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Active Plasma Membrane P-type H+-ATPase Reconstituted into Nanodiscs Is a Monomer*

  • Bo Højen Justesen
    Affiliations
    Centre for Membrane Pumps in Cells and Disease-PUMPKIN

    Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C and
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  • Randi Westh Hansen
    Affiliations
    Bionanotechnology and Nanomedicine Laboratory, Department of Chemistry and Nano-science Center, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen, Denmark
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  • Helle Juel Martens
    Affiliations
    Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C and
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  • Lisa Theorin
    Affiliations
    Centre for Membrane Pumps in Cells and Disease-PUMPKIN

    Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C and
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  • Michael G. Palmgren
    Affiliations
    Centre for Membrane Pumps in Cells and Disease-PUMPKIN

    Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C and
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  • Karen L. Martinez
    Affiliations
    Bionanotechnology and Nanomedicine Laboratory, Department of Chemistry and Nano-science Center, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen, Denmark
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  • Thomas Günther Pomorski
    Correspondence
    To whom correspondence may be addressed: Dept. of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871, Frederiksberg C, Denmark.
    Affiliations
    Centre for Membrane Pumps in Cells and Disease-PUMPKIN

    Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C and
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  • Anja Thoe Fuglsang
    Correspondence
    To whom correspondence may be addressed: Dept. of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871, Frederiksberg C, Denmark.
    Affiliations
    Centre for Membrane Pumps in Cells and Disease-PUMPKIN

    Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C and
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  • Author Footnotes
    * This work was supported by the UNIK research initiative of the Danish Ministry of Science, Technology, and Innovation through the “Center for Synthetic Biology” at University of Copenhagen, by the Danish National Research Foundation through the PUMPKIN Center of Excellence, and by an equipment grant from the Carlsberg Foundation.
    This article contains supplemental Figs. S1 and S2.
Open AccessPublished:July 08, 2013DOI:https://doi.org/10.1074/jbc.M112.446948
      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.
      Background: The plasma membrane H+-ATPase generates electrochemical gradients in plants and fungi. The minimal subunit organization required for activity is not known.
      Results: We developed a protocol for reconstitution of active H+-ATPase in nanodiscs.
      Conclusion: The minimal functional unit of the H+-ATPase is a monomer.
      Significance: The plasma membrane H+-ATPase functions like well characterized cation pumping P-type ATPases.

