Reconstitution of the protein insertion machinery of the mitochondrial inner membrane.

We have reconstituted the protein insertion machinery of the yeast mitochondrial inner membrane into proteoliposomes. The reconstituted proteoliposomes have a distinct morphology and protein composition and correctly insert the ADP/ATP carrier (AAC) and Tim23p, two multi-spanning integral proteins of the mitochondrial inner membrane. The reconstituted system requires a membrane potential, but not Tim44p or mhsp70, both of which are required for the ATP-driven translocation of proteins into the matrix. The protein insertion machinery can thus operate independently of the energy-transducing Tim44p-mhsp70 complex.     

Solubilization and reconstitution of hepatic System A-mediated amino acid transport. Preparation of proteoliposomes containing glucagon-stimulated transport activity

System A-mediated amino acid transport activity from rat liver plasma membrane vesicles has been solubilized and reconstituted into proteoliposomes using a freeze-thaw-dilution technique. The presence of cholate, at a cholate to protein ratio of 1:1, during the freeze-thaw step resulted in an enhancement in recoverable transport activity. The carrier required both phosphatidylcholine and phosphatidylethanolamine for optimal activity, but the addition of cholesterol to the reconstitution procedure appeared to have no significant effect on the resulting activity. A lipid to protein ratio of 20:1 yielded maximal transport activity. Sonication of the proteoliposomes provided some improvement in the accuracy of replicate assays for a given proteoliposome preparation. Isolated liver plasma membrane vesicles prepared from rats treated in vivo with glucagon in combination with dexamethasone contained stimulated System A activity. This enhanced transport activity could be solubilized and recovered in proteoliposomes generated from these plasma membranes. The data support the proposal that hormone regulation of the hepatic System A gene results in the de novo synthesis and plasma membrane insertion of the carrier protein itself.     

Preparation of reconstituted acetylcholine receptor membranes suitable for AFM imaging of lipid–protein interactions

The nicotinic acetylcholine receptor (nAChR) has been reconstituted in POPC vesicles at high lipid–protein (L/P) ratios for the preparation of supported lipid bilayers with a low protein density for studies of protein–lipid interactions using atomic force microscopy (AFM). Initial reconstitutions using a standard dialysis method with bulk L/P ratios ranging from 20:1 to 100:1 (w/w) gave heterogeneous samples that contained both empty vesicles and proteoliposomes with a range of L/P ratios. This is problematic because empty vesicles adsorb and rupture to form bilayer patches more rapidly than do protein-rich vesicles, resulting in the loss of protein during sample washing. Although it was not possible to find reconstitution conditions that gave homogeneous populations of vesicles with high L/P ratios, an additional freeze–thaw cycle immediately after dialysis did reproducibly yield a fraction of proteoliposomes with L/P ratios above 100:1. These proteoliposomes were separated by sucrose gradient centrifugation and used to prepare supported bilayers with well-separated individual receptors and minimal adsorbed proteoliposomes. AFM images of such samples showed many small features protruding from the bilayer surface. These features range in height from 1 to 5 nm, consistent with the smaller intracellular domain of the protein exposed, and have lateral dimensions consistent with an individual receptor. Some bilayers with reconstituted protein also had a small fraction of higher features that are assigned to nAChR with the larger extracellular domain exposed and showed evidence for aggregation to give dimers or small oligomers. This work demonstrates the importance of using highly purified reconstituted membranes with uniform lipid–protein ratios for AFM studies of integral membrane protein–lipid interactions.     

