THE SUBCELLULAR SITES OF SPHINGOMYELIN SYNTHESIS IN BHK CELLS

The subcellular distributions of the enzymes which synthesise sphingom yelin (SM) and glucosylceramide (GluCer) from ceramide have been asses sed in BHK cells, On a sucrose density gradient GluCer synthase (a mar ker of the cis/medial Golgi apparatus) and the trans-Golgi marker gala ctosyltransferase showed an similar monotonic distribution. In contras t, SM synthase showed two peaks of activity, a minor one which migrate d with the Golgi markers and a major one which had a density close to that of plasma membrane markers (sphingomyelin, cholesterol, PtdSer, g anglioside GM3 and alkaline phosphodiesterase). When cell homogenates were treated with digitonin, the sedimentation characteristics of the Golgi markers was largely unaffected whereas the plasma membrane marke rs and the main peak of SM synthase activity were shifted to higher de nsity. In contrast, when cells were treated with brefeldin A (BFA) the Golgi markers were shifted to higher density but not the plasma membr ane markers or the main peak of SM synthase. These results suggest tha t the bulk of SM synthase activity in BHK cells is not associated with the Golgi cisternae but with a cell compartment which is relatively r ich in cholesterol (e,g., plasma membrane, endosomes or trans-Golgi ne twork.) Further experiments in which cells were treated with sphingomy elinase provided evidence that SM synthase activity was in an internal compartment and not at the plasma membrane. (C) 1997 Elsevier Science B.V.