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. 1989 Jan;86(2):539–543. doi: 10.1073/pnas.86.2.539

A possible role for Na+,K+-ATPase in regulating ATP-dependent endosome acidification.

R Fuchs 1, S Schmid 1, I Mellman 1
PMCID: PMC286507  PMID: 2536167

Abstract

Endosomes maintain a slightly acidic internal pH, which is directly responsible for their ability to ensure proper sorting of incoming receptors and ligands during endocytosis. At least two distinct subpopulations of endosomes can be distinguished, designated "early" and "late" on the basis of their kinetics of labeling with endocytic tracers. The subpopulations differ not only in their functions (rapid receptor recycling and transport to lysosomes, respectively) but also in their capacities for acidification in intact cells and in vitro. To investigate the possible basis for pH regulation in endosomes, we have studied the transport properties and ion permeabilities of early and late endosomes isolated from Chinese hamster ovary cells. Using endosomes selectively labeled with pH-sensitive endocytic tracers, we found that ATP-dependent acidification is electrogenic, being accompanied by the generation of an interior-positive membrane potential which opposes further acidification. While membrane potential and, consequently, acidification was controlled by the influx of permeant anions and efflux of protons and alkali cations, acidification was further modulated in Na+ and K+-containing buffers by the ouabain- and vanadate-sensitive Na+,K+-ATPase, which appears to be a functional component of the endosomal membrane. The data suggest that electrogenic Na+ transport due to Na+,K+-ATPase activity contributes to the interior-positive membrane potential, thereby reducing ATP-dependent H+ transport. Importantly, inhibition of acidification by Na+,K+-ATPase activity was found only in early endosomes, consistent with their limited acidification capacity relative to late endosomes and lysosomes.