      Introduction

      The plasma membrane (PM)
      The abbreviations used are: PM, plasma membrane; AHA2, A. thaliana H+-ATPase isoform 2; MSP, membrane scaffold protein; SPR, surface plasmon resonance; DDM, n-dodecyl β-d-maltoside; aha2Δ73, 73 amino acid C-terminal truncated A. thaliana plasma membrane H+-ATPase isoform 2; ACMA, 9-amino-6-chloro-2-methoxyacridine; SEC, size exclusion chromatography; ND-AHA2, nanodiscs containing aha2Δ73; DMS, dimethyl suberimidate; BN-PAGE, Blue native-PAGE; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; POPG, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol); DMS, dimethyl suberimidate; PC, phosphocholine.
      H+-ATPase is a prominent member of the P-type ATPases, a large superfamily of proteins pumping ions and lipids across cellular membranes. This family of proteins forms a phosphorylated intermediate during the catalytic cycle, hence P-type, and is divided into five subfamilies, P1 to P5, each having different substrate specificities (
      • Axelsen K.B.
      • Palmgren M.G.
      Evolution of substrate specificities in the P-type ATPase superfamily.
      ,
      • Palmgren M.G.
      • Nissen P.
      P-type ATPases.
      ). The PM H+-ATPase belongs to the P3 subfamily only found in plant and fungi and is responsible for proton extrusion out of the cell. The resulting proton gradient is used by proton-coupled transporters and secondary active transport of nutrients and metabolites across the PM. In addition to the fundamental role in nutrient uptake, the plant PM H+-ATPase is involved in a number of processes during plant growth and development, such as stomatal movement and cell elongation. Also, responses to both biotic and abiotic stresses often require the activation of the PM H+-ATPase (
      • Hager A.
      Role of the plasma membrane H+-ATPase in auxin-induced elongation growth: historical and new aspects.
      ,
      • Duby G.
      • Poreba W.
      • Piotrowiak D.
      • Bobik K.
      • Derua R.
      • Waelkens E.
      • Boutry M.
      Activation of plant plasma membrane H+-ATPase by 14-3-3 proteins is negatively controlled by two phosphorylation sites within the H+-ATPase C-terminal region.
      ,
      • Fuglsang A.T.
      • Paez-Valencia J.
      • Gaxiola R.A.
      ).
      A large body of information on the structure, function, and regulation of PM H+-ATPases from the P3 subfamily has been compiled during the last decade. Cryo-electron microscopic images of the Neurospora crassa H+-ATPase (
      • Cyrklaff M.
      • Auer M.
      • Kühlbrandt W.
      • Scarborough G.A.
      2-D structure of the Neurospora crassa plasma membrane ATPase as determined by electron cryomicroscopy.
      ) and the recently solved crystal structure of the Arabidopsis thaliana H+-ATPase isoform 2 (AHA2) (
      • Pedersen B.P.
      • Buch-Pedersen M.J.
      • Morth J.P.
      • Palmgren M.G.
      • Nissen P.
      Crystal structure of the plasma membrane proton pump.
      ) revealed the presence of 10 transmembrane helices and three large cytoplasmic domains, including the phosphorylation and ATP-binding sites. The overall structure of AHA2 resembles the one known for the well studied sarco/endoplasmic reticulum Ca2+-ATPase pumps, which belongs to the P2 subfamily (
      • Axelsen K.B.
      • Palmgren M.G.
      Evolution of substrate specificities in the P-type ATPase superfamily.
      ).
      Oligomerization is a common feature of members of the P-type ATPase family of pumps. Thus, representative members of the P-ATPase family, such as the sarcoplasmic reticulum Ca2+ pump (
      • Andersen J.P.
      Monomer-oligomer equilibrium of sarcoplasmic reticulum Ca-ATPase and the role of subunit interaction in the Ca2+ pump mechanism.
      ) and the Na+/K+ pump (
      • Blanco G.
      • Koster J.C.
      • Mercer R.W.
      The α subunit of the Na,K-ATPase specifically and stably associates into oligomers.
      ), self-associate. The monomer of the sarcoplasmic reticulum Ca2+-ATPase and the α-β protomers of the Na+/K+- and H+/K+-ATPases are capable of performing all the steps of the reaction cycle (
      • Andersen J.P.
      Monomer-oligomer equilibrium of sarcoplasmic reticulum Ca-ATPase and the role of subunit interaction in the Ca2+ pump mechanism.
      ,
      • Vilsen B.
      • Andersen J.P.
      • Petersen J.
      • Jørgensen P.L.
      Occlusion of 22Na+ and 86Rb+ in membrane-bound and soluble protomeric α β-units of Na,K-ATPase.
      ,
      • Dach I.
      • Olesen C.
      • Signor L.
      • Nissen P.
      • le Maire M.
      • Møller J.V.
      • Ebel C.
      Active detergent-solubilized H+,K+-ATPase is a monomer.
      ). The human PM Ca2+-ATPase is a P2B-ATPase that resembles PM H+-ATPases by having an extended C-terminal regulatory domain. PM Ca2+-ATPase isolated from human erythrocytes undergoes reversible, enzyme concentration-dependent oligomerization (
      • Kosk-Kosicka D.
      • Bzdega T.
      Activation of the erythrocyte Ca2+-ATPase by either self-association or interaction with calmodulin.
      ,
      • Kosk-Kosicka D.
      • Bzdega T.
      • Wawrzynow A.
      Fluorescence energy transfer studies of purified erythrocyte Ca2+-ATPase. Ca2+-regulated activation by oligomerization.
      ). This oligomerization process involves the C-terminal calmodulin-binding domain of the pump (
      • Kosk-Kosicka D.
      • Bzdega T.
      • Wawrzynow A.
      Fluorescence energy transfer studies of purified erythrocyte Ca2+-ATPase. Ca2+-regulated activation by oligomerization.
      ,
      • Kosk-Kosicka D.
      • Bzdega T.
      Effects of calmodulin on erythrocyte calcium ATPase activation and oligomerization.
      ,
      • Levi V.
      • Rossi J.P.
      • Castello P.R.
      • González Flecha F.L.
      Structural significance of the plasma membrane calcium pump oligomerization.
      ) and likely results in an activated high affinity state of the pump (
      • Sackett D.L.
      • Kosk-Kosicka D.
      The active species of plasma membrane Ca-ATPase are a dimer and a monomer-calmodulin complex.
      ).
      An early study concluded that the functional unit of the fungal Neurospora PM H+-ATPase reconstituted with excess lipid in liposomes might be a monomer (
      • Goormaghtigh E.
      • Chadwick C.
      • Scarborough G.A.
      Monomers of the Neurospora plasma membrane H+-ATPase catalyze efficient proton translocation.
      ). Subsequent structural studies have revealed the presence of PM H+-ATPase dimers and hexamer complexes, but their functional roles remain to be elucidated (
      • Kanczewska J.
      Activation of the plant plasma membrane H+-ATPase by phosphorylation and binding of 14-3-3 proteins converts a dimer into a hexamer.
      ,
      • Ottmann C.
      • Marco S.
      • Jaspert N.
      • Marcon C.
      • Schauer N.
      • Weyand M.
      • Vandermeeren C.
      • Duby G.
      • Boutry M.
      • Wittinghofer A.
      • Rigaud J.-L.
      • Oecking C.
      Structure of a 14-3-3 coordinated hexamer of the plant plasma membrane H+-ATPase by combining x-ray crystallography and electron cryomicroscopy.
      ). A distinct characteristic of P-type PM H+-ATPases is the presence of a C-terminal regulatory domain (R-domain) (
      • Pedersen B.P.
      • Buch-Pedersen M.J.
      • Morth J.P.
      • Palmgren M.G.
      • Nissen P.
      Crystal structure of the plasma membrane proton pump.
      ), and activation of pump activity occurs by phosphorylation-dependent binding of 14-3-3 regulatory proteins to this domain (
      • Jelich-Ottmann C.
      • Weiler E.W.
      • Oecking C.
      Binding of regulatory 14-3-3 proteins to the C terminus of the plant plasma membrane H+-ATPase involves part of its autoinhibitory region.
      ,
      • Fuglsang A.T.
      • Borch J.
      • Bych K.
      • Jahn T.P.
      • Roepstorff P.
      • Palmgren M.G.
      The binding site for regulatory 14-3-3 protein in plant plasma membrane H+-ATPase: Involvement of a region promoting phosphorylation-independent interaction in addition to the phosphorylation-dependent C-terminal end.
      ). A three-dimensional reconstruction of purified PM H+-ATPase/14-3-3 complex suggested a hexameric arrangement (
      • Ottmann C.
      • Marco S.
      • Jaspert N.
      • Marcon C.
      • Schauer N.
      • Weyand M.
      • Vandermeeren C.
      • Duby G.
      • Boutry M.
      • Wittinghofer A.
      • Rigaud J.-L.
      • Oecking C.
      Structure of a 14-3-3 coordinated hexamer of the plant plasma membrane H+-ATPase by combining x-ray crystallography and electron cryomicroscopy.
      ) in line with the reported structure from x-ray crystallography on two-dimensional crystals (
      • Huang L.-S.
      • Berry E.A.
      Purification and characterization of the proton translocating plasma membrane ATPase of red beet storage tissue.
      ,
      • Auer M.
      • Scarborough G.A.
      • Kühlbrandt W.
      Three-dimensional map of the plasma membrane H+-ATPase in the open conformation.
      ). Whether the activation of the pump correlates with its oligomerization status is not known. Likewise, it remains uncertain whether the monomeric state is active like it is in other P-type ATPases.
      Reconstitution of membrane-embedded proteins in soluble nanoscale lipid bilayers, also termed nanodiscs, is a novel technique in the study of membrane proteins (
      • Bayburt T.H.
      • Grinkova Y.V.
      • Sligar S.G.
      Self-assembly of discoidal phospholipid bilayer nanoparticles with membrane scaffold proteins.
      ). Assembled from membrane scaffold proteins (MSPs), a nanodisc consists of two MSPs encircling a planar lipid bilayer in a double-belt configuration. The amphiphilic helical structure of the MSPs shields the hydrophobic edge of the lipid bilayer and stabilizes discrete disc sizes determined by the length of the MSPs, resulting in diameters ranging from 10 to 17 nm (
      • Denisov I.G.
      • Grinkova Y.V.
      • Lazarides A.A.
      • Sligar S.G.
      Directed self-assembly of monodisperse phospholipid bilayer nanodiscs with controlled size.
      ,
      • Ritchie T.K.
      • Grinkova Y.V.
      • Bayburt T.H.
      • Denisov I.G.
      • Zolnerciks J.K.
      • Atkins W.M.
      • Sligar S.G.
      Reconstitution of membrane proteins in phospholipid bilayer nanodiscs.
      ). Advantages in using this system include water solubility, monodispersity, flexible lipid composition, access to both sides of the bilayer simultaneously, and controlled stoichiometry of disc to target protein. Despite still being a relative novel approach for membrane protein study, several examples of membrane proteins reconstituted into nanodiscs exist, e.g. G-protein-coupled receptors (
      • Bayburt T.H.
      • Leitz A.J.
      • Xie G.
      • Oprian D.D.
      • Sligar S.G.
      Transducin activation by nanoscale lipid bilayers containing one and two rhodopsins.
      ,
      • Bayburt T.H.
      • Vishnivetskiy S.A.
      • McLean M.A.
      • Morizumi T.
      • Huang C.-C.
      • Tesmer J.J.
      • Ernst O.P.
      • Sligar S.G.
      • Gurevich V.V.
      Monomeric rhodopsin is sufficient for normal rhodopsin kinase (GRK1) phosphorylation and arrestin-1 binding.
      ,
      • Leitz A.J.
      • Bayburt T.H.
      • Barnakov A.N.
      • Springer B.A.
      • Sligar S.G.
      Functional reconstitution of β2-adrenergic receptors utilizing self-assembling nanodisc technology.
      ,
      • Whorton M.R.
      • Bokoch M.P.
      • Rasmussen S.G.
      • Huang B.
      • Zare R.N.
      • Kobilka B.
      • Sunahara R.K.
      A monomeric G protein-coupled receptor isolated in a high density lipoprotein particle efficiently activates its G protein.
      ,
      • Whorton M.R.
      • Jastrzebska B.
      • Park P.S.
      • Fotiadis D.
      • Engel A.
      • Palczewski K.
      • Sunahara R.K.
      Efficient coupling of transducin to monomeric rhodopsin in a phospholipid bilayer.
      ) and cytochrome P450s (
      • Baas B.J.
      • Denisov I.G.
      • Sligar S.G.
      Homotropic cooperativity of monomeric cytochrome P450 3A4 in a nanoscale native bilayer environment.
      ,
      • Civjan N.R.
      • Bayburt T.H.
      • Schuler M.A.
      • Sligar S.G.
      Direct solubilization of heterologously expressed membrane proteins by incorporation into nanoscale lipid bilayers.
      ,
      • Denisov I.G.
      • Baas B.J.
      • Grinkova Y.V.
      • Sligar S.G.
      Cooperativity in Cytochrome P450 3A4: Linkages in substrate binding, spin state, uncoupling, and product formation.
      ,
      • Duan H.
      • Civjan N.R.
      • Sligar S.G.
      • Schuler M.A.
      Co-incorporation of heterologously expressed Arabidopsis cytochrome P450 and P450 reductase into soluble nanoscale lipid bilayers.
      ). Nanodisc-reconstituted membrane proteins can essentially be handled as soluble proteins in aqueous solution. This facilitates the application of analytical techniques that are normally difficult to use in the study of membrane proteins such as surface plasmon resonance (SPR), nuclear magnetic resonance spectroscopy, as well as electron paramagnetic resonance spectroscopy.
      Here, we demonstrate the reconstitution of the A. thaliana PM P-type proton ATPase isoform 2 into the nanodisc. Using native gel analysis, cross-linking, and transmission electron microscopy, we demonstrate that the nanodisc-embedded AHA2 is an active monomer. Reconstitution of the PM H+-ATPase into nanodiscs enables further functional and structural analysis of this family of pumps in complex with regulatory proteins. This is demonstrated by the ability of soluble nanodiscs with reconstituted PM H+-ATPase to be selectively immobilized for SPR analysis.

