Control of N-linked carbohydrate unit synthesis in thyroid endoplasmic reticulum by membrane organization and dolichyl phosphate availability.
Thyroid rough endoplasmic reticulum (ER) has been shown to contain a highly organized multienzyme system capable of carrying out the N-glycosylation of newly synthesized proteins. These reactions were studied in isolated ER vesicles and found to be controlled to a large extent by the availability of a key substrate, dolichyl phosphate (Dol-P), as well as by the amount of endogenous polypeptide acceptor present. Although in intact vesicles UDP-Glc was utilized in an efficient manner to form Dol-P-Glc and glucosylated oligosaccharide-lipid, after disruption of vesicle integrity, even with low concentrations of Triton X-100, the coupling of Dol-P-Glc formation to lipid-linked oligosaccharide assembly and subsequent N-glycosylation was substantially impaired. Increased incubation temperatures also resulted in a decreased effectiveness of glucose transfer from Dol-P-Glc to lipid-oligosaccharide, presumably because of a decline in the extent of structural organization of the ER membranes. The limited availability of endogenous Dol-P was demonstrated by the pronounced stimulation in Dol-P-Glc formation resulting from the addition of this lipid acceptor to Triton-disrupted ER membranes as well as by its generation in intact vesicles. The latter was accomplished by stimulating recycling of endogenous Dol-P through the addition of a peptide (Tyr-Asn-Leu-Thr-Ser-Val) which is an N-glycosylation substrate. The inhibition of Dol-P-Glc synthesis from UDP-Glc observed in the presence of elevated levels of GDP-Man which could be relieved in Triton-disrupted or intact ER vesicles by the addition or generation, respectively, of Dol-P, is considered to be the result of a competing requirement for Dol-P by the mannosyltransferase. Moreover GTP, by selectively inhibiting the mannosyltransferase, prevented the decrease of Dol-P-Glc formation caused by GDP-Man. Since addition of the acceptor peptide to intact vesicles stimulated Dol-P-P-GlcNAc as well as Dol-P-Glc and Dol-P-Man synthesis it would appear that a pool of Dol-P available in common to all three enzymes responsible for dolichol-linked monosaccharide synthesis exists in the ER membranes.
D000818:Animals; D002417:Cattle; D003902:Detergents / Q000494:pharmacology; D004288:Dolichol Phosphates / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006023:Glycoproteins / Q000096:biosynthesis* / Q000235:genetics; D006155:Guanosine Diphosphate Mannose / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000494:pharmacology; D011106:Polyisoprenyl Phosphates / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D013961:Thyroid Gland / Q000378:metabolism*; D014316:Tritium; D014532:Uridine Diphosphate Glucose / Q000378:metabolism
D003902:Detergents; D004288:Dolichol Phosphates; D006023:Glycoproteins; D011106:Polyisoprenyl Phosphates; D014316:Tritium; D006155:Guanosine Diphosphate Mannose; D011092:Polyethylene Glycols; D006160:Guanosine Triphosphate; D017830:Octoxynol; D014532:Uridine Diphosphate Glucose