Processes such as endo- or exocytosis, membrane recycling, fertilization and enveloped viruses infection require one or more critical membrane fusion reactions. A key feature in viral and cellular fusion phenomena is the involvement of specific fusion proteins. Among the few well-characterized fusion proteins are viral spike glycoproteins responsible for penetration of enveloped viruses into their host cells, and sperm proteins involved in sperm-egg fusion. In their sequences, these proteins possess a "fusion peptide, " a short segment (up to 20 amino acids) of relatively hydrophobic residues, commonly found in a membrane-anchored polypeptide chain. To simulate protein-mediated fusion, many studies on peptide-induced membrane fusion have been conducted on model membranes such as liposomes and have employed synthetic peptides corresponding to the putative fusion sequences of viral proteins, or de novo synthesized peptides. Here, the application of peptides as a model system to understand the molecular details of membrane fusion will be discussed in detail. Data obtained from these studies will be correlated to biological studies, in particular those that involve viral and sperm-egg systems. Structure-function relationships will be revealed, particularly in the context of protein-induced membrane perturbations and bilayer-to-nonbilayer transition underlying the mechanism of fusion. We will also focus on the involvement of lipid composition of membranes as a potential regulating factor of the topological fusion site in biological systems.

Guinea pig reticulocytes lose their transferrin (Tf) binding activity during maturation, in the form of vesicles (exosomes) released into the extracellular medium. Vesicles were prepared from cultures of reticulocytes to study the possible externalization of a particular membrane-associated activity, i.e., that of "aminophospholipid translocase." Analysis of the peptide composition of these vesicles revealed that the major proteins are the Tf receptor and another peptide (70kDa), which is probably the "clathrin-uncoating ATPase" described by Johnstone et al. (1987). The exosome had a lipid composition similar to erythrocyte membrane, although with a lightly but significantly lower phosphatidylethanolamine content. The aminophospholipid distribution in the vesicle membrane was determined by fluorescamine labeling. The exosomes showed an asymmetric aminophospholipid distribution similar to that of erythrocytes. "Aminophospholipid translocase" activity was absent, as no transverse diffusion of spin-labeled phospholipids occurred over more than 2 hours at 37 degrees C.

After incorporation of spin-labeled phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine analogues in the outer leaflet of the plasma membrane in resting platelets, more than 90% amino-head analogues accumulated within 30 min in the inner leaflet by aminophospholipid translocase activity, while choline analogues mostly remained on the outer leaflet. Platelets were then activated by thrombin or Ca2+ ionophore A23187. No outward movement of internally located spin-labeled aminophospholipids was observed during thrombin-induced activation, whereas the influx of externally located probes increased slightly. During A23187-mediated activation, similar slightly increased influx was observed, while 40-50% of the initially internally located aminophospholipids could then be extracted from the outer leaflet. This sudden exposure on the outer face was dependent on an increase in intracellular Ca2+ and achieved in less than 2 min at 37 degrees C. Inhibition of translocase activity by N-ethylmaleimide did not induce any aminophospholipid outflux. When probes were incorporated on the outer face of the plasma membrane in resting platelets, they were still fully accessible from the extracellular medium after A23187-induced activation. Moreover, they were distributed between the vesicles and remnant platelets in proportion to the external membrane phospholipidic content in each structure. This suggested that no scrambling of plasma membrane leaflets occurred during the vesicle blebbing. Moreover, the spin-labeled aminophospholipids exposure rate and amplitude were unchanged when vesicle formation was inhibited by the calpain inhibitor calpeptin. These results indicate that loss of asymmetry thus inducing generation of a catalytic surface is not the consequence of vesicle formation. Conversely, we propose that vesicle shedding is an effect of PL transverse redistribution and calpain-mediated proteolysis during activation.

Spin-labeled analogues of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin were added to human platelet suspensions. Due to the partial water solubility of these spin-labeled lipids which possess a relatively short beta-chain (C5), they incorporate rapidly in membranes. The orientation of the spin-labels within the platelet plasma membrane was assessed by following the spontaneous reduction at 37 and 4 degrees C due to endogenous reducing agents present in the cytosol. The rate of spontaneous reduction showed unambiguously that the labels incorporated initially in the outer leaflet of the plasma membrane and that the rate of outside-inside translocation of the aminophospholipids was faster than that of the choline derivatives. For example, at 37 degrees C, the half-time for the transverse diffusion of a phosphatidylcholine analogue was found to be of the order of 40 min, while it was less than 7 min for the phosphatidylserine analogue. At low temperatures, a fraction of the labels gave rise to a strongly immobilized ESR component. This fraction, which corresponded to 20-30% of the initial spin-label concentration, was found resistant to chemical reduction from the inner side of the membrane and also to externally added reducing agents such as ascorbate. Presumably these immobilized lipids are trapped in a gel phase formed in the outer leaflet at 4 degrees C. Cell aging, which depletes the cells of ATP, resulted in the progressive inhibition of the fast transport of the aminophospholipids from the outer to inner leaflet. Treatment of the cells with iodoacetamide completely blocked the transverse diffusion of the spin-labels.(ABSTRACT TRUNCATED AT 250 WORDS)

