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A New Method for the Reconstitution of Membrane Proteins into Giant Unilamellar Vesicles

Philippe Girard ^*, Jacques Pécréaux ^*, Guillaume Lenoir , Pierre Falson , Jean-Louis Rigaud ^*  and Patricia Bassereau ^*

^* Laboratorie Physico Chimie Cioue, Unité Mixte de Recherche 168 Centre National de la Recherche Scientifique/Institut Curie, 75231 Paris Cedex 05, France; and Laboratoire de Recherche Correspondant 34V Commissariat à l'Energie Atomique, Institut Curie, 75231 Paris Cedex 05, France; Commissariat à l'Énergie Atomique, Centre d'Etudes de Saclay, Direction des Sciences du Vivant, Département de Biologie Joliot Curie, Service de Biophysique des Fonctions Membranaires, Unité de Recherche Associée 2096 Centre National de la Recherche Scientifique, 91191 Gif-sur-Yvette Cedex, France

Correspondence: Address reprint requests to Philippe Girard, E-mail: philippe.girard{at}curie.fr.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION AND CONCLUSION ACKNOWLEDGEMENTS REFERENCES

 
In this work, we have investigated a new and general method for the reconstitution of membrane proteins into giant unilamellar vesicles (GUVs). We have analyzed systematically the reconstitution of two radically different membrane proteins, the sarcoplasmic reticulum Ca²⁺-ATPase and the H⁺ pump bacteriorhodopsin. In a first step, our method involved a detergent-mediated reconstitution of solubilized membrane proteins into proteoliposomes of 0.1–0.2 µm in size. In a second step, these preformed proteoliposomes were partially dried under controlled humidity followed, in a third step, by electroswelling of the partially dried film to give GUVs. The physical characteristics of GUVs were analyzed in terms of morphology, size, and lamellarity using phase-contrast and differential interference contrast microscopy. The reconstitution process was further characterized by analyzing protein incorporation and biological activity. Both membrane proteins could be homogeneously incorporated into GUVs at lipid/protein ratios ranging from 5 to 40 (w/w). After reconstitution, both proteins retained their biological activity as demonstrated by H⁺ or Ca²⁺ pumping driven by bacteriorhodopsin or Ca²⁺-ATPase, respectively. This constitutes an efficient new method of reconstitution, leading to the production of large unilamellar membrane protein-containing vesicles of more than 20 µm in diameter, which should prove useful for functional and structural studies through the use of optical microscopy, optical tweezers, microelectrodes, or atomic force microscopy.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION AND CONCLUSION ACKNOWLEDGEMENTS REFERENCES

 
Biological membranes are differentially permeable barriers separating two compartments. These membranes are formed by a mixture of lipid molecules organized as a bilayer in which are embedded amphiphilic proteins involved in a wide range of important biological processes such as transport phenomena, energy transduction, signaling, cell recognition, and motility (Alberts et al., 1994). However, the study at the molecular level of the roles of membrane proteins in these processes is often hampered by the complexity of the native membranes. Thus, as a first step toward a better understanding of complex biological membranes, it is often necessary to focus on more simple systems, consisting of membrane proteins reconstituted into artificial lipid membranes. In this context, incorporation of purified membrane proteins into proteoliposomes by detergent-mediated reconstitutions has allowed for the acquisition of a large amount of information about the functions and the structures of different membrane proteins (Rigaud et al., 1995, 2000). Despite the usefulness of these detergent-mediated reconstitutions, one main limitation is often related to the relative small size (0.1–0.2 µm) of the reconstituted proteoliposomes. Indeed, this small size impedes the analysis of structure/function relationships of membrane proteins through optical microscopy techniques such as fluorescence correlation spectroscopy, dynamic force spectroscopy, and single particle tracking or through proteoliposomes manipulation using pipettes, tweezers, electrodes, or atomic force microscopy (AFM).

Giant unilamellar vesicles (GUVs) with sizes ranging from 10 to 100 µm are potentially attractive models and are becoming objects of intense scrutiny in diverse areas focusing on biological functions such as adhesion, fusion and fission, or motility (Luisi and Walde, 2000). Several methods to produce giant vesicles have been developed and reported in the literature. One of the methods that has been applied successfully to produce large proteoliposomes amenable to patch-clamp recording and electrophysiological measurements is based on a dehydration-rehydration procedure described by Criado and Keller (1987). Such a method consists of the dehydration of biological membranes or preformed proteoliposomes in the presence of added exogenous lipids followed by a rehydration process in the desired buffer (Ajouz et al., 2000). As a variant of this method, the rehydration of thin dried films obtained after the evaporation of the solvent from lipid-protein complexes solubilized in organic solvents has been used to produce giant proteoliposomes (Darszon et al., 1980). The main advantage of this dehydration/rehydration method is that these vesicles can be prepared in a large range of buffer compositions and more specifically in buffers with high ionic strengths. However, this method produces a very heterogeneous population of proteoliposomes with a large proportion of multilamellar vesicles related to the uncontrolled rehydration process. This bottleneck has been overcome by the electroformation technique (Angelova et al., 1992), in which a lipid film is rehydrated in the presence of an alternating current (AC) electric field. Thus, GUVs can be produced with a more homogeneous distribution in size, centered around 20 µm in diameter. The use of this electroformation technique was first described by Manneville and co-workers to reconstitute an integral membrane protein, bacteriorhodopsin (BR) (Manneville et al., 2001). The GUVs were formed by rehydration under an AC electrical field of a dried film prepared from a solution of lipids and bacteriorhodopsin in diethyl ether. Unfortunately, such a reconstitution of membrane proteins by electroformation is very limited, due to the use of organic solvents that denature most amphiphilic membrane proteins. Another approach has been recently reported in which lipidic GUVs prepared by electroformation were fused with small preformed proteoliposomes containing bacteriorhodopsin. However, such a procedure limits the amount of protein that can be incorporated and leads to the insertion of foreign molecules, namely the fusogenic peptides that have been covalently attached to the preformed proteoliposomes to allow their fusion with GUVs (Kahya et al., 2001).

