Conversion of dihydroceramide to ceramide occurs at the cytosolic face of the endoplasmic reticulum

Dihydroceramide desaturase is responsible for the introduction of the 4,5-trans double bond into ceramide. Here, we describe the localization of this enzyme in the endoplasmic reticulum (ER) using ER- and Golgi-enriched fractions from rat liver. Furthermore, enzyme topology was studied. Mild proteolysis of ER-derived vesicles under conditions which assure membrane integrity (latency of mannose 6-phosphatase was at least 91%) resulted in an up to 90% inactivation of dihydroceramide desaturase activity. This indicates a cytosolic orientation of dihydroceramide desaturase activity in the ER membrane.