Characterization of the unidirectional transport of carnitine catalyzed by the reconstituted carnitine carrier from rat liver mitochondria

The carnitine carrier from rat liver mitochondria was purified by chromatography on hydroxyapatite and celite and reconstituted in egg yolk phospholipid vesicles by adsorbing the detergent on polystyrene beads. In the reconstituted system, in addition to the carnitine/carnitine exchange, the purified protein catalyzed a uni-directional transport (uniport) of carnitine measured as uptake into unloaded proteoliposomes as well as efflux from prelabelled proteoliposomes. In both cases the reaction followed a first-order kinetics with a rate constant of 0.023-0.026 min-1. Besides carnitine, also acylcarnitines were transported in the uniport mode. N-Ethylmaleimide inhibited the uni-directional transport of carnitine completely. The uniport of carnitine is not influenced by the delta pH and the electric gradient across the membrane. The activation energy for uniport was 115 kJ/mol and the half-saturation constant on the external side of the proteoliposomes was 0.53 mM. The maximal rate of the uniport at 25 degrees C was 0.2 mumol/min per mg protein, i.e. about 10 times lower than that of the reconstituted carnitine transport in exchange mode.     

The H(+)-ATPase of the plasma membrane from yeast. Kinetics of ATP hydrolysis in native membranes, isolated and reconstituted enzymes

The H(+)-ATPase of the plasma membrane from Saccharomyces cerevisiae has been isolated, purified and reconstituted into asolectin liposomes. The kinetics of ATP hydrolysis have been compared for the H(+)-ATPase in the plasma membrane, in a protein/lipid/detergent micelle (isolated enzyme) and in asolectin proteoliposomes (reconstituted enzyme). In all three cases the kinetics of ATP hydrolysis can be described by Michaelis-Menten kinetics with Km = 0.2 mM MgATP (plasma membranes), Km = 2.4 mM MgATP (isolated enzyme) and Km = 0.2 mM MgATP (reconstituted enzyme). However, the maximal turnover decreases only by a factor of two during isolation of the enzyme and does not change during reconstitution; the activation of the H(+)-ATPase by free Mg2+ is also only slightly influenced by the detergent. The dissociation constant of the enzyme-Mg2+ complex Ka, does not alter during isolation and the dissociation constant of the enzyme-substrate complex, Ks, increases from Ks = 30 microM (plasma membranes) to Ks = 90 microM (isolated enzyme). ATP binding to the H(+)-ATPase ('single turnover' conditions) for the isolated and the reconstituted enzyme resulted in both cases in a second-order rate constant k1 = 2.6 x 10(4) M-1.s-1. From these observations it is concluded that the detergent used (Zwittergent TM 3-14) interacts reversibly with the H(+)-ATPase and that practically all H(+)-ATPase molecules are reconstituted into the liposomes with the ATP-binding site being directed to the outside of the vesicle.     

Reconstitution of the uncoupling protein of brown adipose tissue mitochondria. Demonstration of GDP-sensitive halide anion uniport

The fluorescent anion indicator 6-methoxy-N-(3-sulfopropyl)quinolinium was trapped in proteoliposomes reconstituted with purified 32-kDa uncoupling protein and used to detect GDP-sensitive uniports of Cl-, Br-, and I-. Transport of these halide anions was rapid and potential-dependent. F- and nitrate were found to inhibit Cl- uptake competitively, suggesting that these anions are also substrates for transport. This preparation also exhibited H+(OH-) transport, showing that the reconstituted uncoupling protein possesses both halide and H+ transport functions, as is observed in intact brown adipose tissue mitochondria. Cl- transport was inhibited to the residual level observed in liposomes without protein when GDP was present on both sides of the membrane. Cl- transport was inhibited by about 50% when GDP was present only on one side of the membrane. We infer that uncoupling protein reconstitutes into proteoliposomes with a 1:1 ratio of sidedness orientation. The Km values for Cl- uniport were 100 and 65 mM, respectively, in GDP-loaded and non-GDP-loaded vesicles. Participation of the inner membrane anion channel in the observed transport is rendered unlikely by the fact that this carrier is insensitive to GDP. A variety of additional experiments probing for inner membrane anion channel yielded uniformly negative results, confirming the absence of contamination by this protein. Our results therefore demonstrate that the uncoupling protein mediates anion translocation, a function previously reported as lacking in the reconstituted system.     