In this work, we present a new method, to our knowledge, for the reconstitution of transmembrane proteins into GUVs. This method involves three successive steps: 1), incorporation of the solubilized proteins into the membranes of submicrometer sized unilamellar vesicles by reconstitution upon detergent removal (Rigaud et al., 1995); 2), partial dehydration of these preformed proteoliposomes on conductive glass surfaces; and 3), controlled rehydration in the presence of an AC electric field to form giant vesicles (Angelova et al., 1992). We checked the usefulness of this new method by studying the reconstitution of two radically different transmembrane proteins. One is the Ca²⁺-ATPase from the sarcoplasmic reticulum (SR), an ATP-driven Ca²⁺ pump of 110 kDa consisting of a large hydrophilic domain connected to a hydrophobic domain composed of 10 transmembrane -helices (for review see Lee, 2002). The other protein is the bacteriorhodopsin from Halobacterium salinarium, a light-driven H⁺ pump of 27 kDa that is a very hydrophobic membrane protein formed by seven transmembrane -helices (for review see Oesterhelt and Tittor, 1989). The reconstituted giant vesicles produced by our new procedure were analyzed with respect to size and morphology and with respect to protein incorporation, protein distribution, and biological activity. In summary, our results allow us to define important parameters involved in the efficient and the functional reconstitution into GUVs of two prototypical membrane proteins, which should prove of general use for the reconstitution of other membrane proteins.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION AND CONCLUSION ACKNOWLEDGEMENTS REFERENCES

 
Chemicals
Purified egg yolk L--phophatidylcholine (EYPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), stearolyloleoyl-phosphocholine (SOPC), egg yolk phophatidic acid (EYPA), 1,2-diacyl-sn-glycero-3-(phospho-L-serine) (DOPS), 1,2-diacyl-sn-glycero-3-phosphoethanolamine (DOPE), and 2-(12-(7-nitro2-oxa-1,3-dialzol-4-yl)amino)dodecanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBD-C12-HPC) were purchased from Avanti Polar Lipids (Alabaster, AL). Triton X-100 was from Roche Diagnostics (Mannheim, Germany), and octaethylene glycol mono-n-1-dodecyl ether (C₁₂E₈) was from Sigma-Aldrich (Saint-Quentin-Fallavier, France). Polystyrene Bio-Beads SM-2 (25–50 mesh) were obtained from Bio-Rad (Hercule, CA). The fluorescent probes, fluorescein 5'-isothiocyanate (FITC), Fluo-5N impermeant form, 8-hydroxy-1,3,6-pyrenetrisulfonic acid trisodium salt (pyranine), and 9 amino-acridine (9-AA) were purchased from Molecular Probes (Eugene, OR). All other reagents were of analytical grade.

SR membranes were provided by Dr. P. Champeil (Commissariat à l'Énergie Atomique Saclay, France) and prepared from rabbit white skeletal muscles as described in Champeil et al. (1985). For reconstitution experiments, detergent-solubilized monomeric Ca²⁺-ATPase was prepared by incubating SR vesicles in C₁₂E₈ at a detergent/protein ratio of 2 w/w in a buffer containing 20 mM Mops-Tris, pH 7.0, supplemented with 0.1 mM CaCl₂ to prevent the inactivation of the protein (Levy et al., 1992).

Purple membranes were purified from H. salinarium according to the procedure described in Oesterhelt and Stoeckenius (1974). For reconstitution experiments, detergent-solubilized monomeric bacteriorhodopsin was prepared by incubating overnight purple membrane in Triton X-100 at a detergent/protein ratio of 5 w/w in a buffer containing 20 mM Pipes, pH 6.8 (Rigaud et al., 1988).

Preparation of submicrons proteoliposomes
BR and Ca²⁺-ATPase were reconstituted into proteoliposomes according to the general procedure developed by Rigaud and co-workers (Rigaud et al., 1988; Levy et al., 1992; Rigaud and Levy, 2003). Typically, pure liposomes at 4 mg lipid/ml were prepared by sonication in a buffer containing 2 mM Mops-Tris, pH 7.0, and completely solubilized into mixed micelles at detergent/lipid ratios of 2 (w/w) for Triton X-100 and C₁₂E₈. Then aliquots of solubilized proteins were added to the detergent-lipid mixture under stirring at the desired lipid/protein ratio. After 30 min incubation for micellar equilibration, the detergent was removed at room temperature by adding prewashed SM2 Bio-Beads (Rigaud et al., 1998). For BR reconstitution, Triton X-100 was removed by a slow removal procedure consisting in two successive additions of 10 mg Bio-Beads per milligram of Triton X-100 for 1 h each, followed by a third addition of 20 mg Bio-Beads per milligram of Triton X-100 to ensure full detergent removal. For Ca²⁺-ATPase reconstitution, inhomogeneous protein incorporation (Levy et al., 1992) was avoided by fast detergent removal through the addition of 30 mg Bio-Beads per milligram of C₁₂E₈ at once, followed by an incubation of 4 h. After complete detergent removal, the reconstituted proteoliposomes were separated from the beads by pipetting the supernatant.

