Regulation of synthesis of oligosaccharyl pyrophosphoryl dolichol during seed germination

Subsaturating (approximately physiological) concentrations of [1-14C]dolichol were supplied in vitro to soybean embryo microsomes, to enable the formation of [1-14C]dolichyl phosphate ([1-14C]Dol-P). By including saturating amounts of nonradiolabeled UDP-GlcNAc, GDP-Man, and UDP-Glc individually or in combination in the assay, the extent to which newly formed [1-14C]Dol-P is directed into [1-14C]dolichyl pyrophosphate ([1-14C]Dol-PP) – GlcNAc, [1-14C]Dol-PP-(GlcNAc)2, [1-14C]Dol-P-Man, and [1-14C]Dol-P-Glc could be followed. The addition of UDP-GlcNAc did not appreciably decrease the rate of accumulation of free [1-14C]Dol-P, whereas GDP-Man and UDP-Glc reduced the free [1-14C]Dol-P content of the assays by about 20 and 50%, respectively. The appearance of Dol-P-Man and (or) Dol-P-Glc accounted entirely for the decreased amounts of free Dol-P present. Even in the presence of all three nucleoside diphosphate sugars, about 40–50% of the total [1-14C]Dol-P formed remained in the free phosphate form. The early intermediates Dol-PP-GlcNAc and Dol-PP-(GlcNAc)2 were produced only in trace amounts by microsomes isolated prior to germination and between 2 and 20 h of germination. The availability of Dol-P does not appear to be the rate-limiting factor in Dol-PP-oligosaccharide formation and N-linked glycoprotein formation in this system.Key words: dolichol, dolichyl phosphate, N-oligosaccharide, synthesis regulation.