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In order to fix spin-labeled acids at the boundary layer of membrane-bound proteins, spin-labeled long-chain derivatives (m,n)MSL (general formula, CH,(CH2),R-(CH2),COO(CH2)2-M, where R is an oxazolidine ring containing a nitroxide and M is a maleimide residue) were synthesized. The spin-labeled molecules bind covalently to at least two different classes of sulfhydryl groups on rhodopsin in disc membrane fragments from bovine retina. One class of sites is hydrophilic and corresponds to the two SH groups labeled readily by N-ethylmaleimide; the second class of sites is only reached by hydrophobic probes. (10,3)MSL binds equally well to the two classes of sites on rhodopsin, whereas (1 , I 4)MSL, more hydrophobic, binds preferentially to the hydrophobic sites. Apparently a third class of SH groups can be labeled if a very large excess of (m,n)MSL is employed, but proteins may be denatured in this latter case. Labels not covalently bound are removed from the membranes by incubation with fatty acid free bovine serum albumin. However, it is found that the probes do not bind only to rhodopsin in the disc membranes. (m,n)MSL also binds covalently to phosphatidylethanolamine in the rod outer segments or in liposomes. This covalent binding to phospholipids is demonstrated by lipid extraction and thin-layer chromatographic analysis. In order to obtain the pure EPR spectra of the spin-labeled fatty acids bound to the protein, the spectra corresponding to phospholipid-bound spin labels have been I t is often admitted that intrinsic membrane proteins are surrounded by a boundary layer or "annulus" of rigidly bound lipid. The immobilization of this shell of lipid has been deduced essentially from EPR experiments involving spin-labeled fatty acids incorporated into reconstituted systems containing variable lipid to protein ratios. Jost et al. (1973) were the first to propose from spin-label experiments the model of a boundary layer of lipid surrounding an intrinsic membrane protein, namely cytochrome oxidase. Later, Hesketh et al. (1976) reported similar experiments with Ca2+-ATPase, while Chapman et al. (1977) showed that gramcidin A can lead to the same EPR results, if this polypeptide is dissolved in a small amount of lipid.Rhodopsin was also tested with spin-labeled fatty acids. Pontus and Delmelle (1975) found evidence of a rigid boundary layer around this hydrophobic protein. However, previously, Hong & Hubbell (1972) had reached a different conclusion from spin-label experiments with rhodopsin reincorporated into phospholipid; they suggested that the acerage viscosity of the membranes was dependent on the lipid to protein ratio. This is in good agreement with recent results put foward by Cherry et al. (1977) from very different experiments involving bacteriorhodopsin. Finally, using proton manuscript receiced A'ocember 21, 1978. This investigation was supported by research grants from the Centre National de la Recherche Scientifique (ERA 690) and the DelEgation G&n&rale a la Recherche Scientifique et Technique (Comm...

Pseudomonas exotoxin (PE) is a cytotoxin which, after endocytosis, is delivered to the cytosol where it inactivates protein synthesis. Using diaminobenzidine cytochemistry, we found over 94% of internalized PE in transferrin (Tf) -positive endosomes of lymphocytes. When PE translocation was examined in a cell-free assay using purified endocytic vesicles, more than 40% of endosomal 125 I-labeled PE was transported after 2 h at 37°C, whereas a toxin inactivated by point mutation in its translocation domain was not translocated. Sorting of endosomes did not allow cell-free PE translocation, whereas active PE transmembrane transport was observed after Ͼ 10 min of endocytosis when PE and fluorescent-Tf were localized by confocal immunofluorescence microscopy within a rab5-positive and rab4-and rab7-negative recycling compartment in the pericentriolar region of the cell. Accordingly, when PE delivery to this structure was inhibited using a 20°C endocytosis temperature, subsequent translocation from purified endosomes was impaired. Translocation was also inhibited when endosomes were obtained from cells labeled with PE in the presence of brefeldin A, which caused fusion of translocationcompetent recycling endosomes with translocation-incompetent sorting elements. No PE processing was observed in lymphocyte endosomes, the full-sized toxin was translocated and recovered in an enzymatically active form. ATP hydrolysis was found to directly provide the energy required for PE translocation. Inhibitors of endosome acidification (weak bases, protonophores, or bafilomycin A1) when added to the assay did not significantly affect 125 I-labeled PE translocation, demonstrating that this transport is independent of the endosome-cytosol pH gradient. Nevertheless, when 125