BOTH SPHINGOLIPIDS AND CHOLESTEROL PARTICIPATE IN THE DETERGENT INSOLUBILITY OF ALKALINE-PHOSPHATASE, A GLYCOSYLPHOSPHATIDYLINOSITOL-ANCHORED PROTEIN, IN MAMMALIAN MEMBRANES

SPB-1, a Chinese hamster ovary cell variant defective in serine palmit oyltransferase activity for sphingolipid synthesis, provides a useful system for studying the effects of sphingolipids and/or cholesterol de privation on cellular functions and membrane properties. To investigat e whether there was an interaction among sphingolipids, cholesterol, a nd glycosylphosphatidylinositol (GPI)-anchored proteins in biological membranes, we introduced human placental alkaline phosphatase (FLAP) i n SPB-1 and in wild type cells by stable transfection and examined the effects of sphingolipid and/or cholesterol deprivation on the solubil ity of FLAP in Triton X-100. Although the FLAP solubility of the membr anes isolated from the control cells in Triton X-100 was only 10%, dep rivation of sphingolipid and cholesterol further enhanced the solubili ty, which reached 50% when both sphingolipids and cholesterol were dep rived. The enhanced solubility was suppressed to the control level by metabolic complementation with exogenous sphingosine and cholesterol. The sphingolipid and cholesterol content of the isolated membranes cha nged independently, eliminating the possibility that sphingolipid depr ivation induced a reduction in cellular cholesterol and enhanced FLAP solubility and vice versa. It was also unlikely that the enhanced solu bility was due to structural changes in FLAP molecules since, regardle ss of sphingolipid and cholesterol deprivations, almost all FLAP had t he GPI-anchor moiety and there were no differences in the apparent mol ecular weight of the protein in supernatant and precipitate fractions of the detergent-treated membranes. In addition, the expression level of caveolin in the isolated membranes was not significantly affected b y sphingolipids and/or cholesterol depletion. These results indicated that both sphingolipids and cholesterol were involved in the FLAP inso lubility and suggested that these lipids coordinately played a role in formation of Triton X-100-resistant complexes.