Intracellular Trafficking of Influenza Hemagglutinin and Members of the Low Density Lipoprotein Receptor Family

Abstract

Polarized epithelial cells usually line body cavities, providing a barrier between two dissimilar environments. Distinct apical and basolateral membrane surfaces are maintained by sorting proteins in the biosynthetic and endocytic pathways. Influenza hemagglutinin is sorted to the apical membrane in polarized epithelial cells by a mechanism mediated by an association with lipid microdomains known as lipid rafts. Lipid rafts and their associated proteins are operationally defined by their resistance to detergent solubilization at cold temperatures. By systematic mutagenesis of the transmembrane domain of hemagglutinin, I show that ten consecutive amino acids are required to confer resistance to detergent extraction. Although some of the hemagglutinin transmembrane mutants were sorted apically without incorporation into detergent-resistant membranes, I determined that these mutants were transiently associated with lipid rafts. A small fraction of hemagglutinin coprecipitates with MAL/VIP17, a protein required for apical transport. The hemagglutinin and MAL that co-precipitated were contained in a detergent-resistant vesicle in an orientation consistent with a transport intermediate, suggesting that MAL might sort hemagglutinin into apical vesicles in the Golgi. However, the time course of the association of hemagglutinin and MAL in the biosynthetic pathway indicate that the two proteins do not associate until the majority of the HA reached the cell surface. Both the timing and limited extent of coprecipitation suggest that MAL may not sort hemagglutinin into apical vesicles in the biosynthetic pathway. Megalin, a member of the low density lipoprotein family of receptors, is sorted apically in polarized epithelial cells via a sorting signal present in its cytosolic domain. I show that the cytosolic domain of megalin associates with sorting nexin 17, a protein that interacts with all core members of the low density lipoprotein receptor family. Although sorting nexin 17 may not sort megalin apically in the biosynthetic pathway, I show that overexpression of sorting nexin 17 increases the rate of low density lipoprotein receptor recycling to the plasma membrane. The increase in recycling does not affect the expression of low density lipoprotein receptor at the cell surface, suggesting that sorting nexin 17 decreases the cycling time of the receptor.