      EXPERIMENTAL PROCEDURES

      Materials

      Phospholipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG) were used as received from Avanti Polar Lipids Inc. (Birmingham, AL). Bio-Beads SM-2 absorbent was purchased from Bio-Rad. Detergent n-dodecyl β-d-maltoside (DDM) was obtained from Glycon Biochemicals (Luckenwalde, Germany). Unless indicated otherwise, all other chemicals and reagents were obtained from Sigma.

      Expression and Purification of Plasma Membrane H+-ATPase Isoform 2

      A plasmid based on the multicopy vector YEp-351 (
      • Hill J.E.
      • Myers A.M.
      • Koerner T.J.
      • Tzagoloff A.
      Yeast/E. coli shuttle vectors with multiple unique restriction sites.
      ) was used for expression in yeast, containing the coding sequence of a 73-amino acid C-terminal truncated A. thaliana PM H+-ATPase isoform 2 (aha2Δ73) (plasmid pMP1280) (
      • Buch-Pedersen M.J.
      • Palmgren M.G.
      Conserved Asp684 in transmembrane segment M6 of the plant plasma membrane P-type proton pump AHA2 is a molecular determinant of proton translocation.
      ). The coding sequence is under the control of the PMA1 promoter and in fusion with a C-terminal Met-Arg-Gly-Ser-His6 (MRGSH6) tag. For expression, the Saccharomyces cerevisiae strain RS-72 (MATa ade1-100 his4-519 leu2-3,112) (
      • Cid A.
      • Perona R.
      • Serrano R.
      Replacement of the promoter of the yeast plasma membrane ATPase gene by a galactose-dependent promoter and its physiological consequences.
      ) was transformed and cultured essentially as described previously (
      • Regenberg B.
      • Villalba J.M.
      • Lanfermeijer F.C.
      • Palmgren M.G.
      C-terminal deletion analysis of plant plasma membrane H+-ATPase: yeast as a model system for solute transport across the plant plasma membrane.
      ). In RS-72, the endogenous yeast PM H+-ATPase PMA1 gene is placed under the control of a genomic galactose-dependent promoter. This strain grows in media containing galactose, whereas growth in glucose-based medium requires the complementation of the yeast PM H+-ATPase by the constitutively expressed A. thaliana PM H+-ATPase. The cells were grown and harvested by centrifugation, and membranes were isolated as described previously (
      • Pedersen B.P.
      • Buch-Pedersen M.J.
      • Morth J.P.
      • Palmgren M.G.
      • Nissen P.
      Crystal structure of the plasma membrane proton pump.
      ). All subsequent manipulations were performed at 4 °C, and all buffers contained 0.3 mm phenylmethylsulfonyl fluoride and 3 μg/ml pepstatin A. Membranes were solubilized using DDM at a 3:1 detergent/protein (w/w) ratio in solubilization buffer (50 mm Mes-KOH, pH 6.5, 20% (v/v) glycerol, 1 mm EDTA, 50 mm KCl, 1 mm dithiothreitol) containing 1.2 mm ATP with gentle agitation for 30 min. Insoluble material was removed by centrifugation for 60 min at 100,000 × g. The supernatant containing solubilized A. thaliana H+-ATPase was diluted 1:1 (v/v) with washing buffer WB500 (50 mm Mes-KOH, pH 6.5, 20% (v/v) glycerol, 0.15% (w/v) DDM, 500 mm KCl, 10 mm imidazole) and incubated for 16 h with nickel-nitrilotriacetic acid resin (1 ml of resin, 30 mg of membrane protein) pre-equilibrated in the same buffer. To minimize unspecific binding, the nickel-nitrilotriacetic acid resin was washed with 10 volumes of washing buffer WB250 (as WB500 with 250 mm KCl) followed by 10 volumes of washing buffer WB50 (as WB500 with 50 mm KCl). Bound proteins were eluted with 2 volumes of elution buffer (50 mm Mes-KOH, pH 6.5, 50 mm KCl, 300 mm imidazole, 20% (v/v) glycerol, 0.5 mm dithiothreitol, and 0.075% (w/v) DDM). Centrifugal concentrators (Vivaspin 20, GE Healthcare) with a 30-kDa molecular mass cutoff were used for buffer exchange of the eluted proteins to solubilization buffer containing 0.075% (w/v) DDM in two washing steps. Purified H+-ATPase was finally concentrated to 5–10 g/liter, frozen in liquid nitrogen, and stored at −80 °C.

      Expression and Purification of Membrane Scaffold Proteins

      His7-tagged membrane scaffold protein MSP1D1 (
      • Denisov I.G.
      • Grinkova Y.V.
      • Lazarides A.A.
      • Sligar S.G.
      Directed self-assembly of monodisperse phospholipid bilayer nanodiscs with controlled size.
      ) was expressed in Escherichia coli and purified as described previously (
      • Bayburt T.H.
      • Grinkova Y.V.
      • Sligar S.G.
      Self-assembly of discoidal phospholipid bilayer nanoparticles with membrane scaffold proteins.
      ). The His7 tag on MSP1D1 was removed by treatment with His6-tagged tobacco etch virus protease, overnight at room temperature. After cleavage, the sample was loaded on a nickel-Sepharose column (GE Healthcare), and the cleaved membrane scaffolding protein MSP1D1(−) devoid of the polyhistidine tag was collected in the flow-through, whereas tobacco etch virus protease and any uncleaved protein were retained onto the column.

      Vesicle Reconstitution and Proton Pumping Assay

      Purified PM H+-ATPase was reconstituted into preformed asolectin vesicles as described previously (
      • Lanfermeijer F.C.
      • Venema K.
      • Palmgren M.G.
      Purification of a histidine-tagged plant plasma membrane H+-ATPase expressed in yeast.
      ), using 12 μg of purified protein per 200 μl of Mes-KOH buffer, pH 6.5, containing 50 mm octyl glucoside and 10.6 mm asolectin. Proton pumping was determined using 9-amino-6-chloro-2-methoxyacridine (ACMA) quenching assay (
      • Venema K.
      • Palmgren M.G.
      Metabolic modulation of transport coupling ratio in yeast plasma membrane H+-ATPase.
      ). Briefly, proteoliposomes (20 μl) in 2 ml of buffer (20 mm MOPS-KOH, pH 7.0, 40 mm K2SO4, 25 mm KNO3, 1 μm ACMA, 60 nm valinomycin, 2 mm ATP) were analyzed spectrophotometrically (excitation at 412 nm; emission at 480 nm; bandpass 2 nm, integration time 0.1 s) using a Fluoromax-4 spectrofluorometer (Horiba, Edison, NJ). Proton pumping resulting in ACMA fluorescence quenching was initiated by the addition of MgSO4 to a final concentration of 2 mm and when indicated was dissipated by the addition of 10 μm carbonyl cyanide m-chlorophenylhydrazone.

      Reconstitution and Purification of PM H+-ATPase in Nanodiscs

      Reconstitution of PM H+-ATPase into nanodiscs was based on the method described previously (
      • Bayburt T.H.
      • Grinkova Y.V.
      • Sligar S.G.
      Self-assembly of discoidal phospholipid bilayer nanoparticles with membrane scaffold proteins.
      ). Lipids in chloroform were mixed in glass tubes to achieve the indicated lipid composition. A lipid mixture of POPC/POPG (3:2, molar ratio) was applied in the reconstitution experiments. Excess chloroform was blown off with nitrogen, and lipids were solubilized at a concentration of 50 mm in MEK buffer (Mes-KOH, 1 mm EDTA, 100 mm KCl, pH 6.5) containing 200 mm octyl glucoside and incubated with purified MSP1D1 or MSP1D1(−) with gentle agitation overnight at 4 °C. Purified aha2Δ73 was added to give a final lipid/MSP/H+-ATPase molar ratio of 64:1:0.125 and incubated for 1 h at 4 °C. Detergent was removed by incubation for 4 h at 4 °C with 1 g/ml SM-2 Adsorbent Bio-Beads. Reconstituted nanodiscs were purified and analyzed by size exclusion chromatography (SEC) (Superdex 200 10/300 GL, GE Healthcare) with a flow rate of 0.4 ml/min using MEK buffer as the mobile phase. The column was calibrated according to the manufacturer's instructions using the following known standards: blue dextran (2 MDa); ferritin (400 kDa; 12.2 nm); aldolase (158 kDa; 9.62 nm); conalbumin (75 kDa); ovalbumin (43 kDa; 6.1 nm); carbonic anhydrase (29 kDa); ribonuclease A (13.7 kDa; 3.28 nm), and aprotinin (6.5 kDa) (GE Healthcare). Values for the Stokes diameter of selected standards were plotted at their respective elution volume. Stokes diameter of the reconstituted nanodiscs was estimated by a linear fit of the Stokes diameter of the standard proteins versus the partition coefficient Kav (supplemental Fig. S1). A 1-ml nickel-Sepharose column (GE Healthcare) was used to separate nanodiscs containing histidine-tagged aha2Δ73 from empty nanodiscs assembled with MSP1D1(−). Nanodiscs containing aha2Δ73, from here on denoted ND-AHA2, were eluted using a gradient of 0–300 mm imidazole over a volume of 5 ml in MEK buffer, then buffer-exchanged to MEK using centrifugal concentrators with a 10-kDa molecular mass cutoff (Vivaspin 6, GE Healthcare), and concentrated to a final volume of about 500 μl.

      Cross-linking

      Cross-linking was performed by incubating samples with 10 mm dimethyl suberimidate (DMS; Pierce) in 50 mm MOPS-KOH, pH 7.9, for 2 h at ambient temperature (∼25 °C). This cross-linker was specific for amines and had a spacer arm of 11 Å. The reaction was stopped by addition of SDS-PAGE loading buffer.

      Blue-native PAGE

      Buffer conditions during Blue-native (BN)-PAGE were according to Schagger and von Jagow (
      • Schägger H.
      • von Jagow G.
      Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form.
      ), and a cathode buffer with 0.02% (w/v) Coomassie G-250 was used. Gels were either cast at room temperature using a gravity-based gradient gel maker or bought from Invitrogen (NativePAGETM Novex® 4–16% BisTris gels). For cast gels, the final gel dimensions were 20 × 20 cm with a 1.5-mm spacer and gradient gel from 5–15%. The samples were diluted 1:1–5 in 750 mm amino-n-caproic acid, 50 mm BisTris, pH 7.0, and 0.5 mm Na2EDTA to prevent protein precipitation and facilitate gel loading. NativemarkTM unstained protein standard (Invitrogen) was used for molecular weight estimation. Values for the Stokes diameter of selected standards were obtained and displayed along with the molecular weight (
      • Cavigiolio G.
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      • Ren G.
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      The interplay between size, morphology, stability, and functionality of high density lipoprotein subclasses.
      ,
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      • Bowser S.S.
      The discovery of a novel R-phycoerythrin from an Antarctic red alga.
      ,
      • Radomsky M.L.
      • Whaley K.J.
      • Cone R.A.
      • Saltzman W.M.
      Macromolecules released from polymers: diffusion into unstirred fluids.
      ). For SDS treatment, samples after centrifugation were preincubated with a final concentration of 0.1% SDS for 10 min at room temperature. To account for the apparent size difference between soluble and membrane proteins in BN-PAGE (
      • Wittig I.
      • Beckhaus T.
      • Wumaier Z.
      • Karas M.
      • Schägger H.
      Mass estimation of native proteins by blue native electrophoresis: principles and practical hints.
      ), the running conditions were 0.5 h at 70 V and 5 h at 385 V at 4 °C. After running, the gel was destained in 10% ethanol, 10% glacial acetic acid for 16 h to visualize the bands.

      Transmission Electron Microscopy

      A drop (2 μl) of nanodisc solution, typically diluted to an A280 of about 0.001, was placed on a carbon film-coated, 400 mesh grid and incubated for 30 s. Liquid was wicked away from the grid using a wetted wedge of filter paper, and then negative staining was performed by covering the grid with a drop of aqueous 2.5% uranyl acetate (filtered through a 0.02-μm syringe filter) for 2 min. After wicking away excess stain, the grids were air-dried and placed in a Philips CM 100 transmission electron microscope operated at 80 kV. All images were collected at a magnification of ×64,000 or 92,000. The diameter of 100 nanodiscs from two to three different grids was measured using ImageJ. In the case of nonspherical nanodiscs, the longest side was measured. Contrast adjustments were carried out to improve clarity of images but did not alter overall appearance. Final image processing, cropping, and mounting of the images were done with Adobe Photoshop CS2 and Illustrator CS2. Empty grids, grids with buffer, and grids with tobacco mosaic virus particles were used as control for the technique.

      Surface Plasmon Resonance Analysis

      Binding analysis of ND-AHA2 was detected by SPR using a Biacore X100 instrument at 25 °C. The penta-His antibody (Qiagen) was covalently immobilized to the CM5 sensor chips (GE Healthcare, Biacore) by amino coupling. The penta-His antibody was buffer-exchanged with Bio-Spin 30 columns (Bio-Rad) to 10 mm sodium acetate, pH 5.0, buffer before injection. The surface was activated by 0.2 m 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and 0.05 m N-hydroxysuccinimide for 7 min at a flow rate of 10 μl/min. The penta-His antibody (0.1 mg/ml) was then injected for 7 min at a flow rate of 5 μl/min, and the surface was deactivated by 1 m ethanolamine-HCl for 7 min at a flow rate of 10 μl/min. The immobilization of penta-His antibody was done in a standard running buffer (10 mm HEPES, pH 7.4, 150 mm NaCl, 0.05% surfactant P-20, 3 mm EDTA). The penta-His antibody was immobilized only in flow cell 2; flow cell 1 was left unmodified as a reference surface. To capture aha2Δ73 in nanodiscs on the sensor chip, the running buffer was changed to 50 mm MOPS, pH 7.5, 150 mm KCl, 1 mm DTT, 0.5 mm EDTA. Samples were diluted to different concentrations in running buffer. Each sample was injected for 380 s at a flow rate of 5 μl/min, and dissociation was observed for 600 s. The surface was regenerated by a 30-s injection of 10 mm glycine, pH 2.0, and washed with buffer for 300 s before the next injection. In each case, the bulk effect on the sensorgram was corrected by subtraction of the reference surface sensorgram from the sensorgram of the active surface functionalized with penta-His antibody and subtraction of a blank run.

