Shapes of bilayer vesicles with membrane embedded molecules

The interdependence of the lateral distribution of molecules which are embedded in a membrane (such as integral membrane proteins) and the shape of a cell with no internal structure (such as phospholipid vesicles or mammalian erythrocytes) has been studied. The coupling of the lateral distribution of the molecules and the cell shape is introduced by considering that the energy of the membrane embedded molecule at a given site of the membrane depends on the curvature of the membrane at that site. Direct interactions between embedded molecules are not considered. A simple expression for the interaction of the membrane embedded molecule with the local membrane curvature is proposed. Starting from this interaction, the consistently related expressions for the free energy and for the distribution function of the embedded molecules are derived. The equilibrium cell shape and the corresponding lateral distribution of the membrane embedded molecules are determined by minimization of the membrane free energy which includes the free energy of the membrane embedded molecules and the membrane elastic energy. The resulting inhomogeneous distribution of the membrane embedded molecules affects the cell shape in a nontrivial manner. In particular, it is shown that the shape corresponding to the absolute energy minimum at given cell volume and membrane area may be elliptically non-axisymmetric, in contrast to the case of a laterally homogeneous membrane where it is axisymmetric.     

Heat-shock proteins in membrane vesicles of Bacillus subtilis

Fractionation of B. subtilis cells after heat shock, from 37 degrees C to 54 degrees C, shows an increase in synthesis of proteins localized in cell membranes and a decrease in synthesis of proteins localized in cytosol. There is no such effect of heat shock at temperature of 45 degrees C. Autoradiograms of electrophoretically separated proteins, labelled during heat shock at 54 degrees C, reveal 26 heat-shock proteins (hsps) in membrane vesicles and 11 hsps in cytosol, five of which are common to both fractions. Heat shock at 45 degrees C induces 18 hsps localized in membrane vesicles and 13 hsps localized in cytosol, six of which are common to both fractions. Results are interpreted as showing a relevant role of membrane proteins in cell response to shock at high temperature, pointing to two steps of defense against heat stress.     

Localization of membrane proteins in membrane vesicles of Bacillus subtilis.

Electrons can be transferred to the respiratory chain in whole cells and in membrane vesicles of Bacillus subtilis W 23 by the membrane impermeable electron donor reduced 5-N-methyl-phenazonium-3-sulfonate as efficiently as by the membrane permeable electron donor reduced 5-N-methyl-phenazonium methyl-sulfate, indicating that the respiratory chain is accessible from the outside of the membrane.Succinate is oxidized by whole cells and membrane vesicles at a low rate and does not energize transport of l-glutamate. In the presence of 5-N-methyl-phenazonium-3-sulfonate or 5-N-methyl-phenazonium methyl-sulfate, the oxidation rate and the rate of l-glutamate transport are increased considerably. The electrons are transferred directly from succinic dehydrogenase to these acceptors. Succinic dehydrogenase must therefore be exposed to the outside surface of the membrane in both membrane vesicles and whole cells. The exposure of succinic dehydrogenase to the outside is also indicated by the observations that only a 5% increase in the oxidation rates of succinate-5-N-methyl-phenazonium methylsulfate and succinate-5-N-methyl-phenazonium-3-sulfonate is observed upon solubilization of the membrane with the nonionic detergent Brij-58. Furthermore, treatment of membrane vesicles with trypsin decreases by more than 95% these oxidation rates.NADH is oxidized at a high rate and energizes transport of l-glutamate in whole cells and membrane vesicles effectively. The NADH-oxidation is not effected by trypsin treatment of the vesicles indicating that the oxidation occurs at the inside-surface of the membrane. Trypsin treatment of the vesicles, however, significantly decreases the rate of l-glutamate transport driven by NADH. Therefore component(s) of the transport system for l-glutamate must be effected by trypsin treatment. No apparent differences could be observed in the localization of membrane-bound functions between membrane vesicles and whole cells. This strongly supports the contention that the vesicle membrane of B. subtilis has the same orientation as the cytoplasmic membrane of whole cells.     

