Recombinant V-type proton ATPase subunit e (vha-17)

What is the structural composition of VHA-e (VHA-17) in V-ATPase complexes?

VHA-e is a small 9 kDa subunit consisting of two membrane-integral helices and a cytosolic C-terminal tail. In Arabidopsis, there are two known isoforms of VHA-e . This subunit is part of the membrane-integral V₀ subcomplex of the V-ATPase, which consists of six different subunits (a, d, e, c, c', c'') . The V₀ subcomplex is responsible for proton translocation across membranes, while the cytosolic V₁ subcomplex handles ATP hydrolysis .

How does VHA-e contribute to the functional assembly of V-ATPase?

VHA-e appears to be required for V-ATPase assembly, as its absence results in a Vma– phenotype in yeast, indicating compromised V-ATPase function . Research indicates that VHA-e interacts with Vma21p, an assembly factor, and is suggested to complete the assembly process of the V-ATPase complex .

During assembly, VHA-e likely contributes to the proper organization of other V₀ subunits. The incorporation of VHA-d into the complex appears to follow the interaction between the assembly factor Voa1p, VHA-c, and VHA-c", potentially creating a binding pocket for VHA-d . This sequential assembly process highlights the importance of each subunit, including VHA-e, in building a functional V-ATPase.

What is the subcellular localization pattern of VHA-e in plants?

In plants, VHA-e is notably absent from the vacuole . This contrasts with the distribution of some other V-ATPase subunits that can be found in vacuolar membranes. The specific localization of VHA-e suggests it may be associated with V-ATPase complexes in other endomembrane compartments such as the trans-Golgi network/early endosome (TGN/EE), which is a major site of V-ATPase activity in plants .

When examining the localization of recombinant VHA-e, researchers should consider that tagging the protein (e.g., with GFP) might affect its localization pattern. Controls utilizing known localization markers for different endomembrane compartments are essential for accurate interpretation.

What are the most effective systems for recombinant expression of VHA-e (VHA-17)?

For recombinant expression of VHA-e, several expression systems can be considered based on research objectives:

When expressing VHA-e, it's crucial to consider its small size (9 kDa) and its membrane-integrated nature with two transmembrane domains . Using fusion tags that enhance stability and solubility (such as MBP or SUMO) can improve expression yields. For functional studies, co-expression with interacting partners like Vma21p may enhance proper folding and stability.

How can researchers optimize purification of functionally active recombinant VHA-e?

Purifying membrane proteins like VHA-e presents specific challenges:

• Membrane extraction: Use mild detergents like DDM, LMNG, or digitonin to solubilize VHA-e while preserving native structure.

• Affinity purification: Implement a two-step purification strategy:

  • Initial capture using affinity tags (His, FLAG, or Strep-tag)

  • Secondary purification via size exclusion chromatography

• Quality assessment protocol:

  • SDS-PAGE and western blotting to confirm purity and identity

  • Circular dichroism to assess secondary structure integrity

  • Dynamic light scattering to evaluate homogeneity

• Functional validation: Consider reconstitution into proteoliposomes to assess if purified VHA-e can participate in V-ATPase assembly with other subunits.

It's worth noting that attempts to purify individual V-ATPase subunits have shown that some subunits form stable subcomplexes, such as VHA-E/VHA-G heterodimers . Similar strategies might be applicable when working with VHA-e, potentially co-purifying it with known interacting partners.

What methods are most effective for studying VHA-e interactions with other V-ATPase subunits?

Several complementary approaches can be employed to study VHA-e interactions:

• Co-immunoprecipitation (Co-IP): Using antibodies against VHA-e or potential interacting partners to pull down protein complexes from cellular lysates. This approach has revealed that VHA-e interacts with Vma21p during assembly .

• Yeast two-hybrid (Y2H) screening: While traditional Y2H may be challenging for membrane proteins, split-ubiquitin membrane Y2H systems are better suited for studying VHA-e interactions.

• Förster Resonance Energy Transfer (FRET): By tagging VHA-e and potential binding partners with appropriate fluorophores, interactions can be monitored in living cells based on energy transfer between fluorophores when proteins are in close proximity.

• Bimolecular Fluorescence Complementation (BiFC): This technique involves splitting a fluorescent protein and fusing each half to potential interacting proteins. Fluorescence is reconstituted only when the proteins interact.

• Crosslinking Mass Spectrometry (XL-MS): Chemical crosslinking followed by mass spectrometry can identify interaction interfaces between VHA-e and other subunits, providing structural insights into the complex.

When designing interaction studies, it's important to consider that some interactions may be transient or dependent on the assembly state of the V-ATPase complex.

How can CRISPR/Cas9 be utilized to study VHA-e function in plants?

