Enveloped viruses as model membrane systems: microviscosity of vesicular stomatitis virus and host cell membranes.

The fluorescence probe 1,6-diphenyl-1,3,5-hexatriene was used to study and compare the dynamic properties of the hydrophobic region of vesicular stomatitis virus grown on L-929 cells, plasma membrane of L-929 cells prepared by two different methods, liposomes prepared from virus lipids and plasma membrane lipids, and intact L-929 cells. The rate of penetration of the probe into the hydrophobic region of the lipid bilayer was found to be much faster in the lipid vesicle bilayer as compared with the intact membrane, but in all cases the fluorescence anisotropy was constant with time. The L-cell plasma membranes, the vesicles prepared from the lipids derived from the plasma membranes, and intact cells are found to have much lower microviscosity values than the virus or virus lipid vesicles throughout a wide range of temperatures. The microviscosity of plasma membrane and plasma membrane lipid vesicles was found to depend on the procedure for plasma membrane preparation as the membranes prepared by different methods had different microviscosities. The intact virus and liposomes prepared from the virus lipids were found to have very similar microviscosity values. Plasma membrane and liposomes prepared from plasma membrane lipids also had similar microviscosity values. Factors affecting microviscosity in natural membranes and artificially mixed lipid membranes are discussed.     

Kinetics of phospholipid insertion into monolayers containing the lung surfactant proteins SP-B or SP-C

The lung surfactant proteins SP-B and SP-C are pivotal for fast and reversible lipid insertion at the air/liquid interface, a prerequisite for functional lung activity. We used a model system consisting of a preformed monolayer at the air/liquid interface supplemented with surfactant protein SP-B or SP-C and unilamellar vesicles injected into the subphase of a film balance. The content of SP-B or SP-C was similar to that found in lung lavage. In order to elucidate distinct steps of lipid insertion, we measured the time-dependent pressure increase as a function of the initial surface pressure, the temperature and the phosphatidylglycerol content by means of surface tension measurements and scanning force microscopy (SFM). The results of the film balance study are indicative of a two-step mechanism in which initial adsorption of vesicles to the protein-containing monolayer is followed by rupture and integration of lipid material. Furthermore, we found that vesicle adsorption on a preformed monolayer supplemented with SP-B or SP-C is strongly enhanced by negatively charged lipids as provided by DPPG and the presence of Ca2+ ions in the subphase. Hence, long-range electrostatic interactions are thought to play an important role in attracting vesicles to the surface, being the initial step in replenishment of lipid material. While insertion into the monolayer is independent of the type of protein SP-B or SP-C, initial adsorption is faster in the presence of SP-B than SP-C. We propose that the preferential interaction between SP-B and negatively charged DPPG leads to accumulation of negative charges in particular regions, causing strong adhesion between DPPG-containing vesicles and the monolayer mediated by Ca2+ ions, which eventually causes flattening and rupture of attached liposomes as observed by in situ SFM.     

Small-angle neutron scattering studies of the effects of amphotericin B on phospholipid and phospholipid-sterol membrane structure

Small-angle neutron scattering (SANS) studies have been performed to study the structural changes induced in the membranes of vesicles prepared (by thin film evaporation) from phospholipid and mixed phospholipid-sterol mixtures, in the presence of different concentrations and different aggregation states of the anti-fungal drug, amphotericin B (AmB). In the majority of the experiments reported, the lipid vesicles were prepared with the drug added directly to the lipid dispersions dissolved in solvents favouring either AmB monomers or aggregates, and the vesicles then sonicated to a mean size of ~100 nm. Experiments were also performed, however, in which micellar dispersions of the drug were added to pre-formed lipid and lipid-sterol vesicles. The vesicles were prepared using the phospholipid palmitoyloleoylphosphatidylcholine (POPC), or mixtures of this lipid with either 30 mol% cholesterol or 30 mol% ergosterol. Analyses of the SANS data show that irrespective of the AmB concentration or aggregation state, there is an increase in the membrane thickness of both the pure POPC and the mixed POPC-sterol vesicles-in all cases amounting to ~4 ?. The structural changes induced by the drug's insertion into the model fungal cell membranes (as mimicked by POPC-ergosterol vesicles) are thus the same as those resulting from its insertion into the model mammalian cell membranes (as mimicked by POPC-cholesterol vesicles). It is concluded that the specificity of AmB for fungal versus human cells does not arise because of (static) structural differences between lipid-cholesterol-AmB and lipid-ergosterol-AmB membranes, but more likely results from differences in the kinetics of their transmembrane pore formation and/or because of enthalpic differences between the two types of sterol-AmB complexes.     