Preparation of GUVs
Giant unilamellar vesicles were generated by the electroformation technique (Angelova, 2000). Preformed proteoliposomes were diluted to 0.8 mg lipid/ml and droplets of 2 µl were carefully deposited on indium tin oxide (ITO) treated glass slides. The film was then partially dried overnight in a desiccator under saturated vapor pressure of a saturated NaCl solution. After partial dehydration, the two ITO slides were sealed with paste Sigillum wax (Vitrex, Copenhagen, Denmark) and separated with 1-mm Teflon spacers. About 1 ml buffer containing 0.1 M sucrose, 1 mM Mops-Tris, pH 7.0, and supplemented with either 1 mM MgCl₂ for Ca²⁺-ATPase or 2 mM KCl for BR was added, and the conductive glasses were connected to copper electrodes. For electroformation, an AC electric field provided by a pulse generator was applied for 4 h across the chamber and incremented every 5 min from 20 mV to 1.1 V at 12 Hz frequency. Then, the AC frequency was lowered to 4 Hz, and the voltage was raised to 2 V for 30 min to detach the giant vesicles from the glass slides.

Observation of GUVs
Giant vesicles were observed either by phase contrast or by differential interference contrast (DIC) with an inverted microscope (Axiovert 135, Carl Zeiss, Germany). Twenty microliters of the reconstituted GUVs were transferred into an observation chamber (22 x 22 mm² wide and 1 mm high) containing a buffer in which the sucrose buffer was replaced with an equiosmolar glucose buffer containing 5 mM Mops-Tris, pH 7.0, and supplemented either with 5 mM MgCl₂ and 0.5 mM CaCl₂ for Ca²⁺-ATPase or with 5 mM KCl for BR. The density difference between the outside (glucose) and the inside (sucrose) of the vesicles allowed their sedimentation, and the refractive index gradient enhanced the image contrast. After 10 min, when all liposomes settled down to the bottom area, the whole area was scanned, and the size distribution of GUVs was scored.

Unilamellarity of the GUVs was checked by a fluorescence quenching assay based on the measurement of the distribution of fluorescent lipids between inner and outer monolayers (Gruber and Schindler, 1994). For such measurements, 0.5 mol % NBD-C12-HPC was included in the initial lipidic composition. After electroswelling, GUVs were transferred in a 2-ml cuvette, and the fluorescence was monitored in a Perkin Elmer LS 50B spectrofluorimeter (Foster City, CA) using 2-nm and 4-nm slits for excitation (_ex = 465 nm) and emission (_em = 534 nm), respectively. The contribution of the outer lipid layer was determined by measuring the fluorescence quenching induced by the addition of 20 µl solution containing 1 M sodium hydrosulfite in 5 mM N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (Tes), pH 9.0. Then, addition of a solubilizing Triton X-100 concentration induced a further fluorescence quenching that allowed determining the contribution of inner lipid layers.

Labeling of proteins
Ca²⁺-ATPase was labeled with fluorescein 5'-isothiocyanate by incubating SR vesicles at 2 mg protein/ml for 30 min at 25°C in darkness and in a buffer containing 0.3 M sucrose, 1 mM Mg²⁺, 10 µM Ca²⁺, 50 mM Tricine-Tris, pH 8.0, and a 10-fold excess of fluorescent label. The labeling reaction was stopped by centrifugation at 100,000 x g for 15 min at 4°C. The pellet was suspended in 20 mM Mops-Tris, pH 7.0.

BR was labeled with FITC after a protocol adapted from Heberle and Dencher (1992). The fluorescent labeling was performed by incubating purple membranes in the presence of 1.9 µmol FITC per milligram of protein for 4 h at room temperature in 50 mM Tris-HCl, pH 8.5. The excess of nonbound fluorescein was removed by centrifugation at 60,000 x g for 20 min. The labeled purple membranes were then washed twice in 50 mM Tris-HCl, pH 8.5, and left overnight at 4°C. The day after, membranes were washed three times, first in 0.2 M KCl and then in distilled water.

The concentration of bound fluorescein was measured spectrophotometrically in the presence of 1% sodium dodecyl sulfate, assuming a molar absorption coefficient of 8 x 10⁴ M^–1cm^–1 at 496 nm for the fluorescein bound to the protein (Mitchinson et al., 1982).

Assays for monitoring protein incorporation
Incorporation of FITC labeled proteins into GUVs was analyzed by confocal fluorescence microscopy using a Leica TCS SP2 Confocal Microscope (Leica Lazer Tecknik, Heidelberg, Germany) equipped with a Zeiss HCX-Apochromat 63x, 1.4-numerical-aperture oil immersion lens. An argon ion laser operating at 488 nm was used, close to the maximum of absorption of fluorescein. The epifluorescence was converted into a static beam by an x-y scanner device and focused onto a photomultiplier (PMT). The fluorescence emission, after passing a 505-nm high pass filter, was focused into a pinhole in front of the photomultiplier to eliminate all the out-of-focus light. The pinhole blocking aperture was at 230 µm for BR and at 320 µm for Ca²⁺-ATPase. Commercial confocal software (Leica) was used for image analysis.