CRISPR/Cas9 gene editing offers powerful approaches for studying VHA-e function:

• Complete knockout strategy:

  • Design gRNAs targeting conserved regions of VHA-e genes

  • Screen for homozygous knockout lines

  • Analyze phenotypes related to V-ATPase function, such as altered endomembrane pH or trafficking defects

• Domain-specific mutations:

  • Create targeted mutations in specific domains (transmembrane regions or cytosolic tail)

  • Assess the impact on V-ATPase assembly and function

• Endogenous tagging:

  • Insert fluorescent protein tags at the genomic locus to study native expression levels and localization

  • Add affinity tags for pulldown experiments under native expression conditions

Recent studies have successfully employed CRISPR/Cas9 to generate null alleles of other V-ATPase subunits, revealing essential functions in development . Similar approaches could be applied to VHA-e to determine its specific role in different plant tissues and developmental stages.

How do different isoforms of VHA-e affect V-ATPase assembly and targeting?

In Arabidopsis, two VHA-e isoforms have been identified . Understanding their differential roles requires several research approaches:

• Expression pattern analysis: Determine tissue-specific and developmental expression patterns of each isoform using promoter-reporter constructs or RNA-seq data.

• Isoform-specific localization: Create fluorescently tagged versions of each isoform to determine if they localize to different compartments, similar to how VHA-a isoforms target V-ATPases to specific compartments .

• Complementation studies: Test whether one isoform can functionally replace another in knockout backgrounds.

• Interaction profiling: Determine if different VHA-e isoforms preferentially interact with specific sets of V-ATPase subunits or regulatory proteins.

Research on other V-ATPase subunits has shown that isoform-specific interactions can dictate subcellular targeting. For example, VHA-a1 targets V-ATPase to the TGN/EE while VHA-a2 and VHA-a3 target it to the tonoplast . Similar specificity might exist for VHA-e isoforms, potentially contributing to the functional diversity of V-ATPase complexes.

What role does VHA-e play in V-ATPase disassembly and reassembly during cellular stress?

V-ATPase complexes can undergo reversible disassembly into V₁ and V₀ sectors as a regulatory mechanism, particularly in response to glucose deprivation in yeast. The role of VHA-e in this process could be investigated through:

• Stress response experiments: Monitor the association/dissociation of VHA-e with other V-ATPase subunits under various stress conditions (nutrient limitation, salt stress, pH stress).

• Phosphorylation state analysis: Determine if VHA-e undergoes post-translational modifications during stress responses that might regulate complex stability.

• Time-course imaging: Using fluorescently tagged VHA-e, track its localization and dynamics during stress and recovery phases.

• Mutational analysis: Create phospho-mimetic or phospho-dead mutations at potential regulatory sites to assess their impact on V-ATPase assembly/disassembly.

Understanding how VHA-e contributes to dynamic regulation of V-ATPase activity could provide insights into cellular adaptation to environmental stresses, which is particularly relevant for plants facing variable soil conditions.

How can researchers distinguish between direct and indirect effects of VHA-e mutations?

Distinguishing direct from indirect effects requires multiple complementary approaches:

• Acute inactivation strategies: Use systems like auxin-inducible degron tags to rapidly deplete VHA-e and observe immediate versus delayed effects.

• Structure-function analysis: Create a panel of point mutations or truncations in different domains of VHA-e to identify which regions are critical for specific functions.

• Rescue experiments: Test if wild-type VHA-e expression can restore phenotypes, and compare with rescue using related proteins (e.g., VHA-e from other species).

• Temporal analysis: Monitor the sequence of events following VHA-e disruption to establish cause-effect relationships.

• Systems biology approach: Integrate transcriptomic, proteomic, and metabolomic data to build networks of responses to VHA-e perturbation.

When interpreting results, consider that V-ATPase function affects multiple cellular processes, including pH homeostasis, membrane trafficking, and ion balance. Changes in these parameters can have cascading effects that may be difficult to attribute directly to VHA-e function.

What controls should be included when studying VHA-e localization using fluorescent protein tags?

Proper controls for localization studies include:

• Tag position controls: Compare N- and C-terminal tags to ensure the tag doesn't disrupt localization signals.

• Functionality tests: Verify that tagged VHA-e complements vha-e mutant phenotypes, confirming the fusion protein is functional.

• Expression level controls: Use endogenous promoters when possible, as overexpression can lead to mislocalization.

• Colocalization markers: Include established markers for different compartments (TGN/EE, Golgi, ER, vacuole) to precisely define VHA-e localization.

• Drug treatments: Use trafficking inhibitors (Brefeldin A, Concanamycin A) to test if VHA-e localization depends on active trafficking or V-ATPase activity.

• FRAP (Fluorescence Recovery After Photobleaching) analysis: Determine if VHA-e is stably associated with membranes or undergoes dynamic cycling.

When studying fluorescently tagged proteins, dominant-negative effects of Sar1 GTPase expression can be used to assess ER exit requirements, as demonstrated with other V-ATPase subunits . This approach could reveal whether VHA-e follows similar trafficking pathways to other V-ATPase components.