurements

• Single-molecule pull-down (SiMPull):

  • Immobilization of tagged VMA21 or V₀ components

  • Visualization of stoichiometry and composition of complexes

  • Kinetic analysis of assembly/disassembly events

• Single-particle tracking in live cells:

  • Fluorescent protein tagging of VMA21 for live-cell imaging

  • Tracking of VMA21 movement between ER and Golgi

  • Correlation of mobility changes with assembly state

• Nanodiscs with single-molecule analysis:

  • Reconstitution of VMA21 and V₀ components in nanodiscs

  • Control over lipid composition to study environmental effects

  • Compatible with various single-molecule spectroscopy techniques

• High-speed AFM:

  • Visualization of conformational dynamics at sub-molecular resolution

  • Monitoring of assembly/disassembly processes in real-time

  • Force measurements to probe mechanical stability of complexes

These techniques can provide unique insights into the kinetics and conformational dynamics of VMA21-mediated V-ATPase assembly that complement structural and biochemical approaches.

What emerging technologies hold promise for advancing our understanding of VMA21 function?

Several cutting-edge technologies show potential for deepening our understanding of VMA21:

• Cryo-electron tomography (cryo-ET):

  • Visualization of VMA21 and V-ATPase assembly in situ within cellular membranes

  • Subtomogram averaging to obtain higher resolution of membrane-embedded complexes

  • Correlative light and electron microscopy to target specific cellular regions

• AlphaFold2 and other AI-based structure prediction:

  • Accurate prediction of VMA21 structure and its complexes

  • Integration with experimental data for hybrid structural modeling

  • Prediction of effects of disease-causing mutations

• Time-resolved cryo-EM:

  • Capturing different states of the assembly process

  • Microfluidic mixing devices to initiate assembly before vitrification

  • Classification algorithms to sort different assembly intermediates

• Proximity labeling approaches:

  • BioID or APEX2 fusions with VMA21 to identify transient interactors

  • Spatial and temporal mapping of the VMA21 interactome

  • Compartment-specific interaction profiling

• Optogenetic tools:

  • Light-controlled recruitment or dissociation of VMA21 from assembly sites

  • Perturbation of assembly process with temporal precision

  • Visualization of downstream effects on V-ATPase activity

These technologies promise to overcome current limitations in studying the dynamic aspects of VMA21 function and its interactions within the cellular context.

What are the key unresolved questions about VMA21 that require further investigation?

Despite significant progress, several important questions about VMA21 remain unanswered:

• Structural dynamics:

  • How does VMA21 change conformation during different stages of V₀ assembly?

  • What is the precise binding interface between VMA21 and the proteolipid subunits?

  • How do disease-causing mutations alter these structural dynamics?

• Regulatory mechanisms:

  • Are there post-translational modifications that regulate VMA21 activity?

  • Do cellular stress conditions modulate VMA21-mediated assembly?

  • What factors control the timing of VMA21 dissociation from V₀?

• Interaction with other cellular machinery:

  • How does VMA21 coordinate with the COPII vesicle trafficking machinery?

  • Are there additional quality control checkpoints beyond the known assembly factors?

  • How is VMA21 expression coordinated with other V-ATPase components?

• Tissue-specific functions:

  • Do different cell types have specialized mechanisms involving VMA21?

  • How do these differences contribute to tissue-specific disease manifestations?

  • Are there tissue-specific interaction partners for VMA21?

• Therapeutic targeting:

  • Can VMA21 function be modulated pharmacologically?

  • Would targeting VMA21 offer advantages over direct V-ATPase targeting?

  • How can the structural insights guide development of mutation-specific therapies?

Addressing these questions will require interdisciplinary approaches combining structural biology, cell biology, genetics, and computational methods.

How might systems biology approaches contribute to a more comprehensive understanding of VMA21's role in cellular homeostasis?

Systems biology approaches offer powerful tools for integrating VMA21 function into broader cellular contexts:

• Multi-omics integration:

  • Combining proteomics, transcriptomics, and metabolomics data from VMA21-deficient models

  • Network analysis to identify compensatory pathways and secondary effects

  • Correlation of V-ATPase assembly states with global cellular responses

• Mathematical modeling of assembly kinetics:

  • Ordinary differential equation (ODE) models of the assembly process

  • Stochastic simulations accounting for cellular compartmentalization

  • Parameter estimation from time-resolved experimental data

  • Sensitivity analysis to identify rate-limiting steps

• Genome-scale genetic interaction mapping:

  • Systematic genetic interaction screens with VMA21 mutations

  • Identification of buffering pathways and synthetic lethal interactions

  • Context-specific genetic dependencies in different cell types

• Spatial modeling approaches:

  • Agent-based models of VMA21 trafficking between compartments

  • Reaction-diffusion models of assembly factor gradients

  • Integration with cellular atlas data for spatial context

• Evolutionary systems biology:

  • Comparative analysis of V-ATPase assembly across species

  • Reconstruction of the evolutionary trajectory of assembly mechanisms

  • Identification of conserved principles versus species-specific adaptations

These systems-level approaches can place VMA21 function in the broader context of cellular homeostasis and reveal emergent properties not apparent from reductionist approaches.