Synthesis of Dolichol-P-P-GlcNAc₂Man₉Glc₃. Dolichol (red squiggle) phosphate (Dol-P) located on the cytoplasmic face of the endoplasmic reticulum (ER) membrane receives GlcNAc-1-P from UDP-GlcNAc in the cytoplasm to generate Dol-P-P-GlcNAc. Dol-P-P-GlcNAc is extended to Dol-P-P-GlcNAc₂Man₅ before being “flipped” across the ER membrane to the lumenal side. Subsequently, four Man residues are added from Dol-P-Man and three Glc residues from Dol-P-Glc. Dol-P-Man and Dol-P-Glc are also made on the cytoplasmic side of the ER membrane and “flipped” onto the lumenal side. Yeast mutants defective in an alg (asparagine-linked glycosylation) gene were used to identify the ALG enzyme responsible for many reactions. The 14-sugar oligosaccharide is transferred to the Asn side chain within the sequon N-X-S/T by an oligosaccharyltransferase complex (OST) of 8-9 transmembrane proteins. In mammalian cells, the OSTA complex is associated with the translocon in the ER membrane and preferentially glycosylates nascent polypeptides traversing the translocon, whereas the OSTB complex modifies proteins that have left the translocon and are in the ER lumen. Enzyme names are from the Human Genome Nomenclature Committee (HGNC).