The topological orientation of N,N'-diacetylchitobiosylpyrophosphoryldolichol in artificial and natural membranes.

Purified N,N'-diacetylchitobiosylpyrophosphoryldolichol (chitobiosyl-lipid) in the presence of detergent was shown to act as a substrate for the soluble enzyme galactosyltransferase. The nature of this transfer reaction and the galactose-containing trisaccharide-lipid product have been partially characterized. Using galactosyltransferase as a probe, the topolical arrangement of chitobiosyl-lipid in both artificial and natural membranes has been examined. When incorporated into unilamellar liposomes made from phosphatidylcholine, the disaccharide residue of chitogiosyl-lipid adopts a random transbilayer orientation. Furthermore, no significant mobility in the transverse plane of the membrane (i.e. flip-flop) is detectable. Using both sealed total microsomes or isolated rough microsomes from hen oviduct, the topology of chitobiosyl-lipid after its biosynthesis in the membrane has been determined. The results suggest that, once formed, chitobiosyl-lipid is a relatively static component of the membrane and is oriented with its disaccharide residue facing the lumen of the endoplasmic reticulum.