TRANSPORT FROM LATE ENDOSOMES TO LYSOSOMES, BUT NOT SORTING OF INTEGRAL MEMBRANE-PROTEINS IN ENDOSOMES, DEPENDS ON THE VACUOLAR PROTON PUMP

Endocytosed proteins are sorted in early endosomes to be recycled to t he plasma membrane or transported further into the degradative pathway . We studied the role of endosome acidification on the endocytic traff icking of the transferrin receptor (TfR) as a representative for the r ecycling pathway, the cation-independent mannose 6-phosphate receptor (MPR) as a prototype for transport to late endosomes, and fluid-phase endocytosed HRP as a marker for transport to lysosomes, Toward this pu rpose, bafilomycin A1 (Baf), a specific inhibitor of the vacuolar prot on pump, was used to inhibit acidification of the vacuolar system. Mic rospectrofluorometric measurement of the pH of fluorescein-rhodamine-c onjugated transferrin (Tf)-containing endocytic compartments in living cells revealed elevated endosomal. pH values (pH >7.0) within 2 min a fter addition of Baf. Although recycling of endocytosed Tf to the plas ma membrane continued in the presence of Baf, recycled Tf did not diss ociate from its receptor, indicating failure of Fe3+ release due to a neutral endosomal pH. In the presence of Baf, the rates of internaliza tion 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 Tf R expression at the cell surface was measured during Baf treatment. So rting between endocytosed TfR and MPR was analyzed by the HRP-catalyze d 3,3'-diaminobenzidine crosslinking technique, using transferrin conj ugated to HRP to label the endocytic pathway of the TfR. In the absenc e of Baf, endocytosed surface I-125-labeled MPR was sorted from the Tf R pathway starting at 10 min after uptake, reaching a plateau of 40% a fter 45 min. In the presence of Baf, sorting was initiated after 20 mi n of uptake, reaching similar to 40% after 60 min. Transport of fluid- phase endocytosed HRP to late endosomes and lysosomes was measured usi ng cell fractionation and immunogold electron microscopy. Baf did not interfere with transport of HRP to MPR-labeled late endosomes, but nea rly completely abrogated transport to cathepsin D-labeled lysosomes. F rom these results, we conclude that trafficking through early and late endosomes, but not to lysosomes, continued upon inactivation of the v acuolar proton pump.