Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1989 Feb 1;108(2):377–387. doi: 10.1083/jcb.108.2.377

Low cytoplasmic pH inhibits endocytosis and transport from the trans- Golgi network to the cell surface

PMCID: PMC2115451  PMID: 2918022

Abstract

A fibroblast mutant cell line lacking the Na+/H+ antiporter was used to study the influence of low cytoplasmic pH on membrane transport in the endocytic and exocytic pathways. After being loaded with protons, the mutant cells were acidified at pH 6.2 to 6.8 for 20 min while the parent cells regulated their pH within 1 min. Cytoplasmic acidification did not affect the level of intracellular ATP or the number of clathrin- coated pits at the cell surface. However, cytosolic acidification below pH 6.8 blocked the uptake of two fluid phase markers, Lucifer Yellow and horseradish peroxidase, as well as the internalization and the recycling of transferrin. When the cytoplasmic pH was reversed to physiological values, both fluid phase endocytosis and receptor- mediated endocytosis resumed with identical kinetics. Low cytoplasmic pH also inhibited the rate of intracellular transport from the Golgi complex to the plasma membrane. This was shown in cells infected by the temperature-sensitive mutant ts 045 of the vesicular stomatitis virus (VSV) using as a marker of transport the mutated viral membrane glycoprotein (VSV-G protein). The VSV-G protein was accumulated in the trans-Golgi network (TGN) by an incubation at 19.5 degrees C and was transported to the cell surface upon shifting the temperature to 31 degrees C. This transport was arrested in acidified cells maintained at low cytosolic pH and resumed during the recovery phase of the cytosolic pH. Electron microscopy performed on epon and cryo-sections of mutant cells acidified below pH 6.8 showed that the VSV-G protein was present in the TGN. These results indicate that acidification of the cytosol to a pH less than 6.8 inhibits reversibly membrane transport in both endocytic and exocytic pathways. In all likelihood, the clathrin and nonclathrin coated vesicles that are involved in endo- and exocytosis cannot pinch off from the cell surface or from the TGN below this critical value of internal pH.

Full Text

The Full Text of this article is available as a PDF (3.7 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  2. Ciechanover A., Schwartz A. L., Dautry-Varsat A., Lodish H. F. Kinetics of internalization and recycling of transferrin and the transferrin receptor in a human hepatoma cell line. Effect of lysosomotropic agents. J Biol Chem. 1983 Aug 25;258(16):9681–9689. [PubMed] [Google Scholar]
  3. Dautry-Varsat A., Ciechanover A., Lodish H. F. pH and the recycling of transferrin during receptor-mediated endocytosis. Proc Natl Acad Sci U S A. 1983 Apr;80(8):2258–2262. doi: 10.1073/pnas.80.8.2258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Davoust J., Gruenberg J., Howell K. E. Two threshold values of low pH block endocytosis at different stages. EMBO J. 1987 Dec 1;6(12):3601–3609. doi: 10.1002/j.1460-2075.1987.tb02691.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Doxsey S. J., Brodsky F. M., Blank G. S., Helenius A. Inhibition of endocytosis by anti-clathrin antibodies. Cell. 1987 Jul 31;50(3):453–463. doