Abstract. In our attempt to assess the topology of glucosylceramide biosynthesis, we have employed a truncated ceramide analogue that permeates cell membranes and is converted into water soluble sphingolipid analogues both in living and in fractionated cells. Truncated sphingomyelin is synthesized in the lumen of the Golgi, whereas glucosylceramide is synthesized at the cytosolic surface of the Golgi as shown by (a) the insensitivity of truncated sphingomyelin synthesis and the sensitivity of truncated glucosylceramide synthesis in intact Golgi membranes from rabbit liver to treatment with protease or the chemical reagent DIDS; and (b) sensitivity of truncated sphingomyelin export and insensitivity of truncated glucosylceramide export to decreased temperature and the presence of GTP-'y-S

A fragment of the N-terminal 546 amino acid residues of Clostridium sordellii lethal toxin possesses full enzyme activity and glucosylates Rho and Ras GTPases in vitro. Here we identified several amino acid residues in C. sordellii lethal toxin that are essential for the enzyme activity of the active toxin fragment. Exchange of aspartic acid at position 286 or 288 with alanine or asparagine decreased glucosyltransferase activity by about 5000-fold and completely blocked glucohydrolase activity. No enzyme activity was detected with the double mutant D286A/D288A. Whereas the wild-type fragment of C. sordellii lethal toxin was labeled by azido-UDP-glucose after UV irradiation, mutation of the DXD motif prevented radiolabeling. At high concentrations (10 mM) of manganese ions, the transferase activities of the D286A and D288A mutants but not that of wild-type fragment were increased by about 20-fold. The exchange of Asp 270 and Arg 273 reduced glucosyltransferase activity by about 200-fold and blocked glucohydrolase activity. The data indicate that the DXD motif, which is highly conserved in all large clostridial cytotoxins and also in a large number of glycosyltransferases, is functionally essential for the enzyme activity of the toxins and may participate in coordination of the divalent cation and/or in the binding of UDP-glucose.

We have employed in vitro a truncated ceramide analogue with 8 carbon atoms in the sphingosine and the fatty acyl residue, each, to investigate the activity of various membrane fractions to synthesize truncated sphingomyelin. This shortened ceramide readily diffuses through membranes and therefore can easily find access to the lumina of intact organelles. Sphingomyelin synthase activity resides in the Golgi apparatus, and after sucrose density gradient centrifugation of Golgi‐enriched fractions sphingomyelin synthesis follows a cis Golgi marker enzyme.

Recently, synthesis of lactosylceramide has been described to occur on the cytosolic face of the Golgi [(1991) J. Biol. Chem. 266, 20907-209121. The reactions following in the biosynthesis of higher glycosphingolipids are known to take place in the lumen of the Golgi. For our understanding of the functional organization of the multiglycosyltransferase system of glycosphingolipid synthesis in the Golgi, the knowledge of the topology of individual reactions is a prerequisite. We have developed a simple and quick assay system for sphingolipid biosynthesis and have obtained evidence that lactosylceramide is synthesized in the lumen of the Golgi. Because lactosylceramide is generated by galactosylation of glucosylceramide which, in turn, is synthesized from ceramide and UDP-Glc on the cytosolic surface of the Golgi apparatus, further efforts will be directed to the characterization of a glucosylceramide-translocator in the Golgi membranes rather than a lactosylceramide-translocator.

No abstract