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.

Exosomes are vesicles formed in the endosomal compartment and released in the extracellular medium during reticulocyte maturation into erythrocytes. They have a clearing function because of their enrichment with some proteins known to decrease or disappear from the cell surface during maturation, e.g. acetylcholinesterase and transferrin receptor. We show here that integrin a4b1, present on the surface of erythroid precursors but absent from the mature red cell membrane, is at least partly cleared from the reticulocyte plasma membrane by the exosomal pathway. Using flow cytometry, we found that the a4 subunit disappears from the reticulocyte surface during in vitro maturation. Two different monoclonal antibodies (B-5G10 and HP 2/1) were used to demonstrate the presence of the a4 chain on the exosome surface. Moreover, membrane acetylcholinesterase and lumenal peroxidase-like (i.e. hemoglobin) enzymatic activities were assayed to demonstrate exosome binding to plates coated with increasing fibronectin (FN) concentrations. This interaction was dependent on divalent cations (MnCl 2 . MgCl 2 . CaCl 2 ). Similarly, vesicles bound to plates coated with the chymotryptic 40 K fragment (FN-40) containing the heparin-binding region of FN. This binding was inhibited by exosome preincubation with fibronectin CS1 peptide and with a monoclonal antibody (HP 2/1) against the integrin a4-chain, confirming an a4b1±induced interaction. The importance of the exosome clearance function is highlighted here, since the presence of VLA-4 on reticulocytes often leads to blood circulation complications in some diseases. Moreover, the presence of a4b1 on the exosome surface, by allowing binding to endothelial cells through vascular cell adhesion molecule 1 (VCAM-1), might confer another physiological function to the secreted vesicles.

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.