We have investigated the influence of influenza-induced membrane fusion on the transverse asymmetry of the viral and target membranes. Large unilamellar vesicles containing headgroup-labeled fluorescent phospholipid analogues in both leaflets of the membrane were treated with phospholipase D, converting all outer membrane phospholipids to phosphatidic acid and leading to the release of the fluorescent label from the outside leaflet. After fusion of virus with these liposomes, addition of the enzyme to the fusion product did not release fluorescent label again, indicating that the phospholipid analogues from the inner leaflet of the membranes had not appeared on the outer leaflet. Moreover, the integral membrane protein hemagglutinin, which is present on the outer leaflet of the viral membrane, was quantitatively digested with protease after fusion, indicating that hemagglutinin remained on the outer leaflet of the fusion product. Therefore, there is no merger of the inner with outer leaflets of the viral or the liposomal membrane during fusion, and transverse membrane asymmetry is maintained.The constituents of biological membranes, such as the plasma membranes of cells, are distributed in an asymmetric manner between the two leaflets of the membranes (Devaux, 1991(Devaux, , 1992. Although these membranes continuously undergo membrane fusion and fission, their asymmetry is maintained. Thus, it is likely that membrane asymmetry is maintained even during fusion.One of the most extensively studied membrane fusion mechanisms is that induced by the hemagglutinin (HA) 1 glycoprotein of influenza virus. Fusion mediated by the protein is induced by low pH, leading to a conformational change in HA, which results in the insertion of the N-terminal "fusion peptide" of the HA2 subunit of the protein into the target membrane (Bentz, 1993). Although many details of the conformational change (reviewed by Hughson (1995)) and its role in fusion are now known, it is not clear how the protein achieves the merger of the viral with the target lipid bilayers. For fusion, a non-bilayer structure has to be formed, at least temporarily and locally at the site of fusion . Little is known about these intermediate lipid structures or the role of HA in their formation. One attractive hypothesis proposes that the intermediates formed are stalks, semitoroidal (hourglass-shaped) structures composed of fused outer leaflets, but unfused inner leaflets of both membranes (Siegel, 1993a(Siegel, , 1993b. Stalk formation would be followed by a rupture of the inner membrane leaflets at the site of fusion, followed by their merger, which then completes bilayer fusion. There is little direct evidence for this hypothesis, but a mutant, glycosylphosphatidylinositol-anchored HA, expressed on a cell membrane, was found to fuse the outer membrane leaflets of these cells with those of red blood cells, while not inducing the merger of the inner membrane leaflets (hemifusion) (Kemble et al., 1994). These data suggested that fusion induced by the wild type HA co...

Stationary and kinetic experiments were performed on lipid bilayer membranes to study the mechanism of iodine- and bromine-mediated halide transport in detail. The stationary conductance data suggested that four different 1:1 complexes between I2 and Br2 and the halides I- and Br- were responsible for the observed conductance increase by iodine and bromine (I3-, I2Br-, Br2I-, and Br3-). Charge pulse experiments allowed the further elucidation of the transport mechanism. Only two of three exponential voltage relaxations predicted by the Läuger model could be resolved under all experimental conditions. This means that either the heterogeneous complexation reactions kR (association) and kD (dissociation) were too fast to be resolved or that the neutral carriers were always in equilibrium within the membrane. Experiments at different carrier and halide concentrations suggested that the translocation of the neutral carrier is much faster than the other processes involved in carrier-mediated ion transport. The model was modified accordingly. From the charge pulse data at different halide concentrations, the translocation rate constant of the complexed carriers, kAS, the dissociation constant, kD, and the total surface concentration of charged carriers, NAS, could be evaluated from one single charge pulse experiment. The association rate of the complex, kR, could be obtained in some cases from the plot of the stationary conductance data as a function of the halide concentration in the aqueous phase. The translocation rate constant, kAS, of the different complexes is a function of the image force and of the Born charging energy. It increases 5000-fold from Br3- to I3- because of an enlarged ion radius.

Voltage-clamp experiments were performed on lipid bilayer membranes to study the voltage dependence of the iodine-mediated halide transport. Under all experimental conditions only one exponential current relaxation, apart from the capacitive spike, could be resolved up to a clamp voltage of 200 mV. The current relaxation could be described by an initial conductance, G0, the relaxation time constant, tau, and the relaxation amplitude, alpha, that is the difference between the initial current, I0, and the steady state current, I chi, divided by the steady state current. The occurrence of one single exponential relaxation suggested that one of the different transport steps involved in the carrier-mediated ion transport according to the Lüger-model is always in equilibrium. This is most probably the transport of the free carriers across the membrane. The voltage dependence of G0, tau, and of alpha were used to determine the voltage dependence of the translocation rate constants of the complexed carriers, kAS. In the case of the iodine-mediated iodide transport, G0, tau and alpha were only mediate voltage-dependent, which means the voltage dependent translocation of the complex encounters a trapezoidal barrier shape. For the iodine-mediated bromide translocation G0, tau and alpha exhibited no dependence on the applied clamp-voltage, which suggested that a square Nernst-Planck barrier limits the transport of the corresponding complex.