Abstract
In mammalian cells, inhibition of translation interferes with synthesis of the lipid-linked oligosaccharide (LLO) Glc3Man9GlcNAc2-P-P-dolichol as measured with radioactive sugar precursors. Conflicting hypotheses have been proposed, and the fundamental basis for this regulation has remained elusive. Here, fluorophore-assisted carbohydrate electrophoresis (FACE) was used to measure LLO concentrations directly in cells treated with translation blockers. Further, LLO biosynthetic enzymes were assayed in vitro with endogenous acceptor substrates using either cells gently permeabilized with streptolysin-O (SLO) or microsomes from homogenized cells. In Chinese hamster ovary (CHO)-K1 cells treated with translation blockers, FACE did not detect changes in concentrations of Glc3Man9GlcNAc2-P-P-dolichol or early LLO intermediates. These results do not support earlier proposals for feedback repression of LLO initiation by accumulated Glc3Man9GlcNAc2-P-P-dolichol, or inhibition of a GDP-mannose dependent transferase. With microsomes from cells treated with translation blockers, there was no interference with LLO initiation by GlcNAc-1-P transferase (GPT), mannose-P-dolichol synthase, glucose-P-dolichol synthase, or LLO synthesis in vitro, as reported previously. Surprisingly, inhibition of all of these was detected with the SLO in vitro system. Additional experiments with the SLO system showed that the three transferases shared a limited pool of dolichol-P that was trapped as Glc3Man9GlcNAc2-P-P-dolichol by translation arrest. Overexpression of GPT was unable to reverse the effects of translation arrest on LLO initiation, and experiments with FACE and the SLO system showed that overexpressed GPT was not functional in vivo, although it was highly active in microsomal assays. Thus, the combined use of the SLO in vitro system and FACE showed that LLO biosynthesis depends upon a limited primary pool of dolichol-P. Physical perturbation associated with microsome preparation appears to make available a secondary pool of dolichol-P, masking inhibition by translation arrest, as well as activating a nonfunctional fraction of GPT. The implications of these results for the organization of the LLO pathway are discussed.
Highlights
The lipid-linked oligosaccharide (LLO)1 Glc3Man9GlcNAc2-P-P-dolichol serves as the donor of oligosaccharide units that are transferred by oligosaccharyltransferase (OT) to appropriate asparaginyl residues in nascent polypeptides within the lumen of the endoplasmic reticulum (ER), forming glycoproteins with asparagine-linked (N-linked) Glc3Man9GlcNAc2 glycans [1]
The pathway for LLO synthesis has been elucidated by a combination of biochemical and genetic methods, and to date mutations in seven genes essential for LLO synthesis have been identified as the causes of Congenital Disorders of Glycosylation (CDG) Types Ia-g [2,3]
MPD and GPD are synthesized by the transfer of mannose or glucose from GDP-mannose or UDP-glucose, respectively, to cytoplasmically oriented dolichol-P, and must flip to the lumenal leaflet to participate in LLO synthesis
Summary
The lipid-linked oligosaccharide (LLO) Glc3Man9GlcNAc2-P-P-dolichol serves as the donor of oligosaccharide units that are transferred by oligosaccharyltransferase (OT) to appropriate asparaginyl residues in nascent polypeptides within the lumen of the endoplasmic reticulum (ER), forming glycoproteins with asparagine-linked (N-linked) Glc3Man9GlcNAc2 glycans [1]. LLO synthesis is initiated by the transfer of GlcNAc-1-P from UDP-GlcNAc to dolichol-P by a specific tunicamycin (TN)-sensitive GlcNAc-1-P transferase (GPT) [4] This reaction occurs on the cytoplasmic face of the ER membrane [5]. Cytoplasmically-oriented Man5GlcNAc2-P-P-dolichol flips to the lumenal leaflet [6,7] in a process involving the Rft protein [8] This lumenally-oriented LLO becomes an acceptor for the transfer of four additional mannose residues from mannoseP-dolichol (MPD) and three glucose residues from glucose-P-dolichol (GPD), generating Glc3Man9GlcNAc2-P-P-dolichol. MPD and GPD are synthesized by the transfer of mannose or glucose from GDP-mannose or UDP-glucose, respectively, to cytoplasmically oriented dolichol-P, and must flip to the lumenal leaflet to participate in LLO synthesis. Transferases that use MPD and GPD as donor substrates have an additional requirement for the product of the Lec gene [9]
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