Selective sorting of cargo proteins into bacterial membrane vesicles

In contrast to the well established multiple cellular roles of membrane vesicles in eukaryotic cell biology, outer membrane vesicles (OMV) produced via blebbing of prokaryotic membranes have frequently been regarded as cell debris or microscopy artifacts. Increasingly, however, bacterial membrane vesicles are thought to play a role in microbial virulence, although it remains to be determined whether OMV result from a directed process or from passive disintegration of the outer membrane. Here we establish that the human oral pathogen Porphyromonas gingivalis has a mechanism to selectively sort proteins into OMV, resulting in the preferential packaging of virulence factors into OMV and the exclusion of abundant outer membrane proteins from the protein cargo. Furthermore, we show a critical role for lipopolysaccharide in directing this sorting mechanism. The existence of a process to package specific virulence factors into OMV may significantly alter our current understanding of host-pathogen interactions.     

Signals involved in targeting membrane proteins to synaptic vesicles

1. Synaptic vesicles (SVs) mediate fast regulated secretion of classical neurotransmitters. In order to perform their task SVs rely on a restrict set of membrane proteins. The mechanisms responsible for targeting these proteins to the SV membrane are still poorly understood.2. Likewise, little is known about the intracellular routes taken by these proteins in their way to SV membrane. Recently, several domains and motifs necessary for correct localization of SV proteins have been identified.3. In this review we summarize the sequence motifs that have been identified in the cytoplasmic domains of SV proteins that are involved in endocytosis and targeting of SVs. We suggest that the vesicular acetylcholine transporter, a protein found predominantly in synaptic vesicles, is perhaps a model protein to understand the pathways and interactions that are used for synaptic vesicle targeting.     

Synaptic vesicles interchange their membrane proteins with a large surface reservoir during recycling

During recycling of synaptic vesicles (SVs), the retrieval machinery faces the challenge of recapturing SV proteins in a timely and precise manner. The significant dilution factor that would result from equilibration of vesicle proteins with the much larger cell surface would make recapture by diffusional encounter with the endocytic retrieval machinery unlikely. If SV proteins exchanged with counterparts residing at steady state on the cell surface, the dilution problem would be largely avoided. In this scenario, during electrical activity, endocytosis would be driven by the concentration of a pre-existing pool of SVs residing on the axonal or synaptic surface rather than the heavily diluted postfusion vesicular pool. Using both live cell imaging of endogenous synaptotagmin Ia (sytIa) as well as pHluorin-tagged sytIa and VAMP-2, we show here that synaptic vesicle proteins interchange with a large pool on the cell axonal surface whose concentration is approximately 10-fold lower than that in SVs.     

The anion permeability of vesicles reconstituted with intrinsic proteins from the human erythrocyte membrane

Band 3 protein was reconstituted with lipid vesicles consisting of 94:6 (molar ratio) egg phosphatidylcholine-bovine heart phosphatidylserine in a 2500:1 phospholipid:protein molar ratio by means of a Triton X-100/beads method. The SO2-4 permeability of the resulting vesicles was measured using an influx assay procedure in which the vesicles were sampled and subsequently eluted over Sephadex columns at appropriate time intervals. The accuracy of the assay was greatly increased by using an internal standard in order to correct for vesicle recovery. In agreement with previous work, it could be demonstrated that incorporation of band 3 in the vesicles caused an increase in SO2-4 permeability, which could be (partially) inhibited by high concentrations of DIDS or a competitive anion such as thiocyanate. However, the magnitude of the increased SO2-4 permeability was highly variable, even when vesicles were reconstituted using band 3 isolated from one batch of ghosts. In addition, the SO2-4 influx curves showed complex kinetics. These results are related to the existence of vesicle heterogeneity with respect to protein content and vesicle size as revealed by stractan density gradient centrifugation and freeze-fracture electron microscopy. Band 3 incorporation also increased the L-glucose permeability of the vesicles which could also be inhibited by DIDS. Glycophorin, which has no known transport function, reconstituted with lipid vesicles consisting of 94:6 (molar ratio) egg phosphatidylcholine-bovine heart phosphatidylserine in a 400:1 phospholipid:protein molar ration increased the bilayer permeability towards SO2-4 as well as towards L-glucose. Surprisingly, the SO2-4 permeability in the vesicles could also be inhibited by DIDS and thiocyanate. It is concluded that the use of DIDS and a competitive anion, thiocyanate, in order to prove that band 3 is functionally reconstituted, is highly questionable. The increased SO2-4 and L-glucose permeability of band 3-lipid as well as glycophorin-lipid vesicles and the inhibitory action of DIDS are discussed in the light of the presence of defects at the lipid/protein interface and protein aggregation, which may induce the formation of pores. Since the band 3-lipid vesicles are more permeable for SO2-4 than for L-glucose, in contrast to the glycophorin-containing vesicles, it is suggested that some anion specificity of the increased bilayer permeability in the band 3-lipid vesicles is still preserved.     

