Effect of anion-specific inhibitors on the utilization of sugar nucleotides for N-linked carbohydrate unit assembly by thyroid endoplasmic reticulum vesicles.

The effect of anion-specific inhibitors on the utilization of the sugar nucleotides (UDP-glucose, GDP-mannose, and UDP-N-acetylglucosamine) required for the formation of the oligosaccharide-lipid involved in N-glycosylation has been studied in intact endoplasmic reticulum (ER) vesicles from thyroid. Of the reagents tested, the nonpenetrating probe DIDS (4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid) and its dihydro derivative (H2DIDS) were the most effective, causing a pronounced impairment in the synthesis from UDP-Glc of dolichyl phosphate (Dol-P) glucose (50% reduction at 60 microM DIDS) and in the incorporation of glucose into oligosaccharide-lipid and N-glycosylated protein; in contrast, no inhibition was observed in the formation from UDP-Glc of a glycogen-like proteoglucan. The specificity of the DIDS effect was indicated by the finding that methyl isothiocyanate, a nonanionic amino-reactive agent, demonstrated negligible inhibition. While DIDS also effected a block in the formation of Dol-P-P-GlcNAc from UDP-GlcNAc, no impairment in the utilization of GDP-Man for Dol-P-Man synthesis was observed. Since the DIDS inhibition of UDP-Glc and UDP-GlcNAc utilization was maintained after disruption of the ER vesicles with Triton, even when the incubations were supplemented with Dol-P, it appears that this reagent does not interact with sugar nucleotide translocator proteins but rather with the cytoplasmically oriented anion binding sites of glycosyltransferases (UDP-Glc- and UDP-GlcNAc:Dol-P glucosyl- and GlcNAc-1-P transferases). This is consistent with the protease sensitivity of these enzymes in the intact ER vesicles. Incubation of the vesicles with tritiated H2DIDS (8 microM) introduced radioactivity into membrane polypeptides with molecular weights of about 52,000 and 31,000 as observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that this inhibitor may prove useful as an affinity label in further studies of some of the glycosyltransferases involved in the synthesis of lipid-monosaccharide intermediates.