TOPOLOGY OF SPHINGOLIPID GALACTOSYLTRANSFERASES IN ER AND GOLGI - TRANSBILAYER MOVEMENT OF MONOHEXOSYL SPHINGOLIPIDS IS REQUIRED FOR HIGHERGLYCOSPHINGOLIPID BIOSYNTHESIS

Glucosylceramide (GlcCer) is synthesized at the cytosolic surface of t he Golgi complex while enzymes acting in late steps of glycosphingolip id biosynthesis have their active centers in the Golgi lumen. However, the topology of the ''early'' galactose-transferring enzymes is large ly unknown. We used short-chain ceramides with either a 2-hydroxy fatt y acid (HFA) or a normal fatty acid (NFA) to determine the topology of the galactosyltransferases involved in the formation of HFA- and NFA- galactosylceramide (Gal-Cer), lactosylceramide (LacCer), and galabiosy lceramide (Ga(2)Cer). Although the HFA-GalCer synthesizing activity co localized with an ER marker, the other enzyme activities fractionated at the Golgi density of a sucrose gradient. In cell homogenates and pe rmeabilized cells, newly synthesized short-chain GlcCer and GalCer wer e accessible to serum albumin, whereas LacCer and Ga(2)Cer were protec ted. From this and from the results obtained after protease treatment, and after interfering with UDP-Gal import into the Golgi, we conclude that (a) GlcCer and NFA-GalCer are synthesized in the cytosolic leafl et, while LacCer and Ga(2)Cer are synthesized in the lumenal leaflet o f the Golgi. (b) HFA-Gal-Cer is synthesized in the lumenal leaflet of the ER, but has rapid access to the cytosolic leaflet, (c) GlcCer, NFA -GalCer, and HFA-GalCer translocate from the cytosolic to the lumenal leaflet of the Golgi membrane. The transbilayer movement of GlcCer and NFA-GalCer in the Golgi complex is an absolute requirement for higher glycosphingolipid biosynthesis and for the cell surface expression of these monohexosyl sphingolipids.