Glycoproteins of Sendai virus (HVJ) have a critical ratio for fusion between virus envelopes and cell membranes

The biological activity of two glycoproteins, hemagglutinin and neuraminidase (HN) and fusion (F) proteins, of Sendai virus (HVJ) were studied using purified proteins. The proteins were purified by chromatography on DEAE and CM cellulose in the presence of Nonidet P-40 (NP40). The glycoproteins were reconstituted at various ratios of F to HN into lipid vesicles containing fragment A of diphtheria toxin. The association of HN and F proteins with the vesicles was confirmed by electron microscopy and sucrose density gradient centrifugation. The cytotoxic activity of vesicles containing fragment A on fusion with L cells was determined by measuring colony formation of the cells. It was found that for maximum cytotoxic activity of the vesicles, there was an optimal ratio of F to HN of two. This suggests that HN is not merely the initial binding site to the cell surface, and that interactions between HN and F proteins on the virus surface may be important for the biological activities of these proteins on the cells.     

Immunocytochemical study of the partition and distribution of Sindbis virus glycoproteins in freeze-fractured membranes of infected baby hamster kidney cells

Sindbis virus-infected baby hamster kidney cells were analyzed by thin section fracture-label. Specific immunolabel with antiviral glycoprotein antibodies or with conventional lectin label (wheat germ agglutinin) were used in conjunction with colloidal gold-conjugated protein A or ovomucoid, respectively. In addition, intact infected cells were analyzed with both labeling procedures. Experiments with Sindbis infected-chick embryo fibroblast cells were carried out as controls. Viral transmembrane glycoproteins appeared present in freeze-fractured inner and outer nuclear membrane, endoplasmic reticulum, Golgi stacks and vesicles, and plasma membranes; a clear preferential partition with the exoplasmic faces of all intracellular membranes was observed. By contrast, at the plasma membrane level, Sindbis glycoproteins were found to partition preferentially with the protoplasmic face. It seems likely that this protoplasmic partition is related to the binding with the nucleocapsid that takes place during the budding of the virus. At the cell surface, viral glycoproteins always appeared clustered and were predominantly associated with budding figures: moreover, large portions of the plasma membrane were devoid of both glycoproteins and budding viruses.     

Reconstitution of a protein into lipid vesicles using natural detergents

A method is described for reconstitution of a protein into lipid vesicles using one of the natural detergents lysophosphatidylcholine or lysophosphatidic acid. The intestinal microvillus enzyme, aminopeptidase N (EC 3.4.11.2) is incorporated into lipid vesicles prepared from a total lipid extract of the microvillus membrane. The method is based on fusion of aminopeptidase-lysophospholipid micelles with liposomes prepared by sonication. The incorporation of the protein into the lipid bilayer is analyzed by gel permeation chromatography and sucrose density gradient centrifugation. The coincidence of the protein and lipid profiles is used to evaluate protein incorporation. The incorporation is visualized by electron microscopy with negative staining. The method has the advantage of using natural detergents, lysophospholipids, which are minor but natural constituents of biological membranes. The method could be of value as a tool in studies of mechanisms of insertion of newly synthesized proteins into biological membranes.     

