ABCG25 Antibody

What is ABCG25 and what is its primary function in plants?

ABCG25 is an ATP-binding cassette (ABC) transporter belonging to the G subfamily in Arabidopsis thaliana. It functions as an abscisic acid (ABA) exporter, transporting ABA synthesized in vascular tissues to guard cells, thus regulating stomatal closure and water conservation in plants . The protein utilizes ATP hydrolysis to drive ABA efflux across cell membranes, a function that has been established through various heterologous expression systems including Xenopus oocytes and Sf9 insect cells .

Since ABA is synthesized inside cells, the efflux of ABA to the extracellular space represents the first step in its hormonal signaling pathway, except when moving through plasmodesmata. ABCG25 was the first ABA exporter identified in plants and plays a crucial role in plant responses to environmental stresses, particularly drought conditions .

What is the structural organization of ABCG25?

ABCG25 exists as a homodimeric structure with two-fold symmetry, consisting of nucleotide-binding domains (NBDs) and transmembrane domains (TMDs). Recent cryo-EM studies have revealed the three-dimensional architecture of ABCG25 in different conformational states . In the apo and ABA-bound states, ABCG25 adopts an inward-facing conformation with a cone-shaped cavity formed by opposing TMDs .

The protein undergoes significant conformational changes during its transport cycle. In the ATP-bound state, dramatic rearrangements occur in both NBD and TMD domains. The ATP-binding-induced NBD closure is transduced to the TMD via a three-helix bundle connecting these domains, resulting in substrate translocation .

Which experimental systems are most effective for studying ABCG25?

ABCG25 is effectively studied using heterologous expression systems including:

• Xenopus oocytes

• Spodoptera frugiperda 9 (Sf9) insect cells

• Membrane vesicles derived from ABCG25-expressing Sf9 cells

These systems allow researchers to assess ABA transport activity through radioisotope-labelled ABA ([³H]-ABA) assays under controlled conditions. The baculovirus expression system in Sf9 cells has proven particularly valuable for producing sufficient quantities of protein for both functional studies and structural analysis by cryo-EM .

How is ABCG25 typically expressed and purified for experimental studies?

ABCG25 expression and purification typically follows this protocol:

• The full-length Arabidopsis thaliana ABCG25 cDNA is cloned into a pFastBac vector with an N-terminal Flag tag (DYKDDDDK)

• Bacmids are generated in DH10Bac cells and baculoviruses are produced and amplified in Sf9 cells

• Cells are harvested 48 hours after viral infection and resuspended in buffer containing 25 mM HEPES-NaOH (pH 7.4) and 150 mM NaCl

• The suspension is supplemented with 1.5% (w/v) DDM (n-dodecyl-β-D-maltopyranoside) with or without 0.3% (w/v) CHS (cholesteryl hemisuccinate) and protease inhibitor cocktail

• After incubation at 4°C for 2 hours, the supernatant is isolated by centrifugation and incubated with anti-Flag M2 affinity gel

• Further purification may include size exclusion chromatography to ensure protein homogeneity

This approach allows researchers to obtain highly purified ABCG25 suitable for functional and structural studies.

What methods can effectively detect and quantify ABCG25 expression?

ABCG25 expression can be detected and quantified through Western blotting analysis using the following procedure:

• Whole cell lysate samples expressing ABCG25 (wild-type or mutants) are run on precast PAGE gels (typically 8-20%) for 120 minutes at 100V

• Proteins are transferred to polyvinylidene fluoride (PVDF) membranes using semi-dry electrophoretic transfer systems

• Membranes are blocked with 5% non-fat milk for 1 hour at room temperature

• Primary antibody incubation uses anti-FLAG tag mouse monoclonal antibody (1:3,000 dilution) for 1 hour

• After washing with TBST buffer, membranes are incubated with HRP-conjugated secondary antibody

• Signal detection follows standard chemiluminescence protocols

This approach allows researchers to verify expression levels before proceeding with functional assays or protein purification.

How can ABCG25 transport activity be measured in cellular assays?

ABCG25 transport activity can be assessed using two complementary cell-based assays:

[³H]-ABA Loading Assay:

• Cells expressing ABCG25 or control cells are resuspended in PBS citrate buffer (pH 5.5) containing 6.6 nM [³H]-ABA

• The loading process is stopped by centrifugation at specific timepoints

• Cells are washed twice with ice-cold buffer and resuspended with buffer plus 1% Triton X-100 for cell lysis

• Radioactivity is determined using liquid scintillation counting

ABA Efflux Assay:

• Cells are first loaded with [³H]-ABA for 10 minutes

• After washing, cells are resuspended in [³H]-ABA-free buffer

• Aliquots are taken at various timepoints, and remaining cellular radioactivity is measured

• Cells expressing ABCG25 show substantially decreased residual [³H]-ABA compared to control cells

These complementary approaches allow researchers to quantitatively measure ABCG25-mediated ABA transport activity and evaluate the effects of mutations or inhibitors.

How does ABCG25 recognize and bind to its substrate ABA?

ABCG25 recognizes ABA through a specific binding site located in the middle of the transmembrane region, consisting mainly of hydrophobic and polar residues - a conserved feature observed in human ABCG1, ABCG2, and ABCG5/G8 structures . Cryo-EM studies reveal that ABA lies at the middle of the TMDs, with the carboxylate tail pointing to the bottom of the cavity and the ring head group pointing to the cytoplasmic side .

A positively charged lysine residue (K545) at the entrance of the cavity plays an important role in recruiting the negatively charged ABA substrate, as K545A mutation reduces ABA efflux activity . Interestingly, the ABA binding site in ABCG25 overlaps with positions where sterol molecules have been observed in related ABCG transporters, suggesting potential evolutionary relationships in substrate recognition mechanisms .

