Human erythrocytes were fused by incubation with 0.5-2 mM-chlorpromazine hydrochloride at pH 6.8-7.6. Fusogenic preparations of chlorpromazine were cloudy suspensions of microdroplets, and below pH 6.8 chlorpromazine gave clear solutions that were inactive. Unlike control cells, the lateral mobility of the intramembranous particles of the PF-fracture face of chlorpromazine-treated cells was relatively unrestricted, since the particles were partly clustered at 37 degrees C and they exhibited extensive cold-induced clustering. Ca2+ stimulated fusion, but fusion was only very weakly inhibited by EGTA (10 mM) and by N-ethylmaleimide (50 mM); pretreatment of the cells with Tos-Lys-CH2Cl (7-amino-1-chloro-3-L-tosylamidoheptan-2-one) (7.5 mM) markedly inhibited fusion. Changes in the membrane proteins of erythrocytes fused by chlorpromazine, before and after treatment with chymotrypsin to remove band 3 protein, were investigated. The several observations made indicate that the Ca2+-insensitive component of fusion is associated with degradation of ankyrin (band 2.1 protein) to band 2.3-2.6 proteins and to smaller polypeptides by a serine proteinase that is inhibited by Tos-Lys-CH2Cl, and that the component of fusion inhibited by EGTA and N-ethylmaleimide is associated with degradation of band 3 protein to band 4.5 protein by a Ca2+-activated cysteine proteinase. Proteolysis of ankyrin appeared to be sufficient to permit the chlorpromazine-induced fusion of human erythrocytes, but fusion occurred more rapidly when band 3 protein was also degraded in the presence of Ca2+. Since other cells have structures comparable with the spectrin-actin skeleton of the erythrocyte membrane, the observations reported may be relevant to the initiation of naturally occurring fusion reactions in biomembranes. It is also suggested that, should polypeptides with fusogenic properties be produced from integral and skeletal membrane proteins by endogenous proteolysis, their formation would provide a general mechanism for the fusion of lipid bilayers in biomembrane fusion reactions.

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Sheep erythrocyte plasma membrane monolayers were formed on positively charged supports by means of controlled lysis and squirting of cells so that the original protoplasmic or inner surfaces (PS) were exposed. The appearance of the surface was studied by transmission electron microscopy of platinum/carbon replicas following freeze-drying of the membranes. After gentle washing with water or dilute buffer, a network of filaments and particles was found to cover the surface. Whole cells bound to positively charged supports were treated with Triton X-100 and hypertonic KCl, which left the protein components of the cytoskeleton on the support. Stereo electron microscopy of Pt/C replicas of these residues showed complex networks of filaments, similar to those seen on the cytoplasmic surfaces of the intact membranes. In both cases the lengths of the filaments correspond to that of the spectrin dimer. Removal of spectrin, actin and other proteins by alkaline treatment led to loss of this network and revealed 15.4-nm particles on the membranes PS. These particles, which were also visible after negative staining, could be removed by treatment with trypsin and were found to correspond to band 4 protein (the equivalent of human erythrocyte band 3 protein). Membranes freeze-fractured following treatment with alkali showed normal intramembranous particles with frequencies similar to those of native membranes. This indicates that band 4 protein spans the sheep erythrocyte membrane and forms a very high proportion of the intramembranous particles. The protoplasmic portions of these particles may be membrane binding sites for the cytoskeleton in sheep erythrocytes.

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