Designing transmembrane alpha-helices that insert spontaneously

Direct measurement of the free energies of transfer of hydrophobic membrane-spanning alpha-helices from water to membranes is important for the determination of an accurate experiment-based hydrophobicity scale for membrane proteins. An important objective of such a scale is to account for the presently unknown thermodynamic cost of partitioning hydrogen-bonded peptide bonds into the membrane hydrocarbon core. We describe here the physical properties of a transmembrane (TM) peptide, TMX-1, designed to test the feasibility of engineering peptides that spontaneously insert across bilayers but that have the important property of measurable monomeric water solubility. TMX-1, Ac-WNALAAVAAAL-AAVAAALAAVAAGKSKSKS-NH(2), is a 31-residue sequence with a 21-residue nonpolar core, N- and C-caps to favor helix formation, and a highly polar C-terminus to improve solubility and to control directionality of insertion into lipid vesicles. TMX-1 appeared to be soluble in water up to a concentration of at least 1 mg/mL (0.3 mM). However, fluorescence spectroscopy, fluorescence quenching, and circular dichroism (CD) spectroscopy indicated that the high solubility was due to the formation of molecular aggregates that persisted at peptide concentrations down to at least 0.1 microM peptide. Nevertheless, aqueous TMX-1 partitioned strongly into membrane vesicles with apparent mole-fraction free-energy values of -7.1 kcal mol(-1) for phosphatidylcholine (POPC) vesicles and -8.2 kcal mol(-1) for phosphatidylglycerol (POPG) vesicles. CD spectroscopy of TMX-1 in oriented multilayers formed from either lipid disclosed a very strong preference for a transmembrane alpha-helical conformation. When TMX-1 was added to preformed vesicles, it was fully helical. A novel fluorescence resonance energy transfer (FRET) method demonstrated that at least 50% of the TMX-1 insered spontaneously across the vesicle membranes. Binding and insertion were found to be fully reversible for POPC vesicles but not POPG vesicles. TMX-1 was thus found to have many of the properties required for thermodynamic measurements of TM peptide insertion. Importantly, the results obtained delineate the experimental problems that must be considered in the design of peptides that can partition spontaneously and reversibly as monomers into and across membranes. Our success with TMX-1 suggests that these problems are not insurmountable.     

Hyaluronate-binding protein of simian virus 40-transformed 3T3 cells: membrane distribution and reconstitution into lipid vesicles

Hyaluronate-binding protein (HABP) has been extracted in detergent from the membranes of simian virus 40-transformed 3T3 (SV-3T3) cells (Underhill et al, J Biol Chem 258:8086-8091, 1983). When SV-3T3 cells were treated with trypsin prior to isolation and dissolution of the membranes, no hyaluronate-binding activity could be detected. This indicates that all of the detectable HABP of SV-3T3 cells is located on the external surface of the plasma membrane rather than on internal membranes, which would be inaccessible to the trypsin. The detergent-extracted HABP from SV-3T3 membranes was reconstituted into the membrane of lipid vesicles, which were formed by addition of exogenous phosphatidylcholine and cholic acid to the extracts followed by removal of detergent by dialysis against 0.02 M Tris pH 8.0 in the presence of protease inhibitors. Reconstitution was assessed by sedimentation in a discontinuous sucrose gradient and by gel filtration on Sepharose 4B in the presence and absence of detergent. The characteristics of binding of hyaluronate to the reconstituted HABP were then compared with those studied previously for the original membrane-bound HABP and the detergent-extracted HABP (Underhill et al, J Biol Chem 258:8086-8091, 1983). It was observed previously that binding of hyaluronate to HABP in the cell membranes was of higher affinity and specificity than to HABP in the detergent extracts of these membranes. It was found here that reconstitution of the extracted HABP into the membranes of lipid vesicles led to restoration of affinity of binding to the level observed in the original cell membranes. However, whereas chondroitin sulfate does not compete significantly for binding of hyaluronate to cell membrane-bound HABP, partial competition was observed for the reconstituted HABP as well as for detergent-extracted HABP. Thus, it is concluded that the high affinity of binding of hyaluronate to the plasma membrane of SV-3T3 cells is in part dependent on insertion of the HABP in the membrane, but that other interactions, not duplicated in our reconstitution experiments, must be necessary for the specificity of the HABP.     

