ER N-linked glycosylation

N-linked glycosylation begins at the cytosolic leaflet of the ER membrane with the assembly of the oligosaccharide GlcNAc₂Man₉Glc₃ on Dol. The pathway is initiated by the transfer of GlcNAc-P from UDP-GlcNAc to Dol-P, yielding Dol-PP-GlcNAc. The elongation proceeds sequentially through the action of glycosyltransferases anchored at the ER membrane. Once reaching Dol-PP-GlcNAc₂Man₅ the dolichol-linked oligosaccharide is translocated across the membrane and subsequently elongated by Man- and Glc-transferase enzymes using Dol-P-Man and Dol-P-Glc as donor substrates. The membrane-embedded protein RFT1 is required for the translocation of Dol-PP-GlcNAc₂Man₅, although it is unclear if RFT1 acts as a bona fide flippase. Yeast has been instrumental as a genetic model in the characterization of the ER pathway of oligosaccharide assembly. Several yeast N-linked glycosylation mutants (named alg for asparagine-linked glycosylation) led to the identification of the underlying glycosyltransferase genes, which were accordingly abbreviated ALG1 to ALG14.

Figure 30. Assembly of Dol-PP-oligosaccharides at the ER membrane and transfer of complete oligosaccharide to nascent protein.

The family of alg yeast mutants has also greatly contributed to the elucidation of several congenital disorders of glycosylation (CDG). Because of the strong conservation of the dolichol-linked oligosaccharide pathway among eukaryotes, the impact of mutations identified in human CDG patients can be investigated by expressing human ALG genes in the corresponding alg yeast strains. Without the availability of alg yeasts, the detrimental effect of mutations can only be confirmed by assessing the enzymatic activity of the mutant protein. Such analysis is however extremely difficult considering the transmembraneous nature of the alg enzymes and the requirement for complex Dol linked substrates for the assays.