UDP-GALACTOSE-CERAMIDE GALACTOSYLTRANSFERASE IS A CLASS-I INTEGRAL MEMBRANE-PROTEIN OF THE ENDOPLASMIC-RETICULUM

UDP-galactose:ceramide galactosyltransferase (CGalT) transfers UDP-gal actose 60 ceramide to form the glycosphingolipid galactosylceramide, G alactosylceramide is the major constituent of myelin and is also highl y enriched in many epithelial cells, where it is thought to play an im portant role in lipid and protein sorting. Although the biochemical pa thways of glycosphingolipid biosynthesis are relatively well understoo d, the localization of the enzymes involved in these processes has rem ained controversial. We here have raised antibodies against CGalT and shown by immunocytochemistry on ultrathin cryosections that the enzyme is localized to the endoplasmic reticulum and nuclear envelope but no t to the Gels apparatus or the plasma membrane. In pulse-chase experim ents, we have observed that newly synthesized CGalT remains sensitive to endoglycosidase H, confirming the results of the morphological loca lization experiments. In protease protection assays, we show that the largest part of the protein, including the amino terminus, is oriented toward the lumen of the endoplasmic reticulum. CGalT enzyme activity required import of UDP-galactose into the lumen of the endoplasmic ret iculum by a UDP-galactose translocator that is present in the Golgi ap paratus of CHO cells but absent in CHOlec8 cells. Finally, we show tha t CGalT activity previously observed in Golgi membrane fractions in vi tro, in the absence of UDP-glucose, is caused by UDP-glucose:ceramide glucosyltransferase. Therefore all galactosylceramide synthesis occurs by CGalT in vivo in the lumen of the endoplasmic reticulum.