What structural changes occur during the ABCG25 transport cycle?

ABCG25 undergoes a series of conformational changes during its transport cycle:

• Resting state: NBDs of opposing subunits are separated, and TMDs form a cytosol-facing cavity ready for substrate binding

• ABA binding: When ABA enters from the cytosolic side, subtle conformational changes occur in either the TMD or NBD regions

• ATP binding: When ATP binds to the NBDs, they close together, causing rotational and translational changes that transmit to the TMDs through the connecting helix bundles

• Substrate translocation: The inner parts of the TMs from opposing subunits move to close the cytosolic cavity and push bound ABA to translocate along the channel mainly formed by TM2 and TM5

• Substrate release: The extracellular gate transiently opens, releasing ABA to the extracellular space

• Reset: ATP hydrolysis and product release from the NBDs reset ABCG25 to the resting state, ready for the next transport cycle

This alternating access mechanism is consistent with the established transport cycle of other ABC transporters across all kingdoms of life.

How do ABCG25 structures compare with other ABCG family transporters?

Despite functional differences, ABCG25 shares significant structural similarities with human ABCG transporters:

• The ATP-bound state of Arabidopsis ABCG25 aligns well with the structures of human ABCG1 EQ or ABCG2 EQ mutants in the same state (RMSDs of 1.97 Å and 1.83 Å, respectively)

• TMDs from these proteins align well except for changes in TM5b and the extracellular loop between TM5c and TM6

• NBDs also align well with similar ATP binding profiles, and key residues involved in ATP-binding and hydrolysis are highly conserved among Arabidopsis ABCG25, hABCG1, and hABCG2

• The ABA binding site in ABCG25 is similar to the observed cholesterol binding site (site 2) in human ABCG5/ABCG8 and hABCG2, suggesting evolutionary conservation in substrate binding mechanisms

These structural similarities provide valuable insights into the evolutionary relationships between plant and human ABC transporters despite their diverse functions.

What are the major challenges in generating effective antibodies against ABCG25?

Generating effective antibodies against ABCG25 presents several challenges:

• As a membrane protein, ABCG25 has limited exposed epitopes when embedded in the lipid bilayer

• The high degree of conservation between ABCG family members may lead to cross-reactivity

• The conformational changes during the transport cycle mean certain epitopes may only be accessible in specific states

• Expression levels in native tissues may be relatively low

Researchers often circumvent these challenges by using epitope tags like the Flag tag (DYKDDDDK) and corresponding commercially available antibodies for detection and purification, as demonstrated in the structural studies . For studies requiring native protein detection, antibodies against unique, accessible regions of ABCG25 would need to be carefully designed and validated.

How can cryo-EM be effectively applied to study ABCG25 structure and function?

Cryo-EM has proven highly effective for studying ABCG25 structure through a methodical approach:

• Sample preparation: Purified ABCG25 protein (4 μl aliquots) is applied to glow-discharged holey carbon grids, blotted, and plunge-frozen in liquid ethane

• Capturing functional states: Different states are captured by:

  • Apo state: Using purified ABCG25 WT protein without additives

  • ABA-bound state: Incubating purified protein with 2 mM ABA before grid preparation

  • ATP-bound state: Using the E232Q mutant incubated with 5 mM ATP and 10 mM MgCl₂

• Data acquisition: Images are collected on a Titan Krios electron microscope operating at 300 kV, equipped with energy filter and direct electron detector, using specific parameters for magnification, defocus, and dose fractionation

• Model building: Initial models are built by docking AlphaFold2 predictions into cryo-EM maps, followed by manual adjustments and de novo building based on the density

• Refinement and validation: Structure refinements are performed in real space with overfitting monitored through cross-validation approaches

This comprehensive approach has successfully yielded ABCG25 structures at resolutions of 3.0-3.2 Å, revealing critical insights into its transport mechanism .

How might understanding ABCG25 structure contribute to improving plant stress tolerance?

Understanding ABCG25 structure has significant implications for agricultural applications:

• The detailed ABA binding site information could enable rational design of small molecules that modulate ABCG25 activity, potentially enhancing plant drought response

• Knowledge of the conformational changes during transport could inform genetic engineering strategies to optimize ABA distribution in plants under stress conditions

• The identification of key functional residues provides targets for precision breeding approaches using CRISPR/Cas9 or other genome editing technologies

• Understanding the structural basis of sterol interactions with ABCG25 might reveal new regulatory mechanisms that could be exploited to enhance stress tolerance

Since ABCG25 exports ABA from vascular tissues to guard cells to regulate stomatal closure, targeted modifications to enhance its activity during drought conditions could improve water use efficiency in crops.

What are the current knowledge gaps regarding ABCG25 regulation and interaction partners?

Despite recent structural advances, several knowledge gaps remain:

• The mechanisms regulating ABCG25 expression, trafficking, and degradation in response to environmental stresses remain poorly understood

• Potential protein interaction partners that might modulate ABCG25 activity or localization have not been fully characterized

• The role of post-translational modifications in regulating ABCG25 function requires further investigation

• The distinction between ABCG25 and other ABA transporters in Arabidopsis (like ABCG31) remains unclear, as key residues forming the binding site are not highly conserved between them despite similar functions

• The question of whether sterols serve as transport regulators or alternative substrates of ABCG25 in Arabidopsis remains unresolved

Addressing these questions will require integrative approaches combining structural biology with cellular, genetic, and biochemical techniques.