      Other Procedures

      Proteins were analyzed by SDS-PAGE using the system of Fling and Gregerson (
      • Fling S.P.
      • Gregerson D.S.
      Peptide and protein molecular weight determination by electrophoresis using a high molarity tris buffer system without urea.
      ). Western blotting was performed using monoclonal anti-penta-His antibodies (Qiagen), polyclonal antibodies raised against the N-terminal region of AHA2 (AHA2N term; number 762) (
      • Palmgren M.G.
      • Sommarin M.
      • Serrano R.
      • Larsson C.
      Identification of an autoinhibitory domain in the C-terminal region of the plant plasma membrane H+-ATPase.
      ), polyclonal antibodies raised against the preserved catalytic domains of AHA2 (AHA2cat) (
      • Hayashi Y.
      • Nakamura S.
      • Takemiya A.
      • Takahashi Y.
      • Shimazaki K.
      • Kinoshita T.
      Biochemical characterization of in vitro phosphorylation and dephosphorylation of the plasma membrane H+-ATPase.
      ), polyclonal antibodies raised against a 19-amino acid peptide near the C terminus of human ApoA-1 for detection of MSP (ApoA-ICterm; Pierce antibodies PA5-21166), and alkaline phosphatase detection. Concentrations of purified proteins were routinely determined by UV spectroscopy (Nanodrop ND-1000 spectrophotometer, Thermo Scientific) using a calculated molar extinction coefficient at 280 nm of 18,450 and 110,350 m−1cm−1 for MSP and aha2Δ73, respectively. For more accurate concentration determinations, samples were subjected to quantitative amino acid analysis (Dept. of Biochemistry and Molecular Biology, University of Southern Denmark). Densitometry analysis of SDS-polyacrylamide gels was performed with a Gel Doc 2000 (Bio-Rad) interfaced with Labworks Image Acquisition and Analysis Software (Ultra-Violet Products, Upland, CA) using known amounts of MSP and aha2Δ73 as standards. ATPase activity was determined essentially as described previously (
      • Regenberg B.
      • Villalba J.M.
      • Lanfermeijer F.C.
      • Palmgren M.G.
      C-terminal deletion analysis of plant plasma membrane H+-ATPase: yeast as a model system for solute transport across the plant plasma membrane.
      ). Activity was measured at pH 7, and unless stated otherwise, the buffer contained 3 mm ATP (AppliChem). When solubilized ATPases were used, 0.05% (w/v) sonicated asolectin or POPC/POPG (3:2, molar ratio) was added. The Michaelis-Menten parameters of maximal velocity (Vmax) and ATP affinity (Km) were obtained from plots of the ATPase activity as a function of nucleotide concentration by nonlinear regression of the following equation: v = (Vmax × [S])/(Km + [S]), where v is enzyme activity (nanomoles of Pi per min/mg); Vmax is maximal ATPase activity (nanomoles of Pi per min/mg); Km is the Michaelis-Menten constant for ATP (nanomolar), and [S] is substrate concentration (nanomolar). For curve fitting, Vmax and Km slope calculations, SOLVER in Excel 2007 was used.

      RESULTS

      Plant PM H+-ATPase Purifies as a Dimer upon Heterologous Expression in Yeast

      The S. cerevisiae strain RS-72 has previously proved to be suitable for the high level expression and purification of A. thaliana PM H+-ATPase AHA2 (
      • Jahn T.
      • Dietrich J.
      • Andersen B.
      • Leidvik B.
      • Otter C.
      • Briving C.
      • Kühlbrandt W.
      • Palmgren M.G.
      Large scale expression, purification, and 2D crystallization of recombinant plant plasma membrane H+-ATPase.
      ). Here, we used this strain for expression and purification of a C-terminally truncated version of AHA2 (aha2Δ73), which lacks 73 amino acid residues at the C terminus. This deletion removes a regulatory autoinhibitory domain of AHA2 and renders it in a constitutively activated state (
      • Regenberg B.
      • Villalba J.M.
      • Lanfermeijer F.C.
      • Palmgren M.G.
      C-terminal deletion analysis of plant plasma membrane H+-ATPase: yeast as a model system for solute transport across the plant plasma membrane.
      ). Furthermore, aha2Δ73 can be expressed in higher yields as compared with AHA2 and is less prone to degradation (data not shown). Under our conditions, a typical purification by Ni2+ affinity batch-binding resulted in ∼1–2 mg of protein from 1 liter of cell culture. The purified PM H+-ATPase had the expected molecular mass of 97 kDa and was homogeneous as assessed by Coomassie Brilliant Blue-stained SDS-PAGE (Fig. 1A). The protein identity was unequivocally confirmed by Western blot analysis using the anti-His and anti-AHA2N term antibodies (Fig. 1A). ATPase analysis of the purified aha2Δ73 in detergent-containing buffer in the presence of asolectin or POPC/POPG (3:2 molar ratio) revealed an average ATPase activity of 10–20 μmol of Pi/min/mg of protein.
      Figure thumbnail gr1
      FIGURE 1Purification 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-AHA2N 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.
      To evaluate the oligomeric state of purified PM H+-ATPase, aha2Δ73 was subjected to BN-PAGE analysis. The protein migrated predominantly as a single band with a size of ∼200 kDa (Fig. 1B, X1) in the range of dimeric aha2Δ73, with weaker bands visible around ∼450 kDa (Fig. 1B, X2) and ∼600 kDa (Fig. 1B, X3). To confirm the oligomeric states of aha2Δ73, cross-linking was performed using the amine-specific cross-linker DMS (Fig. 1C). Following incubation with DMS, PM H+-ATPase migrated as two bands in SDS-PAGE; the PM H+-ATPase monomer at ∼97 kDa and a band migrating above 170 kDa (indicated by an arrow in Fig. 1C) near the molecular mass correspond to dimeric aha2Δ73 of about 194 kDa. Both bands were recognized in Western blots using an anti-AHA2cat antibody (data not shown). 2-Fold dilutions of the starting amount of PM H+-ATPase resulted in similar dimer/monomer ratios, excluding concentration effects as a possible explanation for the presence of dimers. Furthermore, some protein retained at the stacking gel, suggesting the formation of higher oligomers or aggregates too large to enter the gel pores. These data suggest that the majority of DDM-solubilized PM H+-ATPase is present as oligomers.
      Upon reconstitution into asolectin vesicles, aha2Δ73 exhibited ATP-driven proton pumping activity as revealed by ATP-dependent fluorescence quenching of ACMA, a dye that accumulates inside the vesicles upon protonation (Fig. 1D). These results demonstrate successful purification of functionally active PM H+-ATPase.

      Reconstitution of PM H+-ATPase into Nanodiscs

      Purified aha2Δ73 was used for reconstitution into nanodiscs employing the MSP construct MSP1D1 or MSP1D1(−) devoid of the His7 tag. An MSP/aha2Δ73 ratio was chosen to give a 4-fold excess of nanodiscs to PM H+-ATPase, facilitating the reconstitution of monomeric PM H+-ATPase. Assembly of stable PM H+-ATPase-nanodisc complexes from MSP, lipids, and detergent-solubilized PM H+-ATPase was verified by size exclusion chromatography. For nanodisc preparation with PM H+-ATPase, the chromatogram displayed a major elution peak with a shoulder on the leading edge of the peak (Fig. 2A). By contrast, nanodisc preparation without PM H+-ATPase showed a large and symmetrical elution peak. SDS-PAGE analysis revealed that the PM H+-ATPase co-elutes with MSP in the shoulder region, although the major peak corresponds to MSP alone representing empty nanodiscs (Fig. 2B). Analysis of the ATPase activity across the elution fractions revealed that the peak of enzymatic activity coincided with ND-AHA2 complexes in the shoulder region (Fig. 2C). Taken together, these results confirm successful reconstitution of functional PM H+-ATPase into nanodiscs.
      Figure thumbnail gr2
      FIGURE 2Reconstitution 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.