Assays for monitoring protein activities
The calcium-dependent hydrolytic ATPase activity of the Ca²⁺-ATPase was measured at 25°C in an ATI Unicam UV2-100 spectrophotometer (Cambridge, Great Britain), using an enzyme-coupled method (Möller et al., 1979). With this method, 1 mol ADP produced leads to the oxidation of 1 mol -nicotinamide adenine dinucleotide (NADH) and therefore to a decrease of the absorbance at 340 nm. Phosphoenol pyruvate was used to regenerate ATP. Typical experiments were performed in a continuously stirred 1-cm cuvette containing 40 µg Ca²⁺-ATPase in 2 ml reaction buffer containing 50 mM Mops-Tris, pH 7.5, 0.1 M KCl, 5 mM MgCl₂, 0.1 mM CaCl₂, 1 mM phosphoenol pyruvate, 5 units/ml pyruvate kinase, 5 units/ml lactate dehydrogenase, and 1 mg/ml C₁₂E₈. The reaction was started by the addition of 1 mM ATP and stopped by 2.5 mM EGTA. The rate of ATP hydrolysis could be deduced from the difference of the two slopes obtained in the presence and in the absence of EGTA assuming a molar absorption coefficient ₃₄₀(NADH) = 6220 M^–1cm^–1. Protein concentration was determined separately by absorbance measurements at 280 nm ( = 120,480 M^–1cm^–1).

Ca²⁺ pumping activity was measured as changes in the fluorescence intensity of a low-affinity calcium indicator, Fluo-5N, entrapped inside vesicles. The Ca²⁺-ATPase was reconstituted via electroformation in the sucrose buffer supplemented with 20 µM Fluo-5N. After electroswelling, GUVs were transferred into a microchamber containing the equiosmolar glucose buffer. Changes in internal calcium concentration upon addition of 1 mM ATP were followed in a confocal microscope by measuring the fluorescent intensity of the entrapped Fluo-5N (_ex = 495 nm and _em = 515 nm).

Light-induced transmembrane pH gradients by BR reconstituted into proteoliposomes were measured as changes in the fluorescence intensity of the Delta-pH-probe 9-AA (Cladera et al., 1996). Fluorescence was monitored with a Perkin Elmer LS 50B spectrofluorimeter using 400 and 460 nm for excitation and emission, respectively. Illumination was performed with a 250-W xenon lamp through a flexible glass fiber guide equipped with a low wavelength cutoff at 500 nm and a heat filter.

For BR pumping activity in GUVs, we measured the variations in the internal pH as changes in the fluorescence intensity of the membrane-impermeable pH sensitive probe pyranine entrapped into the GUVs lumen (Rigaud et al., 1988). Here, the GUVs containing BR were formed by electroswelling in the sucrose buffer supplemented with 10 µM pyranine. After electroswelling, GUVs were transferred into a microchamber containing the equiosmolar glucose buffer. Changes in internal pyranine fluorescence after 10 min illumination at 458 nm with a xenon lamp were monitored between 505 and 530 nm in a confocal microscope.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION AND CONCLUSION ACKNOWLEDGEMENTS REFERENCES

 
Reconstitution procedure
Our procedure for reconstituting membrane proteins into GUVs involves three successive steps. In the first step, solubilized proteins are reconstituted into the membranes of submicron proteoliposomes according to the detergent-mediated reconstitution protocol described by Rigaud and co-workers (Rigaud et al., 1995; Rigaud and Levy, 2003). This protocol, which consists of removing detergent from lipid-protein-detergent micellar solutions, has been described as very efficient and relevant for the reconstitution of many membrane proteins, including the Ca²⁺-ATPase (Levy et al., 1992) and BR (Rigaud et al., 1988). The use of polystyrene beads as a detergent-removing agent is a highly reproducible way to generate unilamellar proteoliposomes of 0.1–0.2 µm in size and with homogenous protein incorporation in a large range of lipid/protein ratios. In addition, it allows for quasicomplete detergent removal, minimizing the possible interference of residual detergent molecules on the further formation of giant vesicles (for review see Rigaud et al., 1998). Finally, it is of note that, starting from a micellar lipid-protein-detergent mixture, this reconstitution procedure produces proteoliposomes with an asymmetric but preferential protein orientation in the membrane: 80% of the Ca²⁺-ATPase molecules are oriented with their large cytoplasmic domain outside, leading to efficient ATP-dependent Ca²⁺ accumulation (Levy et al., 1992), whereas 75% of the BR molecules are oriented with their C-terminus outside, leading to an efficient light-induced H⁺ accumulation inside the proteoliposomes (Rigaud et al., 1988).

In the second step, these preformed proteoliposomes are dehydrated on ITO treated glass slides. Preliminary experiments indicated that complete dehydration under high vacuum had deleterious effects on the biological activities of BR and Ca²⁺-ATPase. Thus, it is essential to perform a partial dehydration of the proteoliposomes under controlled humidity, i.e., under saturated vapor pressure of a saturated salt solution.

