Minor folding defects trigger local modification of glycoproteins by the ER folding sensor GT.
UDP-glucose:glycoprotein glucosyltransferase (GT) is a key component of the glycoprotein-specific folding and quality control system in the endoplasmic reticulum. By exclusively reglucosylating incompletely folded and assembled glycoproteins, it serves as a folding sensor that prolongs the association of newly synthesized glycoproteins with the chaperone-like lectins calnexin and calreticulin. Here, we address the mechanism by which GT recognizes and labels its substrates. Using an improved inhibitor assay based on soluble conformers of pancreatic ribonuclease in its glycosylated (RNase B) and unglycosylated (RNase A) forms, we found that the protein moiety of a misfolded conformer alone is sufficient for specific recognition by GT in vitro. To investigate the relationship between recognition and glucosylation, we tested a variety of glycosylation mutants of RNase S-Protein and an RNase mutant with a local folding defect [RNase C65S, C72S], as well as a series of loop insertion mutants. The results indicated that local folding defects in an otherwise correctly folded domain could be recognized by GT. Only glycans attached to the polypeptide within the misfolded sites were glucosylated.
Ritter
Christiane
C
Institute of Biochemistry, Swiss Federal Institute of Technology, Zurich, Switzerland.
Quirin
Katharina
K
Kowarik
Michael
M
Helenius
Ari
A
D004721:Endoplasmic Reticulum / Q000201:enzymology / Q000378:metabolism; D005964:Glucosyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D009154:Mutation; D017510:Protein Folding; D012333:RNA, Messenger; D012259:Ribonuclease, Pancreatic / Q000235:genetics / Q000378:metabolism*; D012260:Ribonucleases / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D006023:Glycoproteins; D012333:RNA, Messenger; D005964:Glucosyltransferases; C072423:mannosylglycoprotein 1,3-glucosyltransferase; D012260:Ribonucleases; C029600:ribonuclease B; D012259:Ribonuclease, Pancreatic