      Separation of Empty and Full Nanodisc Complexes

      From initial reconstitution attempts, it was evident that a single size exclusion chromatography step was insufficient to separate ND-AHA2 complexes from empty nanodiscs, i.e. devoid of PM H+-ATPase. To allow for purification of ND-AHA2 complexes, purified His6-tagged aha2Δ73 was reconstituted into nanodiscs using the MSP construct MSP1D1(−) devoid of the polyhistidine tag and subsequently subjected to Ni2+ affinity chromatography (Fig. 3A). Analysis of the proteins eluted from the Ni2+ matrix by SDS-PAGE and Western blotting demonstrated that under this condition, ND-AHA2 bound to the Ni2+ matrix, although the flow-through contained mainly the empty nanodiscs, with residual aha2Δ73 as observed from Western blotting using anti-penta-His antibody (Fig. 3, B and C). Again, analysis of the ATPase activity across the elution profile showed enzymatic activity, indicating that PM H+-ATPase was correctly reconstituted in the nanodiscs (Fig. 3D). Additionally, the ATPase activity confirmed the presence of residual aha2Δ73 in the flow through. Size exclusion chromatography after nickel affinity chromatography on both the flow-through and the eluate confirmed efficient separation of ND-AHA2 from empty nanodiscs; ND-AHA2 eluted in a major peak ahead of the position for empty nanodiscs (Fig. 4A). A small shoulder on the trailing edge of the peak for nickel affinity-purified nanodiscs indicated a small proportion of empty discs, possibly assembled from residual amounts of His7-tagged MSP1D1 still present in the reconstitution mixture. SDS-PAGE analysis on fractions from size exclusion on purified ND-AHA2 (Fig. 4B) revealed a complete co-elution pattern of aha2Δ73 and MSP1D1(−) devoid of the His7 tag, further confirming the correct assembly of PM H+-ATPase containing nanodiscs.
      Figure thumbnail gr3
      FIGURE 3Separation 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 thumbnail gr4
      FIGURE 4Purification 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 . 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.

      Analytical Characterization of PM H+-ATPase-containing Nanodiscs

      To determine Stokes diameter of the phospholipid bilayer nanodiscs, the size exclusion chromatography column was calibrated using a standard set of proteins. Based on this calibration, empty nanodiscs and ND-AHA2 had overall Stokes diameters of 10.4 and 11.8 nm, respectively. The calculated Stokes diameter for empty nanodiscs are in reasonable agreement with earlier results on MSP1D1 assembled nanodiscs, reporting a Stokes diameter of 9.6 nm for nanodiscs assembled using dipalmitoyl-PC lipids and MSP1D1(−) (
      • Bayburt T.H.
      • Grinkova Y.V.
      • Sligar S.G.
      Self-assembly of discoidal phospholipid bilayer nanoparticles with membrane scaffold proteins.
      ,
      • Denisov I.G.
      • Grinkova Y.V.
      • Lazarides A.A.
      • Sligar S.G.
      Directed self-assembly of monodisperse phospholipid bilayer nanodiscs with controlled size.
      ). As illustrated in Fig. 5D, the largest dimension on a model of ND-AHA2 results in values of about 11 nm, consistent with the measured Stokes diameter from SEC analysis.
      Figure thumbnail gr5
      FIGURE 5Visualization 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 (
      • Pedersen B.P.
      • Buch-Pedersen M.J.
      • Morth J.P.
      • Palmgren M.G.
      • Nissen P.
      Crystal structure of the plasma membrane proton pump.
      ); 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 (
      • Humphrey W.
      • Dalke A.
      • Schulten K.
      VMD: Visual molecular dynamics.
      ).
      The dimension and homogeneity of empty nanodiscs and ND-AHA2 were further assayed from analysis of transmission electron microscopy images (Fig. 5, A1 and B1). This revealed most of the discs, whether full or empty, to exhibit diameters in the range of 10–11 nm (Fig. 5C) and to be lying individually on the carbon film. However, within the samples of ND-AHA2, we observed a number of paired or even stacked discs (Fig. 5, B2 and B3). Because such side view positioned paired discs were only found in samples from full discs, it is likely that this phenomenon was due to interaction between the inserted pumps and probably not the process of preparation.
      Homogeneity of the assembled and affinity-purified nanodiscs was further investigated by BN-PAGE analysis. Under these conditions, empty discs migrated at ∼146 kDa (Fig. 6A, lane 1), whereas ND-AHA2 migrated as a single band at ∼361 kDa (Fig. 6A, lane 2). Upon solubilization of ND-AHA2 by SDS, single bands at ∼200 and ∼20 kDa were observed corresponding to free PM H+-ATPase and MSP (Fig. 6A, lane 4), whereas solubilized empty nanodiscs displayed only a single band for MSP at ∼20 kDa (Fig. 6A, lane 5). Consistent with previous BN-PAGE analysis, purified DDM-solubilized PM H+-ATPase was detected at ∼200 kDa (Fig. 6A, lane 3). Based on simple geometrical assumptions when estimating the amount of lipids in the discs, the apparent molecular mass of empty nanodiscs and ND-AHA2 was estimated to be 129 and 213 kDa, respectively. The discrepancy between calculated and apparent molecular weights in BN-PAGE analysis may be explained by the difference in Coomassie staining between the soluble protein standards and nanodiscs, with the latter representing a complex of scaffold proteins, membrane protein, and lipids (
      • Schägger H.
      • Cramer W.A.
      • von Jagow G.
      Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis.
      ). This is further evident from the relatively low degree of Coomassie Brilliant Blue staining of aha2Δ73 observed in densitometry analysis of SDS-polyacrylamide gels (supplemental Fig. S2). The Stokes diameters estimated from the protein standards are in agreement with the values found from SEC and transmission electron microscopy analysis, with ∼9.7 nd ∼11.6 nm for empty nanodiscs and ND-AHA2 respectively.
      Figure thumbnail gr6
      FIGURE 6Analysis 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 AHA2cat (lanes 1–4) and ApoA-ICterm (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.

      PM H+-ATPase Reconstitutes into Nanodiscs as a Monomer

      The MSP/aha2Δ73 stoichiometry was found to be 2:1 from densitometry analysis of Coomassie-stained gels, including a standard curve of purified proteins for which concentrations had been determined by quantitative amino acid analysis (supplemental Fig. S2). Collectively, these results demonstrate predominant formation of stable nanodiscs with a single PM H+-ATPase monomer. To confirm the monomeric state of the reconstituted PM H+-ATPase, cross-linking was performed using the amine-specific cross-linker DMS, previously shown to cross-link DDM-solubilized aha2Δ73 dimers (Fig. 1C). Cross-linking of empty nanodiscs resulted in two additional bands migrating to ∼40 kDa (D1) and ∼55 kDa (D2) in SDS-PAGE (Fig. 6B, lanes 2 and 3). This pattern is consistent with previous reports of cross-linked ApoAI in reconstituted high density lipoprotein particles, resulting in two distinct dimer forms (
      • Bhat S.
      • Sorci-Thomas M.G.
      • Tuladhar R.
      • Samuel M.P.
      • Thomas M.J.
      Conformational adaptation of apolipoprotein A-I to discretely sized phospholipid complexes.
      ). The two dimer forms of MSP1D1(−) were also observed from cross-linking of ND-AHA2 on both Coomassie-stained SDS-PAGE and Western blotting using an ApoA-ICterm antibody (Fig. 6C, lanes 4 and 4*), confirming a similar conformation of MSPs in full and empty nanodisc complexes. From cross-linking of ND-AHA2, three additional bands at ∼115 kDa (C1), ∼150 kDa (C2), and above the 170-kDa marker (C3) were observed in Coomassie-stained SDS-PAGE (Fig. 6C, lane 4 left panel). Only the bands at C1 and C2 are clearly visible from Western blotting using AHA2cat and ApoA-ICterm antibodies (Fig. 6C, lanes 4 and 4*, right panel). Comparison of C1, C2, and C3 to the dimer product of cross-linked DDM-solubilized aha2Δ73 (Fig. 6C, lane 2) reveals all to migrate to lower molecular weights than the apparent aha2Δ73 dimer. Based on these observations, we conclude that C1, C2, and C3 are products of cross-linking between the MSPs and aha2Δ73 and that aha2Δ73 is reconstituted as a monomer in the nanodiscs.