The third step of the reconstitution process is a rehydration step in the presence of an AC electric field. Growing of large GUVs from the rehydrated lipid-protein film was promoted with an AC electric field with increments from 20 mV to 1.1 V at 12 Hz frequency. Once the GUVs were formed, the AC frequency was lowered to 4 Hz and the voltage raised to 2 V for 30 min. This helped the mature giant vesicles to adopt a spherical shape and to separate them from the glass slides. It is important to stress that the buffer composition used for the preparation of the preformed liposomes and for the rehydration step is crucial, considering that low ionic strengths impede the formation of giant vesicles by electroformation (see below).

Morphology and size
The effects of protein content, lipid composition, or buffer composition on the morphology and the size of reconstituted GUVs were monitored by phase contrast microscopy (Fig. 1 a). For Ca²⁺-ATPase and BR reconstitution, GUV formation was dependent upon the lipid/protein ratio of the initial detergent-reconstituted proteoliposomes. Giant vesicles could be directly formed from native SR vesicles (lipid/Ca²⁺-ATPase ratio of 0.8 w/w), but they exhibited tubular, ovoid, and pear-like shapes, probably related to the geometry of Ca²⁺-ATPase. Starting from proteoliposomes reconstituted at lipid/Ca²⁺-ATPase ratios of 3.5 w/w (i.e., 490 mol/mol), spherical GUVs began to form, and the fraction of nonspherical vesicles decreased with increasing lipid/protein ratios. Preparations with only spherical GUVs could be obtained from proteoliposomes with lipid/Ca²⁺-ATPase ratios ranging from 5 to 40 w/w (i.e., from 680 to 5500 mol/mol). As already observed for the electroformation of pure lipidic giant vesicles, the size distribution of the GUVs containing Ca²⁺-ATPase was very broad with diameters varying from 5 to 100 µm, centered around 20 µm (Fig. 1 b). Similar results were obtained for the reconstitution of BR into GUVs. In particular, no GUVs could be formed starting from BR-proteoliposomes reconstituted at lipid/protein ratios smaller than 5 w/w (160 mol/mol), whereas spherical and large GUVs could be formed from proteoliposomes reconstituted at lipid/BR ratios ranging from 5 to 40 w/w (160–1350 mol/mol).

View larger version (64K): [in this window] [in a new window]   FIGURE 1  Size distribution of reconstituted GUVs. (a) Phase-contrast image of EYPC/EYPA (9:1) GUVs containing Ca2+-ATPase at a lipid/protein ratio of 6.5 w/w. Scale bar = 10 µm. (b) Size distribution of the Ca2+-ATPase reconstituted GUVs. Vesicles with a diameter lower than 5 µm were not counted.

The composition of the buffers used for the preparation of the preformed liposomes and for the rehydration buffer is very important since the process of GUV formation by electroformation requires low ionic strengths (Angelova, 2000). Indeed, we have found that GUVs could not be formed with buffers containing more than 5 mM monovalent ions or 1 mM divalent ions. Since these low ionic strengths could be deleterious for biological activities, we have determined the minimal salt concentration needed to maintain the activity of the reconstituted proteins. For BR, we analyzed the amplitudes of the light-induced transmembrane pH gradients at different points in our reconstitution procedure by monitoring the changes in 9-AA fluorescence depending upon the buffer composition. When reconstitutions were performed in pure water, no activities could be detected. However, buffers containing 1 mM Mops-Tris pH 7.0 and 2 mM KCl were sufficient to observe a large light-induced 9-AA fluorescence quenching (F/F = 30%) in the initial preformed proteoliposomes and after the dehydration/rehydration step (data not shown). For Ca²⁺-ATPase, reconstitution and rehydration buffers containing 1 mM Mops-Tris pH 7.0 and 1 mM MgCl₂ were sufficient to retain the hydrolytic ATPase activity in preformed liposomes and after the dehydration/rehydration step (data not shown; see also Fig. 6 a).

View larger version (35K): [in this window] [in a new window]   FIGURE 6  Activity of Ca2+-ATPase into GUVs. Giant vesicles were prepared in EYPC/EYPA (9:1) at a lipid/Ca2+-ATPase ratio of 6.5 w/w. (a) ATPase activity was performed at 25°C as described in Materials and Methods. The reconstituted GUVs were transferred to a cuvette and solubilized with C12E8 (arrow 1). The reaction was started by the addition of ATP and stopped with EGTA. (b) Calcium pumping activity. Confocal images of a Ca2+-ATPase GUV (30 µm in diameter) reconstituted in EYPC/EYPA/NBD-C12-HPC s and encapsulating Fluo-5N. After ATP addition, the fluorescence intensity in the interior of the vesicles increases as a function of time. (b1) t = 5 min; (b2) t = 15 min; and (b3) t = 25 min.

View larger version (79K): [in this window] [in a new window]   FIGURE 2  Unilamellarity of reconstituted GUVs using a fluorescent quenching assay. Giant vesicles were prepared with a lipid mixture containing EYPC/NBD-C12-HPC (99.5:0.5 mol/mol) at a lipid/Ca2+-ATPase ratio of 6.5 w/w. (a) Unilamellarity of the GUVs was checked by measuring the distribution of fluorescent lipids between inner and outer monolayers as described in Materials and Methods. Addition of sodium hydrosulfite to reconstituted GUVs (arrow 1) induced a rapid quenching of the fluorescent lipids present in the outer lipid layer. Solubilization of the vesicles by an excess of Triton X-100 (arrow 2) induced a second decrease in fluorescence due to the quenching of the inner lipids. The I1/I2. ratio is an index of the unilamellarity of the vesicles and should be equal to 0.5 for a perfect unilamellarity. The value of 0.6 found for reconstituted GUVs could be accounted for by the presence of small vesicles (b) or other lipidic structures (arrows in c) that were sometimes visible encapsulated by the GUVs. Scale bars of phase-contrast images correspond to 5 µm.

