The recycling endosome of Madin-Darby canine kidney cells is a mildly acidic compartment rich in raft components

We present a biochemical and morphological characterization of recycling en dosomes containing the transferrin receptor in the epithelial Madin-Darby c anine kidney cell line. We find that recycling endosomes are enriched in mo lecules known to regulate transferrin recycling but lack proteins involved in early endosome membrane dynamics, indicating that recycling endosomes ar e distinct from conventional early endosomes. We also find that recycling e ndosomes are less acidic than early endosomes because they lack a functiona l vacuolar ATPase. Furthermore, we show that recycling endosomes can be rea ched by apically internalized tracers, confirming that the apical endocytic pathway intersects the transferrin pathway. Strikingly, recycling endosome s are enriched in the raft lipids sphingomyelin and cholesterol as well as in the raft-associated proteins caveolin-1 and flotillin-1. These observati ons may suggest that a lipid-based sorting mechanism operates along the Mad in-Darby canine kidney recycling pathway, contributing to the maintenance o f cell polarity. Altogether, our data indicate that recycling endosomes and early endosomes differ functionally and biochemically and thus that differ ent molecular mechanisms regulate protein sorting and membrane traffic at e ach step of the receptor recycling pathway.