Ectoplasmic insertion of a glycosylphosphatidylinositol-anchored protein in glycosphingolipid- and cholesterol-containing phosphatidylcholine vesicles.

Glycosylphosphatidylinositol (glycosyl-PtdIns)-anchored proteins are proposed to be clustered in membrane microdomains enriched in cholesterol and glycosphingolipids (GlySphs). We have prepared biomimetic membranes in order to study the possible phenomena of surface aggregation of these membrane components. Phosphatidylcholine liposomes were treated by octylglucoside to insert a glycosyl-PtdIns-protein, alkaline phosphatase (ALP), some cholesterol, and a GlySph, the lactocerebroside. The association of these compounds was shown by centrifugation on a density gradient. The presence of ALP on the surface of the vesicles was shown by the action of a phospholipase, and the presence of the lactocerebroside was shown by the use of a galactose-specific tetravalent lectin. Our data show that total alkaline phosphatase and half to total lactocerebroside were ectoplasmically inserted in the vesicles membrane. In addition, we observed that the presence of small amounts of ALP in the liposomes led to significant changes in membrane stability with regard to detergent, as shown by the changes in the solubilization process monitored by turbidimetry. Furthermore, we have built an original method to study the cohesion of the vesicles membrane, in which some magnesium ions were trapped in the luminal space of the liposomes during several days. The ALP is magnesium-dependent for its catalytic activity and was inhibited after incubation of ALP-containing liposomes in a magnesium-free buffer. The ALP activity was restored by the addition of detergent to the liposomes, due to the release of the luminal magnesium ions. Surface aggregation phenomena will now be investigated by atomic force microscopy.