Membrane fusion by an RGD-containing sequence from the core protein VP3 of hepatitis A virus and the RGA-analogue: implications for viral infection

The interaction of an RGD-containing epitope from the hepatitis A virus VP3 capsid protein and its RGA-analogue with lipid membranes was studied by biophysical methods. Two types of model membrane were used: vesicles and monolayers spread at the air/water interface, with a composition that closely resembles the lipid moiety of hepatocyte membranes: PC/SM/PE/PC (40:33:12:15; PC: 1-palmitoyl-2-oleoylglycero-sn-3-phosphocholine; SM: sphingomyelin from chicken egg yolk; PE, 1,2-dipalmitoyl-phosphatidylethanolamine; PS: L-alpha-phosphatidyl-L-serine from bovine brain). In addition, zwitterionic PC/SM/PE (47:39:14) and cationic PC/SM/PE/DOTAP (40:33:12:15; DOTAP: 1,2-dioleoyl-3-trimethylammonium-propane) membranes were also prepared in order to dissect the electrostatic and hydrophobic components in the interaction. Changes in tryptophan fluorescence, acrylamide quenching, and resonance energy transfer experiments in the presence of vesicles, as well as the kinetics of insertion in monolayers, indicate that both peptides bind to the three types of membrane at neutral and acidic pH; however, binding is irreversible only at low pH. Membrane-destabilizing and fusogenic activities are triggered by acidification at pH 4-6, characteristic of the endosome. Fluorescence experiments show that VP3-RGD and VP3-RGA induce mixing of lipids and leakage or mixing of aqueous contents in anionic and cationic vesicles at pH 4-6, indicating leaky fusion. Interaction with zwitterionic vesicles (PC/SM/PE) results in leakage without lipid mixing, indicating pore formation. Replacement of aspartic acid in the RGD motif by alanine maintains the membrane-destabilizing properties of the peptide at low pH, but not its antigenicity. Since the RGD tripeptide is related to receptor-mediated cell adhesion and antigenicity, results suggest that receptor binding is not a molecular requirement for fusion. The possible involvement of peptide-induced membrane destabilization in the mechanism of hepatitis A virus infection of hepatocytes by the endosomal route is discussed.     

Interaction of lipid vesicles with monomolecular layers containing lung surfactant proteins SP-B or SP-C

Pulmonary surfactant contains two families of hydrophobic proteins, SP-B and SP-C. Both proteins are thought to promote the formation of the phospholipid monolayer at the air-fluid interface of the lung. The Wilhelmy plate method was used to study the involvement of SP-B and SP-C in the formation of phospholipid monolayers. The proteins were either present in the phospholipid vesicles which were injected into the subphase or included in a preformed phospholipid monolayer. In agreement with earlier investigators, we found that SP-B and SP-C, present in phospholipid vesicles, were able to induce the formation of a monolayer, as became apparent by an increase in surface pressure. However, when the proteins were present in a preformed phospholipid monolayer (20 mN/m) at similar lipid to protein ratios, the rate of surface pressure increase after injection of pure phospholipid vesicles into the subphase at similar vesicle concentrations was 10 times higher. The process of phospholipid insertion from phospholipid vesicles into the protein-containing monolayers was dependent on (1) the presence of (divalent) cations, (2) the phospholipid concentration in the subphase, (3) the size of the phospholipid vesicles, (4) the protein concentration in the preformed monolayer, and (5) the initial surface pressure at which the monolayers were formed. Both in vesicles and in preformed monolayers, SP-C was less active than SP-B in promoting the formation of a phospholipid monolayer. The use of preformed monolayers containing controlled protein concentrations may allow more detailed studies on the mechanism by which the proteins enhance phospholipid monolayer formation from vesicles.     