Incorporation of heterologous outer membrane and periplasmic proteins into Escherichia coli outer membrane vesicles

Gram-negative bacteria shed outer membrane vesicles composed of outer membrane and periplasmic components. Since vesicles from pathogenic bacteria contain virulence factors and have been shown to interact with eukaryotic cells, it has been proposed that vesicles behave as delivery vehicles. We wanted to determine whether heterologously expressed proteins would be incorporated into the membrane and lumen of vesicles and whether these altered vesicles would associate with host cells. Ail, an outer membrane adhesin/invasin from Yersinia enterocolitica, was detected in purified outer membrane and in vesicles from Escherichia coli strains DH5alpha, HB101, and MC4100 transformed with plasmid-encoded Ail. In vesicle-host cell co-incubation assays we found that vesicles containing Ail were internalized by eukaryotic cells, unlike vesicles without Ail. To determine whether lumenal vesicle contents could be modified and delivered to host cells, we used periplasmically expressed green fluorescent protein (GFP). GFP fused with the Tat signal sequence was secreted into the periplasm via the twin arginine transporter (Tat) in both the laboratory E. coli strain DH5alpha and the pathogenic enterotoxigenic E. coli ATCC strain 43886. Pronase-resistant fluorescence was detectable in vesicles from Tat-GFP-transformed strains, demonstrating that GFP was inside intact vesicles. Inclusion of GFP cargo increased vesicle density but did not result in morphological changes in vesicles. These studies are the first to demonstrate the incorporation of heterologously expressed outer membrane and periplasmic proteins into bacterial vesicles.     

Functional mosaicism of membrane proteins in vesicles of Escherichia coli.

Membrane vesicles of Escherichia coli prepared by osmotic lysis of lysozyme ethylenediaminetetracetate (EDTA) spheroplasts have approximately 60% of the total membrane-bound reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase (ED 1.6.99.3) and Mg2+-adenosine triphosphatase (ATPase) (EC 3.6.1.3) activities exposed on the outer surface of the inner membrane. Absorption of these vesicles with antiserum prepared against the purified soluble Mg2+-ATPase resulted in agglutination of approximately 95% of the inner membrane vesicles, as determined by dehydrogenase activity, and about 50% of the total membrane protein. The unagglutinated vesicles lacked all dehydrogenase activity and may consist of outer membrane. Lysozyme-EDTA vesicles actively transported calcium ion, using either NADH or adenosine 5'-triphosphate (ATP) as energy source. However, neither D-lactate nor reduced phenazine methosulfate energized calcium uptake, suggesting that the observed calcium uptake was not due to a small population of everted vesicles. Transport of calcium driven by either NADH or ATP was inhibited by simultaneous addition of D-lactate or reduced phenazine methosulfate. Proline transport driven by D-lactate oxidation was inhibited by either NADH oxidation or ATP hydrolysis. These results suggest that the portion of the total population of vesicles capable of active transport, i.e., the inner membrane vesicles, are functionally a homogeneous population but cannot be categorized as either right-side-out or everted, since activities normally associated with only one side of the inner membrane can be found on both sides of the membrane of these vesicles. Moreover, the data indicate that oxidation of NADH or hydrolysis of ATP by externally localized NADH dehydrogenase or Mg2+-ATPase establishes a protonmotive force of the opposite polarity from that established through D-lactate oxidation.     

Reconstitution of membrane proteins into giant unilamellar vesicles via peptide-induced fusion

