Pivotal role of calnexin and mannose trimming in regulating the endoplasmic reticulum-associated degradation of major histocompatibility complex class I heavy chain.
We have established a mammalian semipermeabilized cell system that faithfully reconstitutes the proteasome-mediated degradation of major histocompatibility complex Class I heavy chain. We show that degradation required unfolding of the protein and was cytosol- and ATP-dependent and that dislocation and degradation required proteasome activity. When the interaction of heavy chain with calnexin was prevented, the rate of degradation was accelerated, suggesting that an interaction with calnexin stabilized heavy chain. Stabilization of heavy chain to degradation was also achieved either by preventing mannose trimming or by removal of the N-linked glycosylation site. This demonstrates that glycosylation and mannose trimming are required to ensure degradation of heavy chain. When degradation or mannose trimming was inhibited, heavy chain formed a prolonged interaction with immunoglobulin heavy chain binding protein, ERp57, and protein disulfide isomerase. Taken together, these results indicate that calnexin association and mannose trimming provide a mechanism to regulate the folding, assembly, and degradation of glycoproteins entering the secretory pathway.
Wilson
C M
CM
School of Biological Sciences, 2.205 Stopford Building, University of Manchester, Manchester M13 9PT, United Kingdom.
Farmery
M R
MR
Bulleid
N J
NJ
D019943:Amino Acid Substitution; D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D037281:Calnexin; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000276:immunology*; D006360:Heat-Shock Proteins / Q000378:metabolism; D015395:Histocompatibility Antigens Class I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007535:Isomerases / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008358:Mannose / Q000378:metabolism*; D018832:Molecular Chaperones / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D014176:Protein Biosynthesis; D011489:Protein Denaturation; D019704:Protein Disulfide-Isomerases / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D002135:Calcium-Binding Proteins; D006360:Heat-Shock Proteins; D015395:Histocompatibility Antigens Class I; D046911:Macromolecular Substances; D018832:Molecular Chaperones; D012333:RNA, Messenger; D011994:Recombinant Proteins; D037281:Calnexin; D007535:Isomerases; D019704:Protein Disulfide-Isomerases; D008358:Mannose