(1)

in which A₀ is the optically measured surface at the initial value ₀ of the tension, A_p is the projected surface for consecutive tension values , and the thermal energy k_BT is 4 x 10^–21 J. As depicted in Fig. 3 b, the tension of a GUV reconstituted at a lipid/Ca²⁺-ATPase ratio of 6.5 w/w (900 mol/mol) increases exponentially with its excess area. These data (solid squares) can be fitted perfectly with Eq. 1 (solid line) using an elastic bending modulus of (9.3 ± 1.1) x k_BT. Fig. 3 c shows the results for 80 independent tests that give an average modulus of 9.0 x k_BT with a standard deviation of 1.5 x k_BT. The order of magnitude of this modulus value is in good agreement with the value of (10.2 ± 1.5) x k_BT found for pure lipid vesicles (Fig. 3 d). Even if the presence of the Ca²⁺-ATPase induces a small decrease in the bending rigidity of the membrane, the vesicles are clearly unilamellar, as no higher modulus multiple of was measured. Moreover, this small decrease may be due to the renormalization of the bending rigidity in the presence of inclusions (endogenous lipids and proteins) (Leibler, 1986). Similar results have been obtained with BR, confirming the unilamellarity of reconstituted GUVs (data not shown).

View larger version (47K): [in this window] [in a new window]   FIGURE 3  Unilamellarity of reconstituted GUVs using elastic bending measurement. Giant vesicles were prepared in EYPC/EYPA (9:1) at a lipid/Ca2+-ATPase ratio of 6.5 w/w. (a) Microscopy image of a reconstituted GUV aspirated with a suction pressure by a micropipette and observed with a 63x differential interference contrast (DIC) oil immersion objective. DP, pipette diameter; Dv, vesicle diameter; and Lp, projection length inside the micropipette. Scale bar = 5 µm. (b) Plot of membrane tension versus the area dilation of a reconstituted vesicle. The solid curve is fitted with an exponential function based on a bending rigidity modulus = (9.3 ± 1.1) x kBT. Histograms of elastic bending moduli from micropipette aspiration experiments on 80 Ca2+-ATPase reconstituted GUVs (c) and on 100 pure lipidic GUVs (d).

View larger version (20K): [in this window] [in a new window]   FIGURE 4  Homogeneity of Ca2+-ATPase incorporation in GUVs. (a) Fluorescence intensity image (false color representation) of a GUV reconstituted at a SOPC/FITC labeled Ca2+-ATPase ratio of 6.5 w/w. Scale bar = 5 µm. (b) Fluorescence distribution of 50 GUVs reconstituted at lipid/Ca2+-ATPase ratios of 3.5 (dark shading), 6.5 (light shading), and 12.5 w/w (open). A normal distribution function has been superimposed for each ratio giving the mean and standard deviation. (c) The average of the normalized fluorescence intensity, obtained in b, is reported as a function of the lipid to Ca2+-ATPase () or lipid/BR () ratios.

View larger version (35K): [in this window] [in a new window]   FIGURE 5  Homogeneity of BR incorporation in GUVs. (a) Confocal image of FITC-labeled BR reconstituted into SOPC vesicles. The fluorescence is homogenously distributed in the membrane of the vesicle. (b) Epifluorescence videomicroscopy frame (courtesy of Dr. J.-B. Manneville, Unité Mixte de Recherche 144 Centre National de la Recherche Scientifique/Institut Curie, Paris) of FITC-labeled BR reconstituted into SOPC vesicles according to the protocol of Manneville et al. (2001). The fluorescence of the membrane is not homogenous suggesting the presence of BR aggregates. Scale bars = 5 µm.

Proteins retain biological activities in GUVs
The efficiency of the reconstitution of Ca²⁺-ATPase into GUVs was analyzed by measuring the calcium-dependent hydrolytic activity and the calcium pumping activity of the reconstituted protein. Using an enzyme coupled assay, which couples ATP hydrolysis to the consumption of NADH (Fig. 6 a), we measured an ATPase activity of 4 µmol ATP/min/mg protein in reconstituted GUVs, whatever the lipid/protein ratio. This value is only 30% lower than that measured in the native SR, indicating that the different steps of our new reconstitution procedure do not significantly affect protein function. In theory, the fraction of Ca²⁺-ATPase accessible from the outside of the GUVs could have been determined by comparing the calcium-dependent ATPase activity with and without added detergent in the medium of measurement. Unfortunately, such a comparison could not be done since the integrity of the GUVs could not be maintained due to the mixing necessary for this cuvette assay. We have also analyzed the calcium pumping activity of Ca²⁺-ATPase reconstituted into GUVs, using a fluorescent calcium indicator, Fluo-5N, encapsulated by GUVs. In the absence of ATP, the fluorescence intensity of the GUV's lumen remained at a basal level. ATP addition in the presence of Ca²⁺ resulted in a significant time-dependent increase in fluorescence, as shown in Fig. 6 b, demonstrating efficient ATP-dependent calcium pumping into the inner compartment of GUVs.