In this work, we present a protocol to reconstitute membrane proteins into giant unilamellar vesicles (GUV) via peptide-induced fusion. In principle, GUV provide a well-defined lipid matrix, resembling a close-to-native state for biophysical studies, including optical microspectroscopy, of transmembrane proteins at the molecular level. Furthermore, reconstitution in this manner would also eliminate potential artifacts arising from secondary interactions of proteins, when reconstituted in planar membranes supported on solid surfaces. However, assembly procedures of GUV preclude direct reconstitution. Here, for the first time, a method is described that allows the controlled incorporation of membrane proteins into GUV. We demonstrate that large unilamellar vesicles (LUV, diameter 0.1 microm), to which the small fusogenic peptide WAE has been covalently attached, readily fuse with GUV, as revealed by monitoring lipid and contents mixing by fluorescence microscopy. To monitor contents mixing, a new fluorescence-based enzymatic assay was devised. Fusion does not introduce changes in the membrane morphology, as shown by fluorescence correlation spectroscopy. Analysis of fluorescence confocal imaging intensity revealed that approximately 6 to 10 LUV fused per microm(2) of GUV surface. As a model protein, bacteriorhodopsin (BR) was reconstituted into GUV, using LUV into which BR was incorporated via detergent dialysis. BR did not affect GUV-LUV fusion and the protein was stably inserted into the GUV and functionally active. Fluorescence correlation spectroscopy experiments show that BR inserted into GUV undergoes unrestricted Brownian motion with a diffusion coefficient of 1.2 microm(2)/s. The current procedure offers new opportunities to address issues related to membrane-protein structure and dynamics in a close-to-native state.     

Unequal distribution of penicillin-binding proteins among inner membrane vesicles of Escherichia coli

Escherichia coli penicillin-binding proteins (PBPs) were associated only with inner membrane vesicles when separated on 30 to 65% or 19 to 49% (wt/wt) sucrose gradients. Fractionation of vesicles through the low-density gradient revealed at least two classes of PBP-inner membrane associations. The first class consisted of PBPs 1 through 4, and the second class consisted of PBPs 5 through 8. These classes were distinguished by the density of vesicles with which they were associated; class 1 PBPs migrated with vesicles of higher density than did class 2 PBPs. Such combinations suggest that PBPs are nonrandomly distributed within the inner membrane, implying potential functional relationships among the PBPs themselves and with particular membrane domains. In addition, in cell lysates and in vesicle fractions, a 60,000-dalton aztreonam-insensitive PBP or protein fragment was observed which could potentially be confused with PBP3.     

bSUM: A bead-supported unilamellar membrane system facilitating unidirectional insertion of membrane proteins into giant vesicles

Fused or giant vesicles, planar lipid bilayers, a droplet membrane system, and planar-supported membranes have been developed to incorporate membrane proteins for the electrical and biophysical analysis of such proteins or the bilayer properties. However, it remains difficult to incorporate membrane proteins, including ion channels, into reconstituted membrane systems that allow easy control of operational dimensions, incorporation orientation of the membrane proteins, and lipid composition of membranes. Here, using a newly developed chemical engineering procedure, we report on a bead-supported unilamellar membrane (bSUM) system that allows good control over membrane dimension, protein orientation, and lipid composition. Our new system uses specific ligands to facilitate the unidirectional incorporation of membrane proteins into lipid bilayers. Cryo–electron microscopic imaging demonstrates the unilamellar nature of the bSUMs. Electrical recordings from voltage-gated ion channels in bSUMs of varying diameters demonstrate the versatility of the new system. Using KvAP as a model system, we show that compared with other in vitro membrane systems, the bSUMs have the following advantages: (a) a major fraction of channels are orientated in a controlled way; (b) the channels mediate the formation of the lipid bilayer; (c) there is one and only one bilayer membrane on each bead; (d) the lipid composition can be controlled and the bSUM size is also under experimental control over a range of 0.2–20 µm; (e) the channel activity can be recorded by patch clamp using a planar electrode; and (f) the voltage-clamp speed (0.2–0.5 ms) of the bSUM on a planar electrode is fast, making it suitable to study ion channels with fast gating kinetics. Our observations suggest that the chemically engineered bSUMs afford a novel platform for studying lipid–protein interactions in membranes of varying lipid composition and may be useful for other applications, such as targeted delivery and single-molecule imaging.     

Interaction of microtubule proteins with phospholipid vesicles

We have examined the interaction of unilamellar dimyristoyl phosphatidylcholine liposomes with the high-speed supernate of brain homogenate and with tubulin purified through one or two cycles of microtubule assembly-disassembly. Tubulin and certian high molecular weight proteins are selectively adsorbed from these mixtures onto liposomes. The composition of adsorbed proteins is similar to that obtained during corresponding cycles of microtubule assembly, suggesting the equivalency of these processes. Adsorption induces stacking and/or fusion of liposomes into multilamellar structures indicating strong protein-lipid interaction. In addition, liposome-adsorbed tubulin forms extensive intermolecular disulfide bridges that are inert to reducing agents in the aqueous medium. The observations form a basis for further study of the distribution, function, and properties of membrane-bound tubulin.     

