Interactions between newly synthesized glycoproteins, calnexin and a network of resident chaperones in the endoplasmic reticulum.
Calnexin is a membrane-bound lectin and a molecular chaperone that binds newly synthesized glycoproteins in the endoplasmic reticulum (ER). To analyze the oligomeric properties of calnexin and calnexin-substrate complexes, sucrose velocity gradient centrifugation and chemical cross-linking were used. After CHAPS solubilization of Chinese Hamster Ovary cells, the unoccupied calnexin behaved as a monomer sedimenting at 3.5 S20,W. For calnexin-substrate complexes the S-values ranged between 3.5-8 S20,W, the size increasing with the molecular weight of the substrate. Influenza hemagglutinin, a well-characterized substrate associated with calnexin in complexes that sedimented at 5-5.5 S20,W. The majority of stable complexes extracted from cells, appeared to contain a single calnexin and a single substrate molecule, with about one third of the calnexin in the cell being unoccupied or present in weak associations. However, when chemical cross-linking was performed in intact cells, the calnexin-substrate complexes and calnexin itself was found to be part of a much larger heterogeneous protein network that included other ER proteins. Pulse-chase analysis of influenza-infected cells combined with chemical cross-linking showed that HA was part of large, heterogeneous, cross-linked entities during the early phases of folding, but no longer after homotrimer assembly. The network of weakly associated resident ER chaperones which included BiP, GRP94, calreticulin, calnexin, and other proteins, may serve as a matrix that binds early folding and assembly intermediates and restricts their exit from the ER.
Tatu
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Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520-8002, USA.
Helenius
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D000818:Animals; D020126:Brefeldin A; D016466:CHO Cells; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D037281:Calnexin; D006224:Cricetinae; D003432:Cross-Linking Reagents; D003517:Cyclopentanes / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000378:metabolism; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000378:metabolism*; D018832:Molecular Chaperones / Q000378:metabolism*; D011817:Rabbits; D013379:Substrate Specificity
D002135:Calcium-Binding Proteins; D003432:Cross-Linking Reagents; D003517:Cyclopentanes; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D018832:Molecular Chaperones; D037281:Calnexin; D020126:Brefeldin A