Abstract
We have investigated the pH-dependent interaction between large unilamellar phospholipid vesicles (liposomes) and membrane vesicles derived from Bacillus subtilis, utilizing a fluorescent assay based on resonance energy transfer (RET) (Struck, D. K., Hoekstra, D., and Pagano, R. E. (1981) Biochemistry 20, 4093-4099). Efficient interaction occurs only with negatively charged liposomes, containing cardiolipin or phosphatidylserine, as revealed by the dilution of the RET probes from the liposomal bilayer into the bacterial membrane. The initial rate of fluorophore dilution increases steeply with decreasing pH. The interaction involves a process of membrane fusion, as indicated by the proportional transfer of cholesteryl-[1-14C]oleate, 14C-labeled egg PC, and the RET probes from the liposomes to the bacterial vesicles, the formation of interaction products with an intermediate buoyant density, and the appearance of colloidal gold, initially encapsulated in the liposomes, in the internal volume of fused structures as revealed by thin-section electron microscopy. Treatment of B. subtilis vesicles with trypsin strongly inhibits the fusion reaction, indicating the protein dependence of the process. Vesicles derived from Streptococcus cremoris or from the inner membrane of Escherichia coli also show low pH-dependent fusion with liposomes. The fusion process described in this paper may well be of considerable importance to studies on the mechanisms of membrane fusion and to studies on the structure and function of bacterial membranes. In addition, the fusion reaction could be utilized to deliver foreign substances into bacterial protoplasts.
Highlights
Somes) and membrane vesicles derived from Bacillus In theabove studies liposomes comsubtilis, utilizing a fluorescent assay based on reso- posed of mixed soybean phospholipids were used as a source nance energy transfer (RET)
Involves a process of membrane fusion, as indicated by In the present studywe have applied a kinetic assay based (i) theproportionaltransfer of cholesteryl-[1-14C] on fluorescence resonance energy transfer (RET’) to study oleate, I4C-labeled egg PC, and the RET probes from the interaction between liposomes of different compositions the liposomes to the bacterialvesicles, (ii) the forma- andmembrane vesicles derivedfrom Bacillussubtilis
Liposomes-Large unilamellar vesicles (LUV) were prepared by Sucrose Density Gradient Centrifugation-B. subtilis membrane reverse-phase evaporation [23, 24] in 100 mM NaCI, 10 mM HEPES vesicles wereincubated for 15min at 25 "Cwith liposomes,containing, sized by extrusion [25] through Unipore polycarbonate 5 mol % N-NBD-PE, at1.2 mM phospholipid phosphorus concentramembranes (Bio-Rad) with a pore size of 0.2 pm and centrifuged in tions each in 100 mM NaC1, 10 mM sodium acetate, 10 mM sodium an Eppendorf microfuge during 15 min to remove any residual larger phosphate at either pH4.0 orpH 8.5
Summary
Low pH-induced Fusion of Liposomes with Membrane Vesicles Derived from Bacillus subtilis DRIESSEN, AJM; HOEKSTRA, D; SCHERPHOF, G; KALICHARAN, RD; WILSCHUT, J. Liposomes-Large unilamellar vesicles (LUV) were prepared by Sucrose Density Gradient Centrifugation-B. subtilis membrane reverse-phase evaporation [23, 24] in 100 mM NaCI, 10 mM HEPES vesicles wereincubated for 15min at 25 "Cwith liposomes,containing (pH 7.4), sized by extrusion [25] through Unipore polycarbonate 5 mol % N-NBD-PE, at1.2 mM phospholipid phosphorus concentramembranes (Bio-Rad) with a pore size of 0.2 pm and centrifuged in tions each in 100 mM NaC1, 10 mM sodium acetate, 10 mM sodium an Eppendorf microfuge during 15 min to remove any residual larger phosphate at either pH4.0 orpH 8.5. Binding Assay-B. subtilis membrane vesicles were incubated for 15min at 25 "Cwith liposomes,containing either0.5 mol % N-NBDP E and 1.0 mol % ~holesteryl-[l-'~C]oleat(e20 Ci/mol) or 0.5 mol % N-NBD-PE and mol % 14C-labeled eggPC (3.4 Ci/mol), in 100 mM NaC1, 10 mM sodium acetate, 10 mM sodium phosphate at either pH 4.0 or pH 7.4. At relatively high probe concentrations, the transfer efficiency does not increase proportionally with the surface density of the fluores-
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
