Folding and oligomerization of influenza hemagglutinin in the ER and the intermediate compartment.
Influenza hemagglutinin (HA) was used to analyze the stepwise folding and oligomeric assembly of glycoproteins in the early secretory pathway of living cells. In addition to mature trimers, six distinct maturation intermediates were identified. Of these, all the incompletely oxidized forms were located in the endoplasmic reticulum (ER) and associated with calnexin, a membrane-bound, lectin-like ER chaperone. Once fully oxidized, the HA dissociated from calnexin as a monomer, which rapidly became resistant to dithiothreitol (DTT) reduction. Part of these extensively folded molecules moved as monomers into the intermediate compartment between the ER and the Golgi complex. Assembly of homotrimers occurred without calnexin-involvement within the ER and in the intermediate compartment. When anchor-free HA molecules were analyzed, it was found that they reach the DTT-resistant monomeric conformation but fail to trimerize. Taken together, the results provide a definition and intracellular localization of several intermediates in the conformational maturation of HA, including the immediate precursor for trimer assembly.
Tatu
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Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06510, USA.
Hammond
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Helenius
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D000818:Animals; D016466:CHO Cells; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D002999:Clone Cells; D006224:Cricetinae; D004229:Dithiothreitol / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism*; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008961:Models, Structural; D011487:Protein Conformation / Q000187:drug effects; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D013997:Time Factors; D014759:Viral Envelope Proteins / Q000378:metabolism*
D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; D004229:Dithiothreitol