No abstract

The amino-terminal extremity of the simian immunodeficiency virus (SlY) transmembrane protein (gp32) has been shown to play a pivotal role in cell-virus fusion and syncytium formation. We provide here evidence of a correlation between the structure and orientation of the modified SIV fusion peptide after insertion into the lipid membrane and its fusogenic activity. The sequence of the wild-type SIV peptide has been modified in such a way that the calculated angles of insertion correspond to an oblique, parallel, or normal orientation with respect to the lipid-water interface. Fourier transform infrared spectroscopy was used to gain experimental informations about the structures and orientations, of the membrane-inserted peptides with respect to the lipid acyl chains. The peptides adopt mainly a ,(-sheet conformation in the absence of lipids. After interaction with large unilamellar liposomes, this 13 sheet is partly converted into a helix. The ability of the modified peptides to promote lipid mixing was assessed by a fluorescence energy transfer assay. The data provide evidence that a-helix formation is not sufficient to induce lipid mixing and that the fusogenic activity of the peptide depends on its orientation in the lipid bilayer.

No abstract

The passage by the low endosomal pH is believed to be an essential step of the diphtheria toxin (DT) intoxication process in vivo. Several studies have suggested that this low pH triggers the insertion of DT into the membrane. We demonstrate here that its insertion into large unilamellar vesicles (LUV) is accompanied by a strong destabilization of the vesicles at low pH. The destabilization has been studied by following the release of a fluorescent dye (calcein) encapsulated in the liposomes. The influence of the lipid composition upon this process has been examined. At a given pH, the calcein release is always faster for a negatively charged (asolectin) than for a zwitterionic (egg PC) system. Moreover, the transition pH, which is the pH at which the toxin-induced release becomes significant, is shifted upward for the asolectin LUV as compared to the egg PC LUV. No calcein release is observed for rigid phospholipid vesicles (DPPC and DPPC/DPPA 9/1 mol/mol) below their transition temperature whereas DT induces an important release of the dye in the temperature range corresponding to the phase transition. The transition pH associated to the calcein release from egg PC vesicles is identical with that corresponding to the exposure of the DT hydrophobic domains, as revealed here by the binding of a hydrophobic probe (ANS) to the toxin. This suggests the involvement of these domains in the destabilization process. Both A and B fragments destabilize asolectin and PC vesicles in a pH-dependent manner but to a lesser extent than the entire toxin.(ABSTRACT TRUNCATED AT 250 WORDS)

The mechanisms regulating the intracellular pH (pHi) in both forms of Trypanosoma brucei brucei (cultured cells) were investigated using the fluorescent probe 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). The pHi values measured were 7.22+/-0.03 in the procyclics and 7.40+/-0.05 in the bloodstream form. In the presence of 24mM HCO3-, pHi values were slightly higher in both forms of trypanosomes suggesting a bicarbonate-linked pH regulation. pHi was more stable in procyclics (between 7.15 and 7.30 in the external pH range 6.4-7.6) than in the bloodstream forms. The amiloride analogue tested decreased pHi, suggesting Na+-driven Na+/H+ antiporters. H+-ATPases also seem to be involved in pHi regulation since the inhibitors N-ethylmaleimide (1 mM) and N,N'-dicyclohexylcarbodiimide (80 microM) induced a rapid acidification in both forms of trypanosomes. Addition of pyruvate caused a cytosol acidification in the bloodstream form only confirming the existence of a diffusion-facilitated carrier for pyruvate, with the cotransport of H+. Our results show that, although similar pH regulation mechanisms seem to exist in both forms of trypanosomes, the procyclics can regulate efficiently their pHi and consequently their plasma membrane potential whereas the bloodstream forms cannot always maintain their pHi and are easily depolarized following a small acid load.