The in vitro insertion of monoamine oxidase B into mitochondrial outer membranes

Bovine monoamine oxidase (MAO) B has been synthesized in vitro using a reticulocyte lysate translation system directed by bovine liver poly(A)+ RNA. The newly synthesized enzyme apparently lacks a cleavable N-terminal extension, but MAO B is readily incorporated into mitochondria or isolated mitochondrial outer membranes prepared from rat liver. ATP is not required for the binding of the newly synthesized enzyme to the outer membranes, but is necessary for the insertion of MAO B into these membrane vesicles. The ATP is not required to generate a mitochondrial membrane potential as assembly occurs under conditions that preclude either the formation or the maintenance of the potential. MAO B will bind to but not become incorporated into outer membrane vesicles which have been treated with trypsin, suggesting that the insertion of MAO B also depends on protein factors present on the outer membranes.     

Fragments from actin binding protein (ABP-280; filamin) insert into reconstituted lipid layers.

Previous computer analyses suggested two possible lipid binding sites, residues 49-71 and 131-155, of the primary amino acid sequence on ABP-280 (filamin), which could facilitate membrane attachment/insertion. We expressed these regions as fusion proteins with schistosomal GST and investigated their interaction with mixtures of zwitterionic (dimyristoyl-l-alpha-phosphatidylcholine, DMPC) and anionic (dimyristoyl-l-alpha-phosphatidylglycerol, DMPG) phospholipids in reconstituted lipid bilayers by differential scanning calorimetry (DSC). Using vesicles of mixed DMPC/DMPG with increasing fusion protein concentrations, we established in calorimetric assays a decrease of the main chain transition enthalpy, DeltaH, and a shift in chain melting temperature. This is indicative of the insertion of these fragments into the hydrophobic region of lipid membranes. We confirmed these findings by the film balance technique using lipid monolayers (DMPG). The binding judged from both methods was of moderate affinity.     

Insertion of influenza M protein into the viral lipid bilayer and localization of site of insertion.

Recent studies with isolated M protein from influenza virus have shown that the protein has a high affinity for lipid. The ability of M to partition into lipid vesicles merely by shaking vesicles and M together is suggestive evidence that the protein could be interacting with the lipid in the virus particle. A more direct analysis was carried our here to determine whether M is in contact with the viral lipid in situ, by using the photoactivatable hydrophobic probe, pyrenesulfonyl azide. Covalent linkage of this probe to M indicated that a segment of M residues with in the virus membrane in contact with the lipid bilayer. M inserted into lipid vesicles at two locations on the molecule. A major insertion into lipid occurred in the middle of the molecule where a large cluster of 20 hydrophobic and neutral amino acids occurs. A second insertion occurred approximately one fourth in from the amino terminus, where a smaller segment of 13 uncharged amino acids is found. Confirmation that M inserted into lipid at these locations came also from results with cyanogen bromide fragments of M. Of the 12 to 13 fragments produced, 3 specifically bound to lipid vesicles. These were the first, second, and third contiguous segments beginnings at the amino terminus and containing the two hydrophobic areas noted above.     

Characterization of lipid insertion into monomolecular layers mediated by lung surfactant proteins SP-B and SP-C