      Enzymatic Properties of Plant Plasma Membrane H+-ATPase in Nanodiscs

      To ascertain that the PM H+-ATPase reconstituted in nanodiscs had maintained its enzymatic properties, we analyzed the ATPase activity of purified and reconstituted PM H+-ATPase. Consistent with previous results, purified PM H+-ATPase in DDM-containing buffer displayed a low basal ATPase activity (about 1.7 μmol Pi/min/mg) that increased ∼10-fold upon addition of lipids in the form of asolectin or POPC/POPG (3:2 ratio). For PM H+-ATPase embedded in nanodiscs with POPC/POPG (3:2), a specific ATPase activity in the range of 10.5 μmol Pi/min/mg was estimated. Kinetic analyses revealed Km(ATP) of ∼180 and ∼60 μm for lipid-activated and nanodisc-reconstituted PM H+-ATPase, respectively. These values are within the range of previous studies on C-terminally truncated deletion mutants, reporting values of 50–500 μm for aha2Δ77 in endoplasmic reticulum vesicles from yeast (
      • Regenberg B.
      • Villalba J.M.
      • Lanfermeijer F.C.
      • Palmgren M.G.
      C-terminal deletion analysis of plant plasma membrane H+-ATPase: yeast as a model system for solute transport across the plant plasma membrane.
      ) and 76 ± 5 μm for lipid-activated aha2Δ73 (
      • Buch-Pedersen M.J.
      • Palmgren M.G.
      Conserved Asp684 in transmembrane segment M6 of the plant plasma membrane P-type proton pump AHA2 is a molecular determinant of proton translocation.
      ). Taken together, our kinetic analysis demonstrated that reconstitution of PM H+-ATPase into nanodiscs did not compromise with its enzymatic properties.
      It is an intrinsic property of the nanodisc system that transport of reconstituted transporters cannot be readily assayed. Thus, in contrast to reconstitution of PM H+-ATPase into liposome vesicles, where transported protons can be made to accumulate from the extravesicular medium into the vesicle lumen, both sides of the nanodisc membrane are freely accessible to the assay medium, and transported protons cannot be trapped and assayed in the same way. In P-type ATPases, transport of ligands from the cytoplasmic to the extra-cytoplasmic side of the membrane takes place during the transition from the E1 conformational state, which hydrolyzes ATP, to the E2 conformational state, more specifically between the phosphorylated E1P and E2P states (
      • Morth J.P.
      • Pedersen B.P.
      • Buch-Pedersen M.J.
      • Andersen J.P.
      • Vilsen B.
      • Palmgren M.G.
      • Nissen P.
      A structural overview of the plasma membrane Na+,K+-ATPase and H+-ATPase ion pumps.
      ). Vanadate is a transition state analog of inorganic orthophosphate, which inhibits P-type ATPases by binding specifically to the E2 conformation (
      • Morth J.P.
      • Pedersen B.P.
      • Buch-Pedersen M.J.
      • Andersen J.P.
      • Vilsen B.
      • Palmgren M.G.
      • Nissen P.
      A structural overview of the plasma membrane Na+,K+-ATPase and H+-ATPase ion pumps.
      ). The vanadate sensitivity of PM H+-ATPase was measured following reconstitution of the enzyme in vesicles as well as in nanodiscs (Fig. 7). Prior to the assay, it was ascertained that the H+-ATPase reconstituted into liposomes actually pumps protons (Fig. 7, inset). PM H+-ATPase reconstituted in both membrane types was found to exhibit the same vanadate sensitivity (Fig. 7). This confirms that PM H+-ATPase reconstituted in both experimental systems has a comparable conformational equilibrium and when reconstituted in both systems are capable of entering the E2 state of the catalytic cycle. Although not direct evidence for proton pumping, this strongly indicates that the H+-ATPase reconstituted into nanodiscs is an active proton pump like the enzyme reconstituted into liposomes.
      Figure thumbnail gr7
      FIGURE 7Vanadate 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).

      Immobilization of PM H+-ATPase-containing Nanodiscs on Penta-His Surface

      We further investigated the potential of nanodiscs for future SPR-mediated investigation of the PM H+-ATPase. Because direct immobilization of proteins and nonspecific cross-linking of the protein to surfaces might impair protein function (
      • Jonkheijm P.
      • Weinrich D.
      • Schröder H.
      • Niemeyer C.M.
      • Waldmann H.
      Chemical strategies for generating protein biochips.
      ), the samples are immobilized on CM5 sensor chips covalently modified with antibodies specifically recognizing His tags (penta-His antibody). A high density of penta-His antibody (15,000–16,000 response units) was covalently bound to the sensor chip surface to facilitate stable binding of nanodiscs containing aha2Δ73 by increasing rebinding of the complex to the antibody.
      Two different reconstitution samples were tested for immobilization onto the surfaces as follows: 1) nanodiscs containing aha2Δ73 assembled from MSP1D1 and purified using a single SEC step (Fig. 8A), and 2) nanodiscs containing aha2Δ73 assembled from MSP1D1(−) purified by nickel affinity purification and SEC to remove excess empty nanodiscs (Fig. 8B, black trace). Comparison with sensorgrams obtained for empty nanodiscs assembled using MSP1D1(−) (Fig. 8B, red trace) confirms that the observed immobilization is due to interaction with the penta-His antibody. The sample containing empty nanodiscs and ND-AHA2 assembled with MSP1D1 has two to three His tags per complex, with one His tag on the PM H+-ATPase and two His tags from the two MSPs. From the avidity effect, the presence of several His tags per complex can promote stable immobilization of the nanodiscs to the surface, although this approach does not allow us to conclude specific immobilization of ND-AHA2 to the surface. For this purpose we applied nickel affinity-purified ND-AHA2 assembled from MSP1D1(−), which confirmed the specific immobilization of ND-AHA2 through the His6 tag on aha2Δ73.
      Figure thumbnail gr8
      FIGURE 8Capture 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(−).

