Glycosyl transferases that participate in the assembly of the lipidlinked oligosaccharide intermediates were solubilized from cultured soybean cells using 0.3% Nonidet P40 (NP40) in the presence of 10% glycerol. The solubilized enzyme preparation was reasonably stable and 50% of the activity still remained after storage at -10°C for I month.The solubilized enzyme synthesized ["C]Man3GlcNAc2-pyrophosphorylpolyprenol and I'4CjMansGlcNAc2-pyrophosphoryl-polyprenol when incubated with GDP-'4Cjmannose plus a partially purified acceptor lipid isolated from calf liver. The formation of these lipid-linked oligosaccharides did not require the addition of dolichyl-phosphate or metal ions. In fact, the addition of 5 to 10 millimolar ethylenediaminetetraacetate stimulated the incorporation of mannose into lipid-linked oligosaccharides 2-to 3-fold. Since little or no dolichyl-phosphoryl-mannose is formed in the presence of ethylenediaminetetraacetate, the results suggest that the mannosyl residues added to form Man3GlcNAcrlipid and MansGlcNAc2-lipid come directly from GDP-mannose without the participation of dolichyl-phosphoryl-mannose. On the other hand, the formation of significant amounts of Man6GlcNAc2-lipid, Man7GlcNAc2-lipid, and MansGlcNAc2-lipid occurred when the above incubations were supplemented with dolichyl-phosphate and metal ions. Based on various time course studies and supplementation studies with various additions, it appears likely that the first five mannose residues to form MansGlcNAcrlipid come directly from GDP-mannose, whereas other mannose units to form larger oligosaccharide-lipids come from dolichylphosphoryl-mannose.In plants as well as animal cells, lipid-linked saccharide intermediates are involved in the formation of the N-linked oligosaccharide chains of membrane and secretory proteins (6,22,23,26 phoma cell line was isolated that had lost the ability to synthesize dolichyl-phosphoryl-mannose, but this mutant could still produce the Man5GlcNAc2-lipid (4). However, when cell-free extracts of this mutant were supplemented with dolichyl-phosphoryl-mannose, they could elongate the Man5GlcNAc2-lipid to larger oligosaccharides. Finally, a number ofthe mannosyl transferases that participate in the formation of the Man5GlcNAc2-lipid have been solubilized (1 1, 16, 17, 24, 25) and the a-1,2 and a-1,3 mannosyl transferases have been partially purified (17,24). These enzymes required GDP-mannose, rather than dolichylphosphoryl-mannose, as the mannosyl donors (16,25).In plants, the lipid-linked saccharide pathway has not been studied in nearly as much detail. The mung bean particulate enzyme catalyzes the incorporation of mannose from GDPmannose into Man1GlcNAc2-lipid, Man2GlcNAc2-lipid, Man3-GlcNAc2-lipid, Man5GlcNAc2-lipid, and Man7GlcNAc2-lipid (13). These oligosaccharides were partially characterized and a pathway of mannose addition was postulated. However, it was not determined whether the various mannose units were donated via GDP-mannose or dolichyl-phosphoryl-mannose. In the present repor...