Processing of N-linked glycans during endoplasmic-reticulum-associated degradation of a short-lived variant of ribophorin I.
Recently, the role of N-linked glycans in the process of ERAD (endoplasmic reticulum-associated degradation) of proteins has been widely recognized. In the present study, we attempted to delineate further the sequence of events leading from a fully glycosylated soluble protein to its deglycosylated form. Degradation intermediates of a truncated form of ribophorin I, namely RI(332), which contains a single N-linked oligosaccharide and is a substrate for the ERAD/ubiquitin-proteasome pathway, were characterized in HeLa cells under conditions blocking proteasomal degradation. The action of a deoxymannojirimycin- and kifunensine-sensitive alpha1,2-mannosidase was shown here to be required for both further glycan processing and progression of RI(332) in the ERAD pathway. In a first step, the Man(8) isomer B, generated by ER mannosidase I, appears to be the major oligomannoside structure associated with RI(332) intermediates. Some other trimmed N-glycan species, in particular Glc(1)Man(7)GlcNAc(2), were also found on the protein, indicating that several mannosidases might be implicated in the initial trimming of the oligomannoside. Secondly, another intermediate of degradation of RI(332) accumulated after proteasome inhibition. We demonstrated that this completely deglycosylated form arose from the action of an N-glycanase closely linked to the ER membrane. Indeed, the deglycosylated form of the protein remained membrane-associated, while being accessible from the cytoplasm to ubiquitinating enzymes and to added protease. Our results indicate that deglycosylation of a soluble ERAD substrate glycoprotein occurs in at least two distinct steps and is coupled with the retro-translocation of the protein preceding its proteasomal degradation.
Kitzmüller
Claudia
C
Max F. Perutz Laboratories, University Departments at the Vienna Biocenter and Institute of Medical Biochemistry, Department of Molecular Genetics, University of Vienna, Austria.
Caprini
Andrea
A
Moore
Stuart E H
SE
Frénoy
Jean-Pierre
JP
Schwaiger
Eva
E
Kellermann
Odile
O
Ivessa
N Erwin
NE
Ermonval
Myriam
M
D003546:Cysteine Endopeptidases / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000201:enzymology* / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008358:Mannose / Q000032:analysis; D008361:Mannosidases / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000378:metabolism; D011134:Polysaccharides / Q000032:analysis / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D021381:Protein Transport
D008565:Membrane Proteins; D009097:Multienzyme Complexes; D011134:Polysaccharides; C034241:ribophorin; D006026:Glycoside Hydrolases; D008361:Mannosidases; C020668:glycanase; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D008358:Mannose