Biophysical Journal 64: 709-715 (1993)
© 1993 the Biophysical Society

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Cholesterol is required for the fusion of single unilamellar vesicles with Mycoplasma capricolum.

Department of Membrane and Ultrastructure Research, Hebrew University-Hadassah Medical School, Jerusalem, Israel.

ABSTRACT

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.(ABSTRACT TRUNCATED AT 250 WORDS)

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