Pulmonary surfactant proteins, SP-B and SP-C, if present in preformed monolayers can induce lipid insertion from lipid vesicles into the monolayer after the addition of (divalent) cations [Oosterlaken-Dijksterhuis, M. A., Haagsman, H. P., van Golde, L. M. G., & Demel, R. A. (1991) Biochemistry 30, 8276-8287]. This model system was used to study the mechanisms by which SP-B and SP-C induce monolayer formation from vesicles. Lipid insertion proceeds irrespectively of the molecular class, and PG is not required for this process. In addition to lipids that are immediately inserted from vesicles into the monolayer, large amounts of vesicles are bound to the monolayer and their lipids eventually inserted when the surface area is expanded. SP-B and SP-C are directly responsible for the binding of vesicles to the monolayer. By weight, the vesicle binding capacity of SP-B is approximately 4 times that of SP-C. For vesicle binding and insertion, the formation of close contacts between monolayer and vesicles is essential. SP-B and SP-C show very similar surface properties. Both proteins form extremely stable monolayers (collapse pressures 36-37 mN/m) of alpha-helical structures oriented parallel to the interface. In monolayers consisting of DPPC and SP-B or SP-C, an increase in mean molecular area is observed, which is mainly attributed to the phospholipid. This will greatly enhance the insertion of new lipid material into the monolayer. The results of this study suggest that the surface properties and the hydrophobic nature of SP-B and SP-C are important for the protein-mediated monolayer formation.     

Interaction of hepatic asialoglycoprotein receptor with dimyristoyl phosphatidylcholine vesicles

The hepatocyte membrane asialoglycoprotein receptor (ASGP-R) was extracted from rabbit liver, purified, and then incubated with preformed vesicles of dimyristoyl phosphatidylcholine. The association of protein with lipid was dependent on vesicle size and the best results were achieved with small vesicles of about 20 nm diameter. The ligand binding capacity of ASGP-R-vesicle complexes was also measured and found to be approximately sevenfold greater than free receptor in aqueous buffer and twofold greater than receptor solubilized in Triton X-100. Most likely, the reconstitution procedure used in these experiments does not result in transmembrane insertion of the receptor. ASGP-R probably resides on the surface of the vesicle, held there primarily by weak hydrophobic forces.     

A mechanism of membrane neutral lipid acquisition by the microsomal triglyceride transfer protein

The microsomal triglyceride transfer protein (MTP) and apolipoprotein B (apoB) belong to the vitellogenin (VTG) family of lipid transfer proteins. MTP is essential for the intracellular assembly and secretion of apoB-containing lipoproteins, the key intravascular lipid transport proteins in vertebrates. We report the predicted three-dimensional structure of the C-terminal lipid binding cavity of MTP, modeled on the crystal structure of the lamprey VTG gene product, lipovitellin. The cavity in MTP resembles those found in the intracellular lipid-binding proteins and bactericidal/permeability-increasing protein. Two conserved helices, designated A and B, at the entrance to the MTP cavity mediate lipid acquisition and binding. Helix A (amino acids 725-736) interacts with membranes in a manner similar to viral fusion peptides. Mutation of helix A blocks the interaction of MTP with phospholipid vesicles containing triglyceride and impairs triglyceride binding. Mutations of helix B (amino acids 781-786) and of N780Y, which causes abetalipoproteinemia, have no impact on the interaction of MTP with phospholipid vesicles but impair triglyceride binding. We propose that insertion of helix A into lipid membranes is necessary for the acquisition of neutral lipids and that helix B is required for their transfer to the lipid binding cavity of MTP.     

CLIC1 inserts from the aqueous phase into phospholipid membranes, where it functions as an anion channel

CLIC1 is a member of the CLIC familyof proteins, which has been shown to demonstrate chloride channelactivity when reconstituted in phospholipid vesicles. CLIC1 exists incells as an integral membrane protein and as a soluble cytoplasmicprotein, implying that CLIC1 might cycle between membrane-inserted andsoluble forms. CLIC1 was purified and detergent was removed, yieldingan aqueous solution of essentially pure protein. Pure CLIC1 was mixedwith vesicles, and chloride permeability was assessed with a chloride efflux assay and with planar lipid bilayer techniques. Soluble CLIC1confers anion channel activity to preformed membranes that isindistinguishable from the previously reported activity resulting fromreconstitution of CLIC1 into membranes by detergent dialysis. Theactivity is dependent on the amount of CLIC1 added, appears rapidly onmixing of protein and lipid, is inhibited by indanyloxyacetic acid-94,N-ethylmaleimide, and glutathione, is inactivated by heat,and shows sensitivity to pH and to membrane lipid composition. Weconclude that CLIC1 in the absence of detergent spontaneously insertsinto preformed membranes, where it can function as an anion-selective channel.

