Regulation of endocytic pH by the Na+,K+-ATPase in living cells.
Acidification of endocytosed ligands destined for lysosomes is biphasic, with a rapid drop to pH 6, followed by a slow decrease to pH 5. Continuous measurements of transferrin acidification have confirmed that the pH minimum in early (presorting) endosomes is approximately pH 6. On the basis of measurements of endosomal acidification in vitro, it has been proposed that the pH in the early endosome is limited by the internalization of the Na+,K+-ATPase, which generates an interior-positive membrane potential in this compartment [Fuchs, R., Schmid, S. & Mellman, I. (1989) Proc. Natl. Acad. Sci. USA 86, 539-543]. We present two lines of evidence that strongly implicate the Na+,K+-ATPase as a major regulatory element of endocytic pH in vivo. First, ouabain, a specific inhibitor of the Na+,K+-ATPase, interferes with the regulation of acidification in early endocytic compartments. Transferrin is normally rapidly acidified to pH 6.0-6.2, followed by alkalinization during recycling. In the presence of ouabain, the minimum pH of transferrin-containing endosomes decreases from 6.0-6.2 to less than 5.3. Second, ouabain eliminates the resistance to both the growth inhibitory and vacuologenic effects of chloroquine in the lysosomal acidification defective cell line CHL60-64. The phenotype of this cell line is consistent with a defect in the removal or inactivation of the early acidification regulatory elements from the late endocytic compartments. The ouabain data suggest that the defect in this cell line is due to improper localization of the Na+,K+-ATPase. A model for pH regulation and vacuolation by weak bases is discussed.
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Department of Biological Sciences, Carnegie-Mellon University, Pittsburgh, PA 15213.
Sipe
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Murphy
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D000818:Animals; D002294:Carcinoma, Squamous Cell; D002455:Cell Division; D002460:Cell Line; D002738:Chloroquine / Q000494:pharmacology; D004705:Endocytosis; D005434:Flow Cytometry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008954:Models, Biological; D010042:Ouabain / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D014168:Transferrin / Q000378:metabolism; D014407:Tumor Cells, Cultured
D014168:Transferrin; D010042:Ouabain; D002738:Chloroquine; D000254:Sodium-Potassium-Exchanging ATPase