Cholesterol is required for the fusion of single unilamellar vesicles with Mycoplasma capricolum.
Small unilamellar vesicles (SUV) were prepared from the total lipid extract of Mycoplasma capricolum. The SUV were labeled with the fluorescent probe octadecylrhodamine B chloride (R18) to a level at which the R18 fluorescence was self-quenched. At pH 7.4 and 37 degrees C, and in the presence of 5% polyethylene glycol, an increase in the R18 fluorescence with time was observed when the R18-labeled SUV were introduced to a native M. capricolum cell suspension. The fluorescence dequenching resulting from dilution of the R18 into the unlabeled membranes of M. capricolum, was interpreted as a result of lipid mixing during fusion between the SUV and the mycoplasma cells. The presence of cholesterol in the SUV was found to be obligatory to allow SUV-mycoplasma fusion to occur. Adaptation of M. capricolum cells to grow in a medium containing low cholesterol concentration provided cells in which the unesterified cholesterol content was as low as 17 micrograms/mg cell protein. The fusion activity of the adapted cells was very low or nonexistent. Nonetheless, when an early exponential phase culture of the adapted cells was transferred to a cholesterol-rich medium, the cells accumulated cholesterol and regained their fusogenic activity. The cholesterol requirement for fusion in the target mycoplasma membrane was met by a variety of planar sterols having a free beta-hydroxyl group, but differing in the aliphatic side chain, e.g., beta-sitosterol or ergosterol, even though these sterols, having a bulky side chain, are preferentially localized in the outer leaflet of the lipid bilayer. It is suggested that the role of cholesterol in mycoplasma-SUV fusion is not at the level of bulk bilayer viscosity but rather, affecting local lipid-lipid or lipid-protein interactions that are relevant to the fusion event.
Tarshis
M
M
Department of Membrane and Ultrastructure Research, Hebrew University-Hadassah Medical School, Jerusalem, Israel.
Salman
M
M
Rottem
S
S
D000222:Adaptation, Physiological; D055592:Biophysical Phenomena; D001703:Biophysics; D002784:Cholesterol / Q000378:metabolism*; D003470:Culture Media; D008081:Liposomes; D008561:Membrane Fusion / Q000502:physiology*; D009174:Mycoplasma / Q000378:metabolism*
D003470:Culture Media; D008081:Liposomes; D002784:Cholesterol