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Selected References

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  1. Basu S. K., Goldstein J. L., Brown M. S. Characterization of the low density lipoprotein receptor in membranes prepared from human fibroblasts. J Biol Chem. 1978 Jun 10;253(11):3852–3856. [PubMed] [Google Scholar]
  2. Cain C. C., Murphy R. F. A chloroquine-resistant Swiss 3T3 cell line with a defect in late endocytic acidification. J Cell Biol. 1988 Feb;106(2):269–277. doi: 10.1083/jcb.106.2.269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cain C. C., Sipe D. M., Murphy R. F. Regulation of endocytic pH by the Na+,K+-ATPase in living cells. Proc Natl Acad Sci U S A. 1989 Jan;86(2):544–548. doi: 10.1073/pnas.86.2.544. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Forgac M., Cantley L., Wiedenmann B., Altstiel L., Branton D. Clathrin-coated vesicles contain an ATP-dependent proton pump. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1300–1303. doi: 10.1073/pnas.80.5.1300. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Galloway C. J., Dean G. E., Marsh M., Rudnick G., Mellman I. Acidification of macrophage and fibroblast endocytic vesicles in vitro. Proc Natl Acad Sci U S A. 1983 Jun;80(11):3334–3338. doi: 10.1073/pnas.80.11.3334. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Glickman J., Croen K., Kelly S., Al-Awqati Q. Golgi membranes contain an electrogenic H+ pump in parallel to a chloride conductance. J Cell Biol. 1983 Oct;97(4):1303–1308. doi: 10.1083/jcb.97.4.1303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Goodno C. C. Myosin active-site trapping with vanadate ion. Methods Enzymol. 1982;85(Pt B):116–123. doi: 10.1016/0076-6879(82)85014-3. [DOI] [PubMed] [Google Scholar]
  8. Harikumar P., Reeves J. P. The lysosomal proton pump is electrogenic. J Biol Chem. 1983 Sep 10;258(17):10403–10410. [PubMed] [Google Scholar]
  9. Hoflack B., Fujimoto K., Kornfeld S. The interaction of phosphorylated oligosaccharides and lysosomal enzymes with bovine liver cation-dependent mannose 6-phosphate receptor. J Biol Chem. 1987 Jan 5;262(1):123–129. [PubMed] [Google Scholar]
  10. Jesaitis A. J., Yguerabide J. The lateral mobility of the (Na+,K+)-dependent ATPase in Madin-Darby canine kidney cells. J Cell Biol. 1986 Apr;102(4):1256–1263. doi: 10.1083/jcb.102.4.1256. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Jørgensen P. L. Structure, function and regulation of Na,K-ATPase in the kidney. Kidney Int. 1986 Jan;29(1):10–20. doi: 10.1038/ki.1986.3. [DOI] [PubMed] [Google Scholar]
  12. Kielian M. C., Marsh M., Helenius A. Kinetics of endosome acidification detected by mutant and wild-type Semliki Forest virus. EMBO J. 1986 Dec 1;5(12):3103–3109. doi: 10.1002/j.1460-2075.1986.tb04616.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Marsh M., Griffiths G., Dean G. E., Mellman I., Helenius A. Three-dimensional structure of endosomes in BHK-21 cells. Proc Natl Acad Sci U S A. 1986 May;83(9):2899–2903. doi: 10.1073/pnas.83.9.2899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Marsh M., Schmid S., Kern H., Harms E., Male P., Mellman I., Helenius A. Rapid analytical and preparative isolation of functional endosomes by free flow electrophoresis. J Cell Biol. 1987 Apr;104(4):875–886. doi: 10.1083/jcb.104.4.875. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Maxfield F. R. Weak bases and ionophores rapidly and reversibly raise the pH of endocytic vesicles in cultured mouse fibroblasts. J Cell Biol. 1982 Nov;95(2 Pt 1):676–681. doi: 10.1083/jcb.95.2.676. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Mellman I., Fuchs R., Helenius A. Acidification of the endocytic and exocytic pathways. Annu Rev Biochem. 1986;55:663–700. doi: 10.1146/annurev.bi.55.070186.003311. [DOI] [PubMed] [Google Scholar]
  17. Merion M., Schlesinger P., Brooks R. M., Moehring J. M., Moehring T. J., Sly W. S. Defective acidification of endosomes in Chinese hamster ovary cell mutants "cross-resistant" to toxins and viruses. Proc Natl Acad Sci U S A. 1983 Sep;80(17):5315–5319. doi: 10.1073/pnas.80.17.5315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Murphy R. F., Powers S., Cantor C. R. Endosome pH measured in single cells by dual fluorescence flow cytometry: rapid acidification of insulin to pH 6. J Cell Biol. 1984 May;98(5):1757–1762. doi: 10.1083/jcb.98.5.1757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ohkuma S., Moriyama Y., Takano T. Identification and characterization of a proton pump on lysosomes by fluorescein-isothiocyanate-dextran fluorescence. Proc Natl Acad Sci U S A. 1982 May;79(9):2758–2762. doi: 10.1073/pnas.79.9.2758. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Ohkuma S., Poole B. Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents. Proc Natl Acad Sci U S A. 1978 Jul;75(7):3327–3331. doi: 10.1073/pnas.75.7.3327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Pollack L. R., Tate E. H., Cook J. S. Turnover and regulation of Na-K-ATPase in HeLa cells. Am J Physiol. 1981 Nov;241(5):C173–C183. doi: 10.1152/ajpcell.1981.241.5.C173. [DOI] [PubMed] [Google Scholar]
  22. Robbins A. R., Oliver C., Bateman J. L., Krag S. S., Galloway C. J., Mellman I. A single mutation in Chinese hamster ovary cells impairs both Golgi and endosomal functions. J Cell Biol. 1984 Oct;99(4 Pt 1):1296–1308. doi: 10.1083/jcb.99.4.1296. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Roff C. F., Fuchs R., Mellman I., Robbins A. R. Chinese hamster ovary cell mutants with temperature-sensitive defects in endocytosis. I. Loss of function on shifting to the nonpermissive temperature. J Cell Biol. 1986 Dec;103(6 Pt 1):2283–2297. doi: 10.1083/jcb.103.6.2283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Schmid S. L., Fuchs R., Male P., Mellman I. Two distinct subpopulations of endosomes involved in membrane recycling and transport to lysosomes. Cell. 1988 Jan 15;52(1):73–83. doi: 10.1016/0092-8674(88)90532-6. [DOI] [PubMed] [Google Scholar]
  25. Stone D. K., Xie X. S., Racker E. An ATP-driven proton pump in clathrin-coated vesicles. J Biol Chem. 1983 Apr 10;258(7):4059–4062. [PubMed] [Google Scholar]
  26. Tanasugarn L., McNeil P., Reynolds G. T., Taylor D. L. Microspectrofluorometry by digital image processing: measurement of cytoplasmic pH. J Cell Biol. 1984 Feb;98(2):717–724. doi: 10.1083/jcb.98.2.717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Yamashiro D. J., Maxfield F. R. Kinetics of endosome acidification in mutant and wild-type Chinese hamster ovary cells. J Cell Biol. 1987 Dec;105(6 Pt 1):2713–2721. doi: 10.1083/jcb.105.6.2713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Yamashiro D. J., Tycko B., Fluss S. R., Maxfield F. R. Segregation of transferrin to a mildly acidic (pH 6.5) para-Golgi compartment in the recycling pathway. Cell. 1984 Jul;37(3):789–800. doi: 10.1016/0092-8674(84)90414-8. [DOI] [PubMed] [Google Scholar]
  29. van Renswoude J., Bridges K. R., Harford J. B., Klausner R. D. Receptor-mediated endocytosis of transferrin and the uptake of fe in K562 cells: identification of a nonlysosomal acidic compartment. Proc Natl Acad Sci U S A. 1982 Oct;79(20):6186–6190. doi: 10.1073/pnas.79.20.6186. [DOI] [PMC free article] [PubMed] [Google Scholar]

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