The transbilayer movement of phosphatidylcholine in vesicles reconstituted with intrinsic proteins from the human erythrocyte membrane

Vesicles have been prepared from 18 : 1_c/18 : 1_c-phosphatidylcholine with or without purified glycophorin or partially purified band 3 (obtained by organomercurial gel chromatography). The vesicles have been characterized by freeze-fracture electron microscopy, binding studies to DEAE-cellulose, ³¹P-NMR and K⁺ trap measurements. Pools of phosphatidylcholine available for exchange have been investigated using phosphatidylcholine exchange protein from bovine liver. The protein-containing vesicles both exhibit exchangeable pools larger than the fraction of phosphatidylcholine in the outer monolayer, whereas in the protein-free vesicles the exchangeable pool is consistent with the outer monolayer. The results indicate that both glycophorin and the partially purified band 3 preparation enhance the transbilayer movement of phosphatidylcholine.     

Immobilization of phospholipid vesicles and protein-lipid vesicles containing red cell membrane proteins on octyl derivatives of large-pore gels

For improved immobilization of phospholipid vesicles and protein-lipid vesicles (cf. Sandberg, M., Lundahl, P., Greijer, E. and Belew, M. (1987) Biochim. Biophys. Acta 924, 185-192) and for chromatographic experiments with vesicles containing membrane protein, we have prepared octyl sulfide derivatives of the large-pore gels Sephacryl S-1000 and Sepharose 2B with ligand concentrations up to 14 and 5 mumol/ml gel, respectively. The Sephacryl derivatives allowed higher flow rates, gave higher rates of adsorption and showed equally high or higher capacities than the Sepharose adsorbents. 'Small', 'medium' and 'large' vesicles of radii approx. 20, 50 and 100 nm showed distribution coefficients on Sephacryl S-1000 of 0.7, 0.5 and 0.05, respectively and could be immobilized on octyl sulfide-Sephacryl S-1000 in amounts corresponding to 110, 40 and 20 mumol of phospholipids per ml gel, respectively. 'Small' vesicles became absorbed onto this gel at a rate of 1.5 mumol of phospholipids per min per ml gel until 60 mumol of phospholipids had become immobilized, whereas the initial adsorption rate was about 0.4 mumol.min-1.ml-1 on octyl sulfide-Sepharose 4B (see reference above) and on octyl sulfide-Sepharose 2B. Lower ligand concentrations gave lower capacities for 'small' vesicles. When vesicles entrapping calcein were immobilized on octyl sulfide-Sephacryl S-1000 some calcein was released during the adsorption process. For 'small' and 'medium' vesicles, respectively, the leakage was 75 and 25% at a ligand concentration of 14 mumol/ml but only 3 and 2% at 5 mumol/ml. The internal volumes of immobilized 'small' and 'medium' vesicles were estimated at 0.97 and 2.9 microliters per mumol of phospholipid by determination of entrapped calcein, which could indicate vesicle radii 20 and 50 nm, respectively. The total volumes of immobilized 'medium' lipid vesicles and 'medium' protein-lipid vesicles containing integral membrane proteins from human red cells, were estimated at 2.9 and 2.0 microliters/mumol, respectively, by chromatography of D- and L-[14C]glucose and calcein on the octyl sulfide-Sephacryl S-1000 column before and after immobilization. These volumes are roughly consistent with the internal volume of the vesicles. A zone of D-glucose eluted 90 microliters later than a zone of L-glucose on a 4- or 5-ml column of octyl sulfide-Sephacryl S-1000 with immobilized 'medium' protein-lipid vesicles containing the glucose transporter from human red cells, probably since part of the internal vesicle volume was accessible to the D-glucose but not to the L-glucose. This indicates that the glucose transporter was active in the immobilized vesicles.(ABSTRACT TRUNCATED AT 400 WORDS)     

Bacterial bug-out bags: outer membrane vesicles and their proteins and functions

Among the major bacterial secretions, outer membrane vesicles (OMVs) are significant and highly functional. The proteins and other biomolecules identified within OMVs provide new insights into the possible functions of OMVs in bacteria. OMVs are rich in proteins, nucleic acids, toxins and virulence factors that play a critical role in bacteria-host interactions. In this review, we discuss some proteins with multifunctional features from bacterial OMVs and their role involving the mechanisms of bacterial survival and defence. Proteins with moonlighting activities in OMVs are discussed based on their functions in bacteria. OMVs harbour many other proteins that are important, such as proteins involved in virulence, defence, and competition. Overall, OMVs are a power-packed aid for bacteria, harbouring many defensive and moonlighting proteins and acting as a survival kit in case of an emergency or as a defence weapon. In summary, OMVs can be defined as bug-out bags for bacterial defence and, therefore, survival.     