The efficiency of the reconstitution of BR into GUVs was analyzed by measuring the light-dependent proton pumping activity, using pyranine as a fluorescent pH-sensitive probe, encapsulated by GUVs. For each vesicle, pictures were recorded with the same settings (e.g., laser intensity, acquisition time, and photomultiplier's sensitivity) to discriminate the effects of photobleaching from the pH changes in the GUV's lumen induced by the H⁺ pumping. Interestingly, the analysis of pyranine fluorescence of 15 independent GUVs revealed the existence of two populations. Fig. 7 depicts the changes observed in two-thirds of the BR containing GUVs. Upon illumination (shaded area in Fig. 7), a large decrease in the pyranine fluorescence intensity is observed, reaching a steady state after about a 5–6-min illumination period. This is indicative of an internal acidification and thus of an inward proton pumping activity mediated by BR. When the illumination is stopped, the fluorescence of pyranine increases as a function of time due to the passive diffusion of the H⁺ previously accumulated in the lumen of the vesicle. However, in one-third of the GUVs, we observed a light-dependent increase of the pyranine fluorescence intensity that reverses when the light is turned off (data not shown). All these data indicate that BR keeps its activity in the reconstituted GUVs. They also indicate that although most of the GUVs have a preferential BR orientation that leads to inward light-dependent H⁺ pumping activity, a significant proportion of the GUVs have an opposite preferential orientation of BR.

View larger version (26K): [in this window] [in a new window]   FIGURE 7  Proton pumping activity of BR into GUVs. Light-induced fluorescence responses of pyranine trapped inside giant vesicles. Giant vesicles containing pyranine were prepared in SOPC at a lipid/BR ratio of 7 (w/w) and analyzed by confocal microscopy. In the shaded part, BR is illuminated and pumps H+. In the open part, the illumination is stopped, and the pH reequilibrates. correspond to the direct measured variation of fluorescence intensities. correspond to the fluorescence intensities corrected for the photobleaching. • correspond to the extrapolated initial fluorescence intensities since the vesicles were already illuminated in the confocal microscope during the installation of the sample and selection of the vesicle, leading to a pumping of protons before the fluorescence measurement.

	DISCUSSION AND CONCLUSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION AND CONCLUSION ACKNOWLEDGEMENTS REFERENCES

The main criteria for a successful reconstitution experiment is the conservation of the protein activity. This was systematically monitored at each step of our reconstitution protocol. Concerning the first step of the protocol, which relies on the incorporation of solubilized membrane proteins into submicron liposomes, we used a detergent-mediated procedure that allows for very efficient reconstitution of Ca²⁺-ATPase and BR (Rigaud and Levy, 2003). Interestingly, the reconstitution of any membrane protein of interest can be rapidly optimized using this procedure, by testing different detergents, different lipids, and analyzing the resulting biological activities. The following steps of our reconstitution protocol involve dehydration of the preformed proteoliposomes followed by rehydration in an electric field. These two steps can be deleterious for membrane proteins. We found that it was necessary to carry out the dehydration step of the preformed proteoliposomes at a low rate and also under a controlled humidity atmosphere to preserve proteoliposomes from complete dehydration. In addition, we took into account that the process of electroformation imposes an ionic strength below 5 mM for the initial buffer used to incorporate the protein into small proteoliposomes as well as for the buffer used during the rehydration. We show that these low ionic conditions do not significantly affect the biological activities of two radically different membrane proteins, the Ca²⁺-ATPase and the bacteriorhodopsin. Noteworthily, reconstituted GUVs were produced that sustained a high level of activity as demonstrated by the light-induced internal pH changes due to the H⁺ pumping activity of BR or the changes in internal Ca²⁺ concentration due to the ATP-dependent Ca²⁺ pumping activity of the Ca²⁺-ATPase.

We also characterized the unilamellarity of the vesicles, together with the quality of protein incorporation in terms of distribution in the reconstituted membranes. Besides preserving the biological activity of the reconstituted proteins, one of the advantages of our method is that it generates spherical unilamellar giant vesicles at a high yield whatever the lipid composition and in a large range of lipid/protein ratios. In particular, it is of note that giant unilamellar vesicles could be reconstituted at lipid/protein ratios as low as 5 w/w (i.e., 160 or 680 mol/mol) for BR and Ca²⁺-ATPase, respectively. At this lipid/protein ratio of 5 w/w, one can estimate that a vesicle of 20 µm in diameter contains 3 x 10⁶ or 6.5 x 10⁵ molecules of BR and Ca²⁺-ATPase, respectively. Also important, it is shown (Figs. 4 a and 5 a) that after reconstitution, fluorescently labeled proteins are smoothly distributed over the entire membrane proteins and do not form aggregates detectable with a microscope. In addition, the analysis of different reconstituted GUVs indicates that their composition is homogeneous in the vesicle suspension, ensuring reproducibility in the experiments. This shows definite improvement compared to previous reports on the reconstitution of bacteriorhodopsin. Using the protocol of Manneville and co-workers (Manneville et al., 2001), by which GUVs were produced by electroformation from a lipid-BR film in organic solvent, BR was found heterogeneously distributed in the vesicle preparation and, more drastically, formed aggregates in the membrane. On the other hand, using the protocol of Kahya et al. (2001) in which preformed proteoliposomes are fused with lipidic GUVs, despite a homogenous protein distribution, the amount of proteins that could be incorporated remained limited due to the presence of the fusogenic agents, which have been reported to induce some lysis at high lipid/protein ratios.