Incorporation of bacterial membrane proteins into liposomes: factors influencing protein reconstitution

Meningococcal and gonococcal outer membrane proteins were reconstituted into liposomes using detergent-mediated dialysis. The detergents octyl glucopyranoside (OGP), sodium cholate and Empigen BB were compared with respect to efficiency of detergent removal and protein incorporation. The rate of OGP removal was greater than for cholate during dialysis. Isopycnic density gradient centrifugation studies showed that liposomes were not formed and hence no protein incorporation occurred during dialysis from an Empigen BB containing reconstitution mixture. Cholate-mediated reconstitution yielded proteoliposomes with only 75% of the protein associated with the vesicles whereas all of the protein was reconstituted into the lipid bilayer during OGP-mediated reconstitution. Essentially complete protein incorporation was achieved with an initial protein-to-lipid ratio of 0.01:1 (w/w) in the reconstitution mixture; however, at higher initial protein-to-lipid ratios (0.02:1) only 75% protein incorporation was achieved. Reconstituted proteoliposomes were observed as large (>300 nm), multilamellar structures using cryo-electron microscopy. Size reduction of these proteoliposomes by extrusion did not result in significant loss of protein or lipid. Extruded proteoliposomes were unilamellar vesicles with mean diameter of about 100 nm.     

A study on the separation of reconstituted proteoliposomes and unincorporated membrane proteins by use of hydrophobic affinity gels, with special reference to band 3 from bovine erythrocyte membranes

Alkyl-Sepharose 4B with octyl, decyl, or dodecyl groups as an alkyl chain was a good adsorbent for any type of detergents and a variety of proteins, but not for phospholipids in a vesicle form. When these gels were added to the mixtures of reconstituted proteoliposomes prepared by using bovine band 3 and the protein unincorporated into liposomes, free band 3 in solution was adsorbed onto the gels and the proteoliposomes could be recovered by filtration, suggesting that this procedure, when applicable, permits a rapid isolation of proteoliposomes without loss and dilution of the sample. In addition, the results indicated that Bio-Beads SM-2 resin, which is virtually nonadsorbing for most proteins, can be used in removing any kind of detergents from those protein-detergent mixtures.     

Reconstitution of the binding protein-dependent galactose transport of Salmonella typhimurium in proteoliposomes.

Binding protein-dependent transport systems mediate the accumulation of diverse substrates in bacteria. The binding protein-dependent galactose transport of Salmonella typhimurium has been reconstituted in proteoliposomes. The proteoliposomes were made with proteins solubilized and renatured from inclusion bodies produced by a bacterial strain containing a plasmid with the mgl (methylgalactose permease) operon of Salmonella typhimurium. Galactose transport is dependent both on the addition of the purified galactose binding protein to the transport assay, and on ATP. The interaction between the liganded galactose binding protein and proteoliposomes displays Michaelis type kinetics with a Km of around 15 microM. Galactose transport is coupled to ATP hydrolysis with a stoichiometry (ATP/galactose) of 2.5:1. Galactose transport in proteoliposomes is not significantly inhibited by the uncoupler carbonylcyanide m-chlorophenylhydrazone, but is inhibited by 0.5 mM vanadate. The present reconstitution of galactose transport in proteoliposomes suggests that the MglA, MglC and MglE proteins have been solubilized and renatured in an active form from the inclusion bodies of the mgl hyperproducing strain.     

Functional Reconstitution of Staphylococcus aureus Truncated AgrC Histidine Kinase in a Model Membrane System

