Membrane fusion, in particular the fusion of enveloped viruses, is often measured with an assay based on octadecylrhodamine (R18) fluorescence dequenching. We have studied the association of R18 with membranes and used the R18 assay to measure virus fusion in model systems and in cultured cells. The results were compared with those of an assay based on the decrease in excimer fluorescence of pyrene-labeled phospholipids. For liposomes made from premixed R18 and phosphatidylcholine (PC), R18 fluorescence quenching was proportional to the concentration of the probe up to about 4 mol %. No quenching was found at very low concentrations of R18. However, various artificial and biological membranes labeled by the addition of R18 from an ethanolic solution showed significant quenching at such low R18 concentrations. Thus, some of the R18 was not randomly distributed but likely was associated with the surface of the membranes in the form of highly quenched clusters or micelles. Moreover, in influenza virus membranes, R18 appeared highly quenched at very low concentrations, indicative of the probe interacting with viral proteins. In contrast, pyrene-labeled PC incorporated in either liposomes or reconstituted viral membranes (virosomes) showed an excimer/monomer fluorescence ratio proportional to the concentration of probe. When intracellular membrane fusion was investigated with R18-labeled influenza virus or Semliki Forest virus (SFV), fluorescence dequenching was observed in the absence of fusion, most likely due to spontaneous probe exchange.(ABSTRACT TRUNCATED AT 250 WORDS)

No abstract

The lining of the maltodextrin-specific maltoporin (LamB) channel exhibits a string of aromatic residues, the greasy slide, part of which has been shown previously by crystallography to be involved in substrate binding. To probe the functional role of the greasy slide, alanine scanning mutagenesis has been performed on the six greasy slide residues and Y118 at the channel constriction. The mutants were characterized by an in vivo uptake assay and sugar-induced-current-noise analysis. Crystallographic analysis of the W74A mutant showed no perturbation of the structure. All mutants showed considerably decreased maltose uptake rates in vivo (<10% of the wild-type value), indicating the functional importance of the investigated residues. Substitutions at the channel center revealed appreciably increased (up to 100-fold) in vitro half-saturation concentrations for maltotriose and maltohexaose binding to the channel. Sugar association rates, however, were significantly affected also by the mutations at either end of the slide (W74A, W358A, and F227A), an effect which became most apparent upon nonsymmetrical sugar addition. The kinetic data are discussed on the basis of an asymmetric one-site two-barrier model, which suggests that, at low substrate concentrations, as are found under physiological conditions, only the heights of the extracellular and periplasmic barriers, which are reduced by the presence of the greasy slide, determine the efficiency of this facilitated diffusion channel.Solute uptake into cells of gram-negative bacteria requires the crossing of two membranes, the outer and the inner membranes, which are separated by the periplasmic space (16). The outer membrane can be considered a defense wall protecting the cell against harmful compounds such as toxins and bile salts. Since a complete seal would impede the import of essential nutrients into the cell, several pore-forming proteins (porins) are located in the outer membrane (19). Transport of solutes through these water-filled channels most commonly proceeds by simple diffusion. For specific porins, diffusional transport is aided by the presence of a binding site.Maltoporin (LamB) specifically facilitates the translocation of malto-oligosaccharides across the outer membrane barrier. Its crystal structure (20) is trimeric, with each monomer consisting of an 18-stranded ␤-barrel enclosing a channel. Long loops protrude into the extracellular space, and short turns face the periplasm. Three loops fold into the ␤-barrel, with loop L3 constricting the channel about halfway through. In the channel lining, there are six contiguous aromatic residues, which form an elongated apolar stripe, the greasy slide, extending from the vestibule through the channel constriction to the periplasmic outlet (Fig. 1). The first of these residues, W74, is provided by L2 of a neighboring subunit. The next three residues, Y41, Y6, and W420, are situated in the middle of the channel. They are followed by W358 and F227, which reside in the wide periplasmic exit hall. A comparable ...

Fusion of influenza virus with liposomes is triggered by low pH, resulting in a conformational change in the fusion protein (HA) and the insertion of fusion peptides from HA into the liposomal membrane. Fusion does not take place immediately after insertion but is preceded by a lag phase, the duration of which, as we have found previously, depends on the presence of ganglioside receptors in the liposomal membrane [Stegmann, T., White, J. M., & Helenius, A. (1990) EMBO J. 9, 4231-4241]. Here we have investigated why that is the case. Surprisingly, the 2-4-fold shorter lag phase observed with phosphatidylcholine (PC)/phosphatidylethanolamine (PE)/ganglioside liposomes was not due to slower or more readily reversible binding of the virus to PC/PE liposomes lacking receptors. Nevertheless, using liposomes with various glycolipids as targets, it was found that specific HA-receptor interactions were required for a shorter lag, and not just the negative charge of the gangliosides, or the presence of ceramide lipid tails in the liposomal membrane. Receptor binding also did not facilitate the conformational change in HA. Surprisingly, however, it was found that after an incubation of the virus at low pH in the absence of target membranes at 0 degrees C for several minutes, the binding and fusion activity of virus using PC/PE liposomes, but not PC/PE/ganglioside liposomes as targets, was decreased. The population of virus that did still bind to and fuse with the PC/PE liposomes after low pH preincubation did so after a significantly increased lag time.(ABSTRACT TRUNCATED AT 250 WORDS)

Membrane fusion induced by the hemagglutinin glycoprotein of influenza virus has been extensively characterized, but the mechanism whereby the protein achieves the merger of the viral and target membrane lipids remains enigmatic. Various lipid intermediate structures have been proposed, and the energies required for their formation predicted. Here, we have analyzed the enthalpies of fusion of influenza with liposomes by titration calorimetry. If a small sample of virus in a weak neutral pH buffer was added to an excess of liposomes at low pH, a two-component reaction was seen, composed of an exothermic reaction and a slower endothermic reaction. The exothermic reaction was the result of acid-base reactions between the neutral pH virus sample and low pH buffer and low-pH-induced changes in the virus. The endothermic reaction was not observed in the absence of liposomes and much reduced if acid-inactivated virus, which had lost its fusion but not its binding activity, was added to liposomes. The endothermic reaction was more temperature dependent than the exothermic reaction; its pH dependence corresponded with that of fusion and its enthalpy was higher if fusion was more extensive. These data indicate that most of the endothermic reaction was due to membrane fusion. The experimentally determined enthalpy of fusion, 0.6-0.7 kcal per mol of viral phospholipids, is much higher than expected on the basis of current theories about the formation of lipid intermediates during membrane fusion.