Transport from late endosomes to lysosomes, but not sorting of integral membrane proteins in endosomes, depends on the vacuolar proton pump (original) (raw)

1995, The Journal of Cell Biology

Endocytosed proteins are sorted in early endosomes to be recycled to the plasma membrane or transported further into the degradative pathway. We studied the role of endosome acidification on the endocytic trafficking of the transferrin receptor (TfR) as a representative for the recycling pathway, the cationindependent mannose 6-phosphate receptor (MPR) as a prototype for transport to late endosomes, and fluidphase endocytosed HRP as a marker for transport to lysosomes. Toward this purpose, bafilomycin A1 (Bar), a specific inhibitor of the vacuolar proton pump, was used to inhibit acidification of the vacuolar system. Microspectrofluorometric measurement of the pH of fluorescein-rhodamine-conjugated transferrin (Tf)-conraining endocytic compartments in living cells revealed elevated endosomal pH values (pH >7.0) within 2 min after addition of Bar. Although recycling of endocytosed Tf to the plasma membrane continued in the presence of Baf, recycled Tf did not dissociate from its receptor, indicating failure of Fe 3+ release due to a neutral endosomal pH. In the presence of Baf, the rates of internalization and recycling of Tf were reduced by a factor of 1.40 +_ 0.08 and 1.57 + 0.25, respectively. Consequently, little if any change in TfR expression at the cell surface was measured during Bar treatment. Sorting between endocytosed TfR and MPR was analyzed by the HRP-catalyzed 3,3'-diaminobenzidine crosslinking technique, using transferrin conjugated to HRP to label the endocytic pathway of the TfR. In the absence of Baf, endocytosed surface 125I-labeled MPR was sorted from the TfR pathway starting at 10 min after uptake, reaching a plateau of 40% after 45 min. In the presence of Bar, sorting was initiated after 20 min of uptake, reaching ~40% after 60 min. Transport of fluid-phase endocytosed HRP to late endosomes and lysosomes was measured using cell fractionation and immunogold electron microscopy. Baf did not interfere with transport of HRP to MPR-labeled late endosomes, but nearly completely abrogated transport to cathepsin D-labeled lysosomes. From these results, we conclude that trafficking through early and late endosomes, but not to lysosomes, continued upon inactivation of the vacuolar proton pump.