Figure 1. The identification of UDP-glucose:glycoprotein glucosyltransferase (UGGT) 1/2 substrates.

(A) The pathway of N-glycosylation in eukaryotic cells is depicted. N-glycan synthesis is initiated in the outer endoplasmic reticulum (ER) membrane leaflet on a dolichol-P-phosphate facing the cytoplasm. Flipping of the precursor N-glycan to the ER luminal leaflet and further synthesis steps mediated by ALG proteins leads to eventual transfer of a Glc₃Man₉GlcNAc₂ N-glycan to a substrate by the oligosaccharyl transferase complex. ALG6 (red lettering) catalyzes the transfer of the initial glucose onto the Man₉ precursor N-glycan. (B) In wild-type (WT) cells, a Glc₃Man₉GlcNAc₂ N-glycan is transferred to substrates. Monoglucosylated substrates may therefore occur via trimming by glucosidases I/II (GlsI/II) or reglucosylation by UGGT1/2. In ALG6^-/- cells, a Man₉GlcNAc₂ N-glycan is transferred to substrates. Therefore, monoglucosylated substrates may only occur through reglucosylation by UGGT1/2. Deoxynojirimycin (500 μM) was added to block the trimming of monoglucosylated substrates by GlsII. ALG6^-/- cells were then lysed and split equally between affinity purifications with either GST-CRT or GST-CRT-Y109A bound to glutathione beads. Affinity-purified samples were then reduced, alkylated, trypsinized, and labeled with tandem mass tag (TMT) labels. Samples were then deglycosylated with PNGaseF, pooled, and analyzed by mass spectrometry. (C) Substrates were identified by dividing the quantification of the TMT label in the GST-CRT condition for each protein by that of the associated GST-CRT-Y109A condition, yielding the fold increase. Localization as predicted by UniprotKB annotation is depicted. A cutoff of threefold increase was applied. Data is representative of two independent experiments. Error bars represent standard error of the mean (SEM). (D) The N-glycoproteome (N-glycopro) was computationally determined by collecting all proteins annotated to contain N-glycans by UniprotKB. Annotated localization information was then used to computationally determine the localization distribution of the N-glycoproteome as well as the identified UGGT substrates.