Rotavirus interaction with isolated membrane vesicles.

To gain information about the mechanism of epithelial cell infection by rotavirus, we studied the interaction of bovine rotavirus, RF strain, with isolated membrane vesicles from apical membrane of pig enterocytes. Vesicles were charged with high (quenching) concentrations of either carboxyfluorescein or calcein, and the rate of fluorophore release (dequenching) was monitored as a function of time after mixing with purified virus particles. Purified single-shelled particles and untrypsinized double-shelled ones had no effect. Trypsinized double-shelled virions induced carboxyfluorescein release according to sigmoid curves whose lag period and amplitude were a function of virus concentration and depended on both temperature and pH. The presence of 100 mM salts (Tris Cl, NaCl, or KCl) was required, since there was no reaction in isoosmotic salt-free sorbitol media. Other membrane vesicle preparations such as apical membranes of piglet enterocyte and rat placenta syncytiotrophoblasts, basolateral membranes of pig enterocytes, and the undifferentiated plasma membrane of cultured MA104 cells all gave qualitatively similar responses. Inhibition by a specific monoclonal antibody suggests that the active species causing carboxyfluorescein release is VP5*. Ca2+ (1 mM), but not Mg2+, inhibited the reaction. In situ solubilization of the outer capsid of trypsinized double-shelled particles changed release kinetics from sigmoidal to hyperbolic and was not inhibited by Ca2+. Our results indicate that membrane destabilization caused by trypsinized outer capsid proteins of rotavirus leads to fluorophore release. From the data presented here, a hypothetical model of the interaction of the various states of the viral particles with the membrane lipid phase is proposed. Membrane permeabilization induced by rotavirus may be related to the mechanism of entry of the virus into the host cell.     

A new method for the reconstitution of membrane proteins into giant unilamellar vesicles

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 Ca(2+)-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 microm 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 Ca(2+) pumping driven by bacteriorhodopsin or Ca(2+)-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 microm in diameter, which should prove useful for functional and structural studies through the use of optical microscopy, optical tweezers, microelectrodes, or atomic force microscopy.     

Incorporation of membrane proteins into large single bilayer vesicles. Application to rhodopsin

A general procedure to incorporate membrane proteins in a native state into large single bilayer vesicles is described. The results obtained with rhodopsin from vertebrate and invertebrate retinas are presented. The technique involves: (a) the direct transfer of rhodopsin-lipid complexes from native membranes into ether or pentane, and (b) the sonication of the complex in apolar solvent with aqueous buffer followed by solvent evaporation under reduced pressure. The spectral properties of rhodopsin in the large vesicles are similar to those of rhodopsin in photoreceptors; furthermore, bleached bovine rhodopsin is chemically regenerable with 9-cis retinal. These results establish the presence of photochemically functional rhodopsin in the large vesicles. Freeze-fracture replicas of the vesicles reveal that both internal and external leaflets contain numerous particles approximately 80 A in diameter, indicating that rhodopsin is symmetrically distributed within the bilayer. More than 75% of the membrane area is incorporated into vesicles larger than 0.5 micron and approximately 40% into vesicles larger than 1 micron.     

Spontaneous transmembrane insertion of membrane proteins into lipid vesicles facilitated by short-chain lecithins

Functional reconstitution of the membrane protein bacteriorhodopsin into lipid vesicles is achieved by mixing aqueous suspensions of long-chain lecithins and purple membrane with the short-chain lecithin diheptanoylphosphatidylcholine (20 mol % of total lipid). The membrane protein is transmembranously inserted in the lipid bilayer of the vesicle and highly active as a light-energized proton pump. This rapid, easy, and gentle procedure might allow functional reconstitution of other membrane systems and isolated membrane proteins as well.