      DISCUSSION

      Nanodisc-embedded Plasma Membrane H+-ATPase Is an Active Monomer

      In this study, we demonstrate that the functional unit of the PM H+-ATPase is a monomer. To do this, we worked out a reconstitution procedure for the plant PM H+-ATPase at the single molecule level into nanodiscs. Expression of His-tagged PM H+-ATPase from a multicopy plasmid under the control of the strong constitutive promoter (PMA1) allowed for effective purification to homogeneity of an C-terminally truncated version of the PM H+-ATPase. To facilitate the formation of nanodiscs with single PM H+-ATPase monomers, a 4-fold excess of nanodiscs to PM H+-ATPase was applied during the reconstitution procedure. Under this condition, the purified, solubilized ATPases efficiently incorporated in nanodiscs. BN-PAGE analysis of ND-AHA2 revealed a single slowly migrating band confirming the homogeneity of the assembled and affinity-purified nanodiscs.
      Despite the oligomeric state of the initially purified detergent-solubilized H+-ATPase, our data show that the pump reconstitutes in nanodiscs as a monomer. This conclusion is based on two independent approaches as follows. 1) Isolation of ND-AHA2 allowed us to estimate the MSP/H+-ATPase stoichiometry by densitometry analysis of SDS-polyacrylamide gels. 2) Cross-linking experiments excluded the presence of dimeric H+-ATPase. Cross-linking experiments also verified a similar conformation of the MSPs in both ND-AHA2 and empty nanodiscs, with a cross-linking product of two distinct dimers in both cases (Fig. 6, B, C, D1, and D2). Furthermore, these experiments confirmed the close proximity of PM H+-ATPase and MSP in the ND-AHA2 complex, giving three different cross-linking complexes after incubation with the DMS cross-linker (Fig. 6C). The three distinct bands with equal spacing likely represent cross-linking products of PM H+-ATPase and one, two, and two in dimer D2 form of MSPs, as also verified from Western blotting against aha2Δ73 and MSP.
      Notably, nanodisc-reconstituted PM H+-ATPase displayed a robust ATPase activity, demonstrating that the pump monomer remains folded in an active conformation. This observation is in line with earlier studies on vesicle-reconstituted N. crassa PM H+-ATPases demonstrating that the pump monomer is likely to be the minimal functional unit (
      • Goormaghtigh E.
      • Chadwick C.
      • Scarborough G.A.
      Monomers of the Neurospora plasma membrane H+-ATPase catalyze efficient proton translocation.
      ). The vanadate sensitivity of the nanodisc-reconstituted PM H+-ATPase confirmed that it is able to alternate between the E1 conformation, which hydrolyzes ATP, to the E2 conformation, which binds vanadate. This indicates that the nanodisc-embedded PM H+-ATPase carries out the complete catalytic cycle, which is associated with proton pumping. Furthermore, the similar vanadate sensitivity of PM H+-ATPase reconstituted in vesicles and nanodiscs suggests that no significant change in conformational equilibrium is introduced when reconstituting the enzyme in nanodiscs as compared with vesicles, where the PM H+-ATPase is shown to pump protons.

      Nanodiscs as a Tool to Study P-type ATPase Function

      In vitro studies involving the H+-ATPases have so far been based on either detergent solubilization or membrane vesicles. The presence of detergent is required throughout purification, manipulation, assays, and structural studies to prevent aggregation. Detergents often perturb interactions and strip the membrane proteins of their endogenous lipids, which may be required to retain their functional state. Reconstitution into vesicles is a powerful model for transport studies and molecular characterization. However, drawbacks in using this system include the heterogeneous nature of the prepared vesicles, orientation of the proteins effectively giving rise to two populations exposed to different solvent phases, and the barrier of the membrane to soluble proteins in membrane protein-protein interaction studies.
      Nanodiscs provide a complementary system for studying membrane pumps under defined experimental conditions. We found that the Vmax values of nanodisc-reconstituted PM H+-ATPase were similar to those measured for asolectin-activated, DDM-solubilized PM H+-ATPase. However, we observed an ∼10-fold higher affinity for ATP of aha2Δ73 in nanodiscs compared with lipid-activated aha2Δ73. This could be due to both the MSPs interacting directly with the PM H+-ATPase or the different lipid environment compared with lipid/DDM micelles. A recent study on empty nanodiscs using small angle x-ray scattering reported area per lipid headgroup values higher for POPC and lower for dilauroyl-PC in nanodiscs compared with free bilayers (
      • Skar-Gislinge N.
      • Simonsen J.B.
      • Mortensen K.
      • Feidenhans'l R.
      • Sligar S.G.
      • Lindberg Møller B.
      • Bjørnholm T.
      • Arleth L.
      Elliptical structure of phospholipid bilayer nanodiscs encapsulated by scaffold proteins: Casting the roles of the lipids and the protein.
      ). Differences in mean bilayer height and phase transition temperature between nanodisc and lamellar dipalmitoyl-PC and dimyristoyl-PC have also been reported using a combined small angle x-ray scattering and differential scanning calorimetry analysis (
      • Denisov I.G.
      • McLean M.A.
      • Shaw A.W.
      • Grinkova Y.V.
      • Sligar S.G.
      Thermotropic phase transition in soluble nanoscale lipid bilayers.
      ). Here, the increase in transition temperature and altered mean bilayer height was explained by the additional lateral pressure and structural perturbations from the MSP. Evidently, the variations in these parameters depend on the applied lipid, although it clearly demonstrates a different lipid environment in nanodiscs compared with other lipid systems, which could explain the different apparent affinity of ATP for PM H+-ATPase in nanodiscs compared with lipid-activated PM H+-ATPase. Natural lipid membranes consist of a large variety of lipids and proteins, with membrane proteins constituting up to 80% of the total weight of membranes (
      • Gennis R.B.
      ). As a result, a large fraction of the lipids in a membrane are in contact with proteins. In this light, it could be speculated that the lipid bilayer in a nanodisc more accurately reflects the natural environment found in membranes, as compared with vesicles of pure lipids and lipid/detergent micelles (
      • Bayburt T.H.
      • Sligar S.G.
      Membrane protein assembly into nanodiscs.
      ). Naturally, the absence of a proton gradient in the nanodisc system is another important difference compared with vesicle-reconstituted PM H+-ATPases, which may influence the kinetics and activity.

      Immobilization of Nanodiscs to Study Regulatory Pump Interactions

      Surface-sensitive techniques allow for real time measurement of biological events and thereby provide thermodynamics and kinetics information on the interaction. For this purpose, SPR is one of the most used techniques. Based on changes of refractive indexes at the surface, SPR does not require any labeling of the proteins. A requirement for the use of SPR is the immobilization of functional proteins onto surfaces. Furthermore, the improved stability of isolated membrane proteins in nanodiscs compared with detergent is a considerable advantage for investigation by surface-sensitive techniques (
      • Glück J.M.
      • Koenig B.W.
      • Willbold D.
      Nanodiscs allow the use of integral membrane proteins as analytes in surface plasmon resonance studies.
      ,
      • Borch J.
      • Torta F.
      • Sligar S.G.
      • Roepstorff P.
      Nanodiscs for immobilization of lipid bilayers and membrane receptors: kinetic analysis of cholera toxin binding to a glycolipid receptor.
      ,
      • Das A.
      • Zhao J.
      • Schatz G.C.
      • Sligar S.G.
      • Van Duyne R.P.
      Screening of type I and II drug binding to human cytochrome P450–3A4 in nanodiscs by localized surface plasmon resonance spectroscopy.
      ). Using both a mixture of empty nanodiscs and ND-AHA2 assembled with MSP1D1 and nickel affinity-purified ND-AHA2, we found stable and specific immobilization to the sensor chip. The stability of the immobilization observed in all cases and the possible regeneration of the surface after treatment at low pH (pH 2) makes thus the proposed strategy suitable for further monitoring protein-protein interactions by SPR.

      Conclusion

      We have demonstrated that the PM H+-ATPases can be embedded into lipid bilayer nanodiscs as a functional monomer. The purification and reconstitution procedures developed here should be useful for the analysis of PM H+-ATPases and other P-type ATPases at the molecular level. The ability to study these pumps embedded in a nanodisc rather than in detergent micelles or vesicles enables the use of a broad arsenal of biochemical and biophysical tools to quantitatively characterize the embedded protein and protein-protein interaction studies between the pump and its soluble interaction partners.

      Acknowledgments

      We are grateful to Anne-Mette Bjerg Petersen for excellent technical assistance, Danny Mollerup Sørensen for fruitful discussion on experimental procedures, and Prof. Toshinori Kinoshita for providing the antibody AHA2cat (
      • Hayashi Y.
      • Nakamura S.
      • Takemiya A.
      • Takahashi Y.
      • Shimazaki K.
      • Kinoshita T.
      Biochemical characterization of in vitro phosphorylation and dephosphorylation of the plasma membrane H+-ATPase.
      ). Imaging data were collected at the Center for Advanced Bioimaging Denmark, University of Copenhagen.

      Supplementary Material

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