Another critical parameter for membrane protein reconstitution is related to the final orientation of the protein within the membrane. Activity measurements performed on BR-reconstituted GUVs reveal the presence of two distinct populations of protein-reconstituted GUVs, which undergo opposite internal pH variations upon illumination (Fig. 7). About two-thirds of the GUVs have a preferential inside-out orientation of BR leading to an inwardly directed H⁺ transport (Fig. 7), whereas about one-third have a preferential right-side out orientation leading to an outwardly directed H⁺ transport. In this context, it has been shown that two orientations of the protein were also present in the initial preformed BR proteoliposomes, with the inside out orientation (75%) predominant. However, by using the pH sensitive fluorescence probe approach at acidic pHs (Seigneuret and Rigaud, 1985, 1988), it was demonstrated that these two orientations were not segregated in different subclasses of proteoliposomes but homogeneously distributed over the entire population of the initial preformed BR proteoliposomes (Rigaud et al., 1988). Thus, the reason for two GUVs populations with opposite preferential protein orientations must be related to the dehydration/rehydration steps. It is difficult to provide an explanation of how two such types of GUVs are formed in parallel, since little is known about the mechanisms of vesicle dehydration and rehydration. Concerning the Ca²⁺-ATPase reconstitution, we have only detected ATP-dependent calcium pumping into the inner compartment of GUVs. However, as opposed to light driven proton pumps, only those Ca²⁺-ATPases oriented right-side out respond to the substrate (ATP) added in the external medium, and, thus, only precise measurements of the Ca²⁺ pumping rates would permit the detection of GUVs with different Ca²⁺-ATPase orientations.

In conclusion, our method of membrane protein reconstitution into giant vesicles fulfills a number of important criteria. This method is relatively simple and also gentle with respect to the protein activity conservation. In addition, it produces giant proteoliposomes of more than 20 µm in diameter, which are spherical and unilamellar, and in which membrane proteins are homogeneously inserted and distributed. Although our method has only been evaluated for the reconstitution of two membrane proteins, the sarcoplasmic reticulum Ca²⁺-ATPase and BR, presumably it will apply to the reconstitution of other transmembrane proteins. Such giant vesicles are critical for studying the mechanisms of transport by membrane proteins as demonstrated here for BR and Ca²⁺-ATPase. More specifically, it will be applicable to single-molecule optical microscopy studies of lateral and rotational mobility and, more generally, folding and association-dissociation equilibriums of individual protein molecules, lipid-protein, and protein-protein interactions. The GUVs' approach could provide an interesting complement to preparative techniques such as, for example, patch-clamp (Walz et al., 2002). High protein incorporation into the membranes of giant vesicles will be of value for high resolution imaging of the surface topography of membrane proteins by atomic force microscopy, keeping in mind that a limitation of this approach is related to the size of the samples to be imaged (Scheuring et al., 2001). Also, the ability to have a controlled concentration of transmembrane proteins in giant vesicles will be of use in the analysis of the adhesion of decorated membranes (Abdelghani-Jacquin et al., 2002), the propulsion of biomimetic bacteria (Giardini et al., 2002), or the effect of the protein activity on the fluctuations of the membrane (Manneville et al., 2001). In this context, using the pipette experiments presented in Fig. 3 a, we have shown that, in the presence of ATP, the activity of the Ca²⁺-ATPase contributed to the amplification of membrane fluctuations, leading to a modulation of the slope of the log(tension) versus relative excess area (our unpublished data).

In the long term, we can also consider interesting biotechnological applications for this technique, for example, reconstituting transport proteins to control the composition inside the GUVs, which could then be used as chemical microreactors (Karlsson et al., 2001). Also, preparations of functionalized GUVs could be used for building up biosensors (Barth et al., 2003). Finally, considering the increasing number of membrane proteins that have been identified in the sequencing of the genomes of different organisms, the reconstitution of many different proteins can be tested in GUVs, and new applications in pharmaceutics or chemistry will emerge.

	ACKNOWLEDGEMENTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION AND CONCLUSION ACKNOWLEDGEMENTS REFERENCES

 
We are very grateful to Dr. Philippe Champeil (Unité de Recherche Associée 2096 Centre National de la Recherche Scientifique /Commissariat à l'Énergie Atomique, Gif-sur-Yvette) for the gift of SR vesicles. We especially thank Dr. Daniel Lévy (Unité Mixte de Recherche 168 Centre National de la Recherche Scientifique/Institut Curie, Paris) for many helpful discussions and Dr. Philippe Méléard (Unité Mixte de Recherche 6052 Centre National de la Recherche Scientifique/Ecole Nationale Supérieure de Chimie de Rennes, Rennes) for his initial suggestion to reconstitute the proteins first into small liposomes. We also thank the staff "Imagerie" of the Curie Institute for confocal microscopy facilities.

This work was supported by grants from the "Fondation pour la Recherche Médicale" (FDT20030627286), from the Délégation Générale à l'Armement/Centre National de la Recherche Scientifique, and from the Centre National de la Recherche Scientifique /Fond National de la Science (Action Concertée Incitative: "Physico-Chimie de la Matière Complexe" 01N40/0020).

Submitted on January 20, 2004; accepted for publication March 31, 2004.

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