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 pathwa y of living cells. In addition to mature trimers, six distinct maturat ion intermediates were identified. Of these, all the incompletely oxid ized forms were located in the endoplasmic reticulum (ER) and associat ed with calnexin, a membrane-bound, lectin-like ER chaperone. Once ful ly oxidized, the HA dissociated from calnexin as a monomer, which rapi dly became resistant to dithiothreitol (DTT) reduction. Part of these extensively folded molecules moved as monomers into the intermediate c ompartment between the ER and the Golgi complex. Assembly of homotrime rs occurred without calnexin-involvement within the ER and in the inte rmediate 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 a nd intracellular localization of several intermediates in the conforma tional maturation of HA, including the immediate precursor for trimer assembly.