Active plasma membrane P-type H+-ATPase reconstituted into nanodiscs is a monomer

Abstract

Plasma membrane H(+)-ATPases form a subfamily of P-type ATPases responsible for pumping protons out of cells and are essential for establishing and maintaining the crucial transmembrane proton gradient in plants and fungi. Here, we report the reconstitution of the Arabidopsis thaliana plasma membrane H(+)-ATPase isoform 2 into soluble nanoscale lipid bilayers, also termed nanodiscs. Based on native gel analysis and cross-linking studies, the pump inserts into nanodiscs as a functional monomer. Insertion of the H(+)-ATPase into nanodiscs has the potential to enable structural and functional characterization using techniques normally applicable only for soluble proteins.

Keywords: H+-ATPase; Monomer; Nanodiscs; Plasma Membrane Proton Pumps; Reconstitution of Membrane Transporters; Scaffold Proteins; Surface Plasmon Resonance (SPR).

FIGURE 1.

Purification and analysis of plant PM H⁺-ATPase aha2Δ73. A, purified aha2Δ73 was subjected to SDS-PAGE (lane 1) and Western blot analysis using anti-His (lane 2) and anti-AHA2_{N term} (lane 3) antibodies. B, BN-PAGE analysis of purified aha2Δ73. Lane M, NativeMark protein standards (Invitrogen); lane 1, DDM-solubilized aha2Δ73. The three major bands X1, X2, and X3 are labeled by arrowheads. C, chemical cross-linking of aha2Δ73. 2-Fold dilutions of aha2Δ73 (starting amount ∼5 μg) were incubated with DMS as indicated and subjected to SDS-PAGE analysis. The cross-linking product is specified by an arrowhead. D, proton pumping of vesicle-reconstituted aha2Δ73 monitored by the proton-dependent fluorophore ACMA. Pumping is initiated by the addition of MgSO₄.

FIGURE 2.

Reconstitution of aha2Δ73 into nanodiscs. A, size exclusion chromatography of nanodiscs assembled in the presence (solid line) or absence (dashed line) of aha2Δ73 using MSP1D1(−). Nine fractions from nanodiscs assembled in the presence of aha2Δ73 were collected as indicated by a stippled bar at top of the chromatogram. Absorption at 280 nm is normalized to ease comparison. B, SDS-PAGE of size exclusion chromatography elution, fractions 1–9. C, ATPase activity of 50-μl aliquots from size exclusion chromatography elution, fractions 1–9.

FIGURE 3.

Separation of empty and aha2Δ73-containing nanodiscs by nickel affinity purification. A, nanodiscs were assembled in the presence of His₆-tagged aha2Δ73 using MSP1D1(−) devoid of His₇ tag and subjected to nickel affinity purification. Nine fractions were collected as indicated by a stippled bar at top of the chromatogram. Flow-through and elution peaks are labeled I and II, respectively. mAU, milli-absorbance units. B, SDS-polyacrylamide gel. C, Western blot analysis on flow-through (Ft) and elution fractions 1–9 using anti penta-His antibody. D, ATPase activity of 20-μl aliquots from corresponding fractions.

FIGURE 4.

Purification of nanodisc-embedded aha2Δ73. Nanodiscs were assembled in the presence of His₆-tagged aha2Δ73 using MSP1D1(−) devoid of His₇ tag and subjected to nickel affinity purification as shown in Fig. 3. A, size exclusion chromatography of flow-through sample I (dashed line) and elution sample II (solid line). Six fractions were collected as indicated by a stippled bar at top of the chromatogram. Absorption at 280 nm is normalized to ease comparison. The column was calibrated using standard molecular weight marker proteins as described under “Experimental Procedures.” B, SDS-polyacrylamide gel of fractions 1–6 obtained after size exclusion chromatography of elution sample II.

FIGURE 5.

Visualization of individual nanodiscs by transmission electron microscopy. Negative staining with uranyl acetate of a field of empty nanodiscs (A1) and ND-AHA2 (B1) is shown. Details from empty nanodiscs (A2 and A3) and ND-AHA2 (B2 and B3) are given. Scale bars represents 100 nm (A1 and B1) and 10 nm (A2, A3, B2, and B3). C, histograms showing the measured diameters for nanodiscs and ND-AHA2. Diameter of empty discs 10.22 ± 1.09 nm, n = 100. Diameter of ND-AHA2 10.76 ± 1.38 nm, n = 100. D, illustration of ND-AHA2 complex with added approximate dimensions. The MSPs are cyan; lipid acyl chains are gray, and lipid head groups are modeled as blue spheres. The structure of aha2Δ73 is colored according to the different domains (7); the 10 transmembrane segments are brown, and the N, P, and A domains are red, blue, and yellow, respectively. The model was prepared using Visual Molecular Dynamics (62).

FIGURE 6.

Analysis of nanodiscs by BN-PAGE and chemical cross-linking. A, BN-PAGE analysis. Lane 1, empty nanodiscs; lane 2, ND-AHA2; lane 3, DDM-solubilized aha2Δ73; lane 4, SDS-solubilized ND-AHA2; lane 5, SDS-solubilized empty nanodiscs. Stokes diameters for select standards are labeled to the right. B, SDS-PAGE analysis of chemical cross-linking of empty nanodiscs with DMS as indicated. Cross-linked MSP dimers (D1 and D2) are specified by arrowheads. Lanes 1 and 2, ∼5 μg of empty nanodiscs; lane 3, ∼2.5 μg of empty nanodiscs. C, SDS-PAGE (left) and Western blot (right) analysis of chemical cross-linking of aha2Δ73 and ND-AHA2 with DMS using anti AHA2_cat (lanes 1–4) and ApoA-I_Cterm (lanes 3* and 4*) antibodies. Cross-linked MSP dimers (D1 and D2) and aha2Δ73/MSP1D1(−) complexes (C1, C2, and C3) are specified by arrowheads. Lanes 1, aha2Δ73; lanes 2, aha2Δ73 incubated with DMS; lanes 3 and 3*, ND-AHA2; lanes 4 and 4*, ND-AHA2 incubated with DMS.

FIGURE 7.

Vanadate sensitivity of ATP hydrolytic activity of aha2Δ73 reconstituted in vesicle (open circles) and nanodisc (filled circles). Data are representative of at least two independent experiments and expressed as percentage of control measured in the absence of vanadate. Straight lines were fitted to the experimental data points to estimate the IC₅₀ values (5.8 μm for vesicles; 4.4 μm for nanodiscs). Inset, confirmation of proton pumping of vesicle-reconstituted aha2Δ73 using the proton-dependent fluorophore ACMA. Pumping was initiated by the addition of MgSO₄ (peak 1) and the resulting proton gradient was dissipated by addition of carbonyl cyanide m-chlorophenylhydrazone (peak 2).

FIGURE 8.

Capture of aha2Δ73 reconstituted in nanodiscs on penta-His antibody sensor chip surface. The arrows mark start and end of injection and start of regeneration, respectively. A, immobilization of a mixed sample of empty nanodiscs and ND-AHA2. Concentrations of 0, 0.5, 1, 5, 10, and 15 μg/ml mixed samples was captured on the surface, and dissociation was observed for 10 min before regeneration. B, capture of 10 μg/ml ND-AHA2 separated from empty nanodiscs (black). Red curve shows sample run of 10 μg/ml empty nanodiscs assembled from MSP1D1(−).