     

Effect of nonbilayer lipids on membrane binding and insertion of the catalytic domain of leader peptidase

Biological membranes contain a substantial amount of "nonbilayer lipids", which have a tendency to form nonlamellar phases. In this study the hypothesis was tested that the presence of nonbilayer lipids in a membrane, due to their overall small headgroup, results in a lower packing density in the headgroup region, which might facilitate the interfacial insertion of proteins. Using the catalytic domain of leader peptidase (delta2-75) from Escherichia coli as a model protein, we studied the lipid class dependence of its insertion and binding. In both lipid monolayers and vesicles, the membrane binding of (catalytically active) delta2-75 was much higher for the nonbilayer lipid DOPE compared to the bilayer lipid DOPC. For the nonbilayer lipids DOG and MGDG a similar effect was observed as for DOPE, strongly suggesting that no specific interactions are involved but that the small headgroups create hydrophobic interfacial insertion sites. On the basis of the results of the monolayer experiments, calculations were performed to estimate the space between the lipid headgroups accessible to the protein. We estimate a maximal size of the insertion sites of 15 +/- 7 A2/lipid molecule for DOPE, relative to DOPC. The size of the insertion sites decreases with an increase in headgroup size. These results show that nonbilayer lipids stimulate the membrane insertion of delta2-75 and support the idea that such lipids create insertion sites by reducing the packing density at the membrane-water interface. It is suggested that PE in the bacterial membrane facilitates membrane insertion of the catalytic domain of leader peptidase, allowing the protein to reach the cleavage site in preproteins.     

Transfer of cytochrome b 5 and NADPH cytochrome c reductase between membranes

NADPH-cytochrome c reductase also reduces cytochrome b 5. The reduction is very slow when the proteins are in solution or bound to different membranes. Only when both proteins share a common membrane, is cytochrome b 5 reduced rapidly by NADPH. The difference in reaction rates indicates recombination on a common membrane of cytochrome b 5 and NADPH reductase originally bound to different vesicles. The recombination of the two proteins occurs with a variety of biological membranes (previously enriched with either reductase or cytochrome b 5) as well as with liposomes. We explain this process as protein transfer rather than vesicle fusion for several reasons: 1. The vesicles do not alter shape or size during incubation. 2. The rate of this process corresponds to the rate of incorporation of the single proteins into liposomes carrying the 'complementary' protein. 3. The exchange of proteins between biological membranes and liposomes occupied by protein does not change the density of either membrane. Protein transfer between membranes appears to be limited to those proteins which had spontaneously recombined with a preformed membrane. In contrast, proteins incorporated into liposomes by means of a detergent were not transferred, nor were endogenous cytochrome b 5 and NADPH-cytochrome c reductase transferred from microsomes to Golgi membranes or lipid vesicles. We conclude that the endogenous proteins and proteins incorporated in the presence of a detergent are linked to the membrane in another manner than the same proteins which had been inserted into a preformed membrane.     

Interaction of Sindbis virus with liposomal model membranes.

Radiolabeled Sindbis virus was found to bind to protein-free lipid model membranes (liposomes) derived from extracts of sheep erythrocytes. The virus interaction was dependent on initial pH, and the range of pH dependence (pH 6.0 to 6.8) was the same as the observed with virus-dependent hemagglutination. After the initial interaction, pH changes no longer influenced the virus binding to liposomes. Virus bound to liposomes prepared from a mixture of erythrocyte phospholipids, but the binding was greatly diminished when either cholesterol or phosphatidylethanolamine was omitted from the liposomal lipid mixture. It was concluded that phospholipids and cholesterol, in a bilayer configuration, may be sufficient for specific virus binding in the absence of membrane protein.