Abstract
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.
Highlights
The limited availability of endogenous dolichyl phosphate (Dol-P) was circumventing potential control points through the addition demonstrated by the pronounced stimulation in Dol-P- of Dol-P and a peptide containing the Asn-X-Thrsequence
We have previously reported that intact thyroid endoplasmic reticulum (ER) vesicles incubated with UDP-[3H]Glc bring about a rapid incorporation of glucose into a lower phase lipid characterized as DolP-Glc [4]
Cubations were terminated by the addition of 2 ml of methanol, and lipid extractions were performed with chloroform/methanol/uffer (3:2:1) and chloroform/methanol/water (10103),in a mannerpreviously described, to yield dolichol-linked monosaccharide and oligosaccharide fractions, respectively [8].The protein-bound oligosaccharides were obtained as glycopeptides after Pronase digestion of the
Summary
The limited availability of endogenous Dol-P was circumventing potential control points through the addition demonstrated by the pronounced stimulation in Dol-P- of Dol-P and a peptide containing the Asn-X-Thrsequence. Dol-P-Man synthesis in ER vesicles; addition of increasing amounts of unlabeled UDP-Glc (up to 100PM)to incubations containing 0.38 PM GDP-[3H]Man resulted in no discernible decrease in mannoseincorporation (data not shown).
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