Proteins targeted to the secretory pathway mediate many biological processes but can only do so when properly folded. Within the endoplasmic reticulum (ER), folding and quality control of secretory proteins is facilitated by N-glycan modifications, which are recognized by a network of chaperones. The gateway to exit from the ER is governed by the enzyme UDP-glucose:glycoprotein glucosyltransferase (UGGT). This enzyme serves as a gatekeeper of glycoprotein quality control cycle where the N-linked glycan serves as a molecular signal of the folding status. UGGT selectively recognizes glycoproteins that have not completed their folding program and transfers a glucose residue to the N-linked glycan. The resulting monoglucosylated species can re-engage with the lectin chaperones for further chances at productive folding or ultimately be targeted for degradation. The folding-sensing mechanism of UGGT has long remained a mystery. Here, we describe physical studies of human UGGT1 and a model glycoprotein substrate, a truncated form of bovine ribonuclease B, S-protein. An in vitro assay of UGGT1 glucosylation shows that S-protein is recognized while intact RNaseB is not. Prior work has suggested that UGGT1 preferentially recognizes molten globule states. Surprisingly, preliminary structural characterization by NMR spectroscopy shows that the S-protein is highly structured. Additional efforts are underway to map UGGT1:S-protein interactions using a combination of single-particle cryo-EM and crosslinking mass spectrometry. We anticipate this work will provide a detailed view into the structure of a UGGT1 substrate as well as key regions that participate in folding-sensing.
Williams et al. (Sun,) studied this question.