The receptor recycling pathway contains two distinct populations of early endosomes with different sorting functions

Receptor recycling involves two endosome populations, peripheral early endo somes and perinuclear recycling endosomes, In polarized epithelial cells, e ither or both populations must be able to sort apical from basolateral prot eins, returning each to its appropriate plasma membrane domain, However, ne ither the roles of early versus recycling endosomes in polarity nor their r elationship to each other has been quantitatively evaluated. Using a combin ed morphological, biochemical, and kinetic approach, we found these two end osome populations to represent physically and functionally distinct compart ments. Early and recycling endosomes were resolved on Optiprep gradients an d shown to be differentially associated with rab4, rab11, and transferrin r eceptor; rab4 was enriched on early endosomes and at least partially deplet ed from recycling endosomes, with the opposite being true for rab11 and tra nsferrin receptor. The two populations were also pharmacologically distinct , with AlF4 selectively blocking export of transferrin receptor from recycl ing endosomes to the basolateral plasma membrane. We applied these observat ions to a detailed kinetic analysis of transferrin and dimeric IEA recyclin g and transcytosis, The data from these experiments permitted the construct ion of a testable, mathematical model which enabled a dissection of the rol es of early and recycling endosomes in polarized receptor transport. Contra ry to expectations, the majority (>65%) of recycling to the basolateral sur face is likely to occur from early endosomes, but with relatively little so rting of apical from basolateral proteins. Instead, more complete segregati on of basolateral receptors from receptors intended for transcytosis occurr ed upon delivery to recycling endosomes.