The integral membrane protein AgrC is a histidine kinase whose sensor domains interact with an autoinducing peptide, resulting in a series of downstream responses. In this study, truncated AgrC_TM5-6C and AgrC_TM5-6C-GFP with GFP as a reporter gene were produced using a bacterial system. Purified AgrC_TM5-6C and AgrC_TM5-6C-GFP were reconstituted into liposomes by a detergent-mediated method. To achieve high-yield protein incorporation, we investigated the effect of different detergents on protein reconstitution efficiency. The highest incorporation was found with N,N-dimethyldode-cylamine N-oxide during complete liposome solubilization, which resulted in a yield of 85±5%. The COOH-terminus of the protein AgrC_TM5-6C was almost exclusively oriented towards the inside of the vesicles. AgrC_TM5-6C in proteoliposomes exhibited approximately a 6-fold increase in constitutive activity compared with AgrC_TM5-6C in detergent micelles. The reconstitution of AgrC_TM5-6C or AgrC_TM5-6C-GFP was characterized using dynamic light scattering, fluorescence microscopy, and transmission electron microscopy. Based on the results, the optimal conditions for protein incorporation were defined. These findings contribute to the study of membrane protein structure and function in vitro using a reconstitution system.     

Fluorescent modified phosphatidylcholine floppase activity of reconstituted multidrug resistance-associated protein MRP1

Multidrug resistance-associated protein (MRP1) may function as a floppase in human red blood cells to translocate phosphatidylserine and/or phosphatidylcholine from inner membrane leaflet to outer leaflet. Here we report that the purified and reconstituted MRP1 protein into asolectin proteoliposomes is mainly in an inside-out configuration and possesses the ability to flop a fluorescent labeled phosphatidylcholine (NBD-PC) from outer leaflet (protoplasmic) to inner leaflet (extracytoplasmic). The reconstituted MRP1 protein retains endogenous ATPase activity. ATP hydrolysis is required for the flopping since removal of ATP and/or Mg2+ inhibits the translocation of NBD-PC. Further evidence to support this conclusion is that the translocation of NBD-PC is inhibited by vanadate, which traps ATP hydrolysis product ADP in the nucleotide binding domains. In addition, the translocation of NBD-PC by proteoliposomes containing MRP1 protein is in a glutathione-dependent manner, similar to the process of translocating anticancer drugs such as daunorubicin. verapamil, vincristine, vinblastine, doxorubicin and oxidized glutathione partially inhibited the translocation of NBD-PC, whereas MK 571, an inhibitor of MRP1 protein, inhibited the translocation almost completely. Taken together, the purified and reconstituted MRP1 protein possesses the ability to flop NBD-PC from outer to inner leaflet of the proteoliposomes.     

Sensitization of differentiated PC12 cells to apoptosis by presenilin-2 is mediated by p38

Recombinant mouse 18 kDa peripheral-type benzodiazepine receptor (PBR) protein was overexpressed in Escherichia coli and isolated using a His. Bind metal chelation resin. Recombinant PBR protein was purified with sodium dodecyl sulfate and reincorporated into liposomes using Bio-Beads SM2 as a detergent removing agent. Negative staining of the reconstituted PBR samples, examined by electron microscopy, showed the formation of proteoliposomes. Freeze-fracture of these proteoliposomes revealed the presence of transmembranous particles of an average size of 3.5 +/- 0.25 nm, consistent with the presence of a monomeric form of the recombinant PBR protein. The reconstituted protein exhibited the ability to bind both the PBR drug ligand isoquinoline carboxamide PK 11195 and cholesterol with nanomolar affinities. These data suggest that a PBR monomer is the minimal functional unit, binding drug ligands and cholesterol.     

Functional reconstitution of a voltage-gated potassium channel in giant unilamellar vesicles

Voltage-gated ion channels are key players in cellular excitability. Recent studies suggest that their behavior can depend strongly on the membrane lipid composition and physical state. In vivo studies of membrane/channel and channel/channel interactions are challenging as membrane properties are actively regulated in living cells, and are difficult to control in experimental settings. We developed a method to reconstitute functional voltage-gated ion channels into cell-sized Giant Unilamellar Vesicles (GUVs) in which membrane composition, tension and geometry can be controlled. First, a voltage-gated potassium channel, KvAP, was purified, fluorescently labeled and reconstituted into small proteoliposomes. Small proteoliposomes were then converted into GUVs via electroformation. GUVs could be formed using different lipid compositions and buffers containing low (5 mM) or near-physiological (100 mM) salt concentrations. Protein incorporation into GUVs was characterized with quantitative confocal microscopy, and the protein density of GUVs was comparable to the small proteoliposomes from which they were formed. Furthermore, patch-clamp measurements confirmed that the reconstituted channels retained potassium selectivity and voltage-gated activation. GUVs containing functional voltage-gated ion channels will allow the study of channel activity, distribution and diffusion while controlling membrane state, and should prove a powerful tool for understanding how the membrane modulates cellular excitability.     

A voltage-activated cation transport pathway associated with the sodium pump.

In proteoliposomes containing reconstituted shark Na,K-ATPase, inside positive potentials open a cation conductance characterized by a voltage-dependence very similar to that found in mammalian erythrocytes. In both proteoliposomes and erythrocytes, the voltage-activated pathway is inhibited by external oligomycin, which traps the Na,K-ATPase in a Na-occluded E1 form. These results indicate that a cation permeable pathway, activated by inside positive potentials, can be ascribed to the Na-K pump--possibly through interaction with its gating mechanism.     

Reconstitution and partial purification of an amiloride-sensitive, cation channel from rabbit kidney

The aim of the present study was to reconstitute and purify an epithelial potassium channel from rabbit kidney. Renal brush border membrane vesicles (BBMV) were found to contain a potassium conductance which was inhibited by amiloride, 5-(N-methyl-N-isobutyl)amiloride (MIA) and by barium. Membrane vesicle proteins were solubilized and reconstituted in proteoliposomes. Channel activity was assayed using Acridine orange and the voltage sensitive dye, 3,3'-diethylthiadicarbocyanine iodide (DiSC2(5)). Both methods yielded similar results which indicated the presence of an amiloride-sensitive, cation channel in the proteoliposomes. This channel was more permeable to K than to Na and its activity was increased in reconstituted proteoliposomes as compared to native brush border membranes. We conclude that rabbit BBMV possess an amiloride sensitive cation channel. Channel activity was successfully reconstituted in proteoliposomes and the protein was partially purified during reconstitution.     

The preparation of cell fusion-inducing proteoliposomes from purified glycoproteins of HVJ (Sendai virus) and chemically defined lipids

Cell fusion-inducing (fusogenic) proteoliposomes of defined chemical composition were reconstituted from purified glycoproteins of hemagglutinating virus of Japan (Sendai virus) either with lipids extracted from the virus particles or with a chemically defined lipid mixture. Cell fusion reactions induced by the reconstituted system have several important characteristics similar to the virus-induced fusion reaction: fusogenic activity of the proteoliposomes depends on the presence of active fusion protein in the vesicles and, in the case of Ehrlich tumor cells, the fusion is almost completely inhibited by adding cytochalasin D to a final concentration of 4 microgram/ml. The only known difference between the original and reconstituted systems is that a greater amount of the latter is necessary for the same degree of fusogenic activity. Thus, the reconstituted system can be used as a model for the Sendai virus-induced fusion reaction. A lipid mixture (phosphatidylcholine:phosphatidylethanolamine:phosphatidylserine:sphingomyelin = 1:2:1:1, by weight, and cholesterol equimolar to the total phospholipids) similar to that of the virion was active for reconstitution, whereas a mixture containing the same composition of phospholipids but no cholesterol, and ones containing cholesterol with only a single species of phospholipid were not reconstitutively active.     

Rapid reconstitution and characterization of highly-efficient sarcoplasmic reticulum Ca pump

The Ca pump was reconstituted from the purified sarcoplasmic reticulum ATPase and excess soybean phospholipids by the freeze-thaw sonication procedure in the presence of cholate. In the absence of Ca precipitating agents, the reconstituted proteoliposomes accumulated Ca2+ at an initial rate of up to 0.7 mumol/mg per min at 25 degrees C, and a value of 1.54 was obtained for the coupling ratio between Ca uptake and Ca2+-dependent ATPase activities. The proteoliposomes were mainly unilamellar vesicles but were heterogeneous with respect to their size. When reconstituted at a lipid/protein ratio of 40, proteoliposomes had a buoyant density of about 1.04 and their average internal volume was 1.4-1.6 microliters/mg of phospholipids. More than 95% of the ATPase was incorporated randomly into these proteoliposomes and the fraction of proteoliposomes that represented about 50% of the total intravesicular isotope space contained right-side-out oriented enzyme. 86Rb efflux from the 86Rb-loaded proteoliposomes was found to be slow even at 25 degrees C. Therefore, the proteoliposomes prepared by the present simple method should be useful for the study of the side-specific interaction of ions such as alkali metal cations with the sarcoplasmic reticulum Ca pump.     

Reconstitution of (Na+ + K+)-ATPase proteoliposomes having the same turnover rate as the membranous enzyme

Membranous (Na+ + K+)-ATPase from the electric eel was solubilized with 3-[3-cholamidopropyl)-dimethylammonio)-1-propanesulfonate (Chaps). 50 to 70% of the solubilized enzyme was reconstituted in egg phospholipid liposomes containing cholesterol by using Chaps. The obtained proteoliposomes consisted of large vesicles with a diameter of 134 +/- 24 nm as the major component, and their protein/lipid ratio was 1.25 +/- 0.07 g protein/mol phospholipid. The intravesicular volume of these proteoliposomes is too small to consistently sustain the intravesicular concentrations of ligands, especially K+, during the assay. The decrease in K+ concentration was cancelled by the addition of 20 microM valinomycin in the assay medium. The low value of the protein/lipid ratio suggests that these proteoliposomes contain one Na+/K+-pump particle with a molecular mass of 280 kDa per one vesicle as the major component. In these proteoliposomes, the specific activity of the (Na+ + K+)-ATPase reaction was 10 mumol Pi/mg protein per min, and the turnover rate of the ATP-hydrolysis was 3500 min-1, the same as the original enzyme under the same assay condition. The ratio of transported Na+ to hydrolyzed ATP was 3, the same as that in the red cell. The proteoliposomes could be disintegrated by 40-50 mM Chaps without any significant inactivation. This disintegration of proteoliposomes nearly tripled the ATPase activity compared to the original ones and doubled the specific ATPase activity compared to the membranous enzyme, but the turnover rate was the same as the original proteoliposomes and the membranous enzyme. This disintegration of proteoliposomes by Chaps suggests the selective incorporation of the (Na+ + K+)-ATPase particle into the liposomes and the asymmetric orientation of the (Na+ + K+)-ATPase particle in the vesicle.     

Studies of cytochrome c oxidase-driven H(+)-coupled phosphate transport catalyzed by the Saccharomyces cerevisiae Pho84 permease in coreconstituted vesicles

The proton-coupled Pho84 phosphate permease of Saccharomyces cerevisiae, overexpressed as a histidine-tagged chimera in Escherichia coli, was detergent-solubilized, purified, and reconstituted into proteoliposomes. Proteoliposomes containing the Pho84 protein were fused with proteoliposomes containing purified cytochrome c oxidase from beef heart mitochondria. Both components of the coreconstituted system were functionally incorporated in tightly sealed membrane vesicles in which the cytochrome c oxidase-generated electrochemical proton gradient could drive phosphate transport via the proton-coupled Pho84 permease. The metal dependency of transport indicates that a metal-phosphate complex is the translocated substrate.     

Chromaffin granule H(+)-ATPase has F1-like structure.

Isolated H(+)-ATPase from chromaffin granules was reconstituted into liposomes and the resultant proteoliposomes were further purified by Ficoll density gradient centrifugation. Studies by electron microscopy showed that proteoliposomes had particle structures (average diameter, about 10 nm) on their outer surface. These particles could be removed from the proteoliposomes by cold treatment. Immuno-electron microscopy showed that these particles were recognized by antibodies against the hydrophilic sector of the enzyme. These results indicate that the H(+)-ATPase has a peripheral membrane structure similar to that of F1-ATPase.