ENDOSOME ACIDIFICATION AND RECEPTOR TRAFFICKING - BAFILOMYCIN A(1) SLOWS RECEPTOR EXTERNALIZATION BY A MECHANISM INVOLVING THE RECEPTORS INTERNALIZATION MOTIF

To examine the relationship between endosome acidification and recepto r trafficking, transferrin receptor trafficking was characterized in C hinese hamster ovary cells in which endosome acidification was blocked by treatment with the specific inhibitor of the vacuolar H+-ATPase, b afilomycin A(1). Elevating endosome pH slowed the receptor externaliza tion rate to approximately one-half of control but did not affect rece ptor internalization kinetics. The slowed receptor externalization req uired the receptor's cytoplasmic domain and was largely eliminated by substitutions replacing either of two aromatic amino acids within the receptor's cytoplasmic YTRF internalization motif. These results confi rm, using a specific inhibitor of the vacuolar proton pump, that prope r endosome acidification is necessary to maintain rapid recycling of i ntracellular receptors back to the plasma membrane. Moreover, receptor return to the plasma membrane is slowed in the absence of proper endo some acidification by a signal-dependent mechanism involving the recep tor's cytoplasmic tyrosine-containing internalization motif. These res ults, in conjunction with results from other studies, suggest that the mechanism for clustering receptors in plasma membrane clathrin-coated pits may be an example of a more general mechanism that determines th e dynamic distribution of membrane proteins among various compartments with luminal acidification playing a crucial role in this process.