Brefeldin A rapidly disrupts plasma membrane polarity by blocking polar sorting in common endosomes of MDCK cells

Recent studies showing thorough intermixing of apical and basolateral endos omes have demonstrated that endocytic sorting is critical to maintaining th e plasma membrane polarity of epithelial cells. Our studies of living, pola rized cells show that disrupting endocytosis with brefeldin-A rapidly destr oys the polarity of transferrin receptors in MDCK cells while having no eff ect on tight junctions. Brefeldin-A treatment induces tubulation of endosom es, but the sequential compartments and transport steps of the transcytotic pathway remain intact. Transferrin is sorted from LDL, but is then missort ed from common endosomes to the apical recycling endosome, as identified by its nearly neutral pH, and association with GFP chimeras of Rabs 11a and 2 5. From the apical recycling endosome, transferrin is then directed to the apical plasma membrane. These data are consistent with a model in which pol arized sorting of basolateral membrane proteins occurs via a brefeldin-A-se nsitive process of segregation into basolateral recycling vesicles. Althoug h disruption of polar sorting correlates with dissociation of gamma -adapti n from endosomes, gamma -adaptin does not appear to be specifically involve d in sorting into recycling vesicles, as we find it associated with the tra nscytotic pathway, and particularly to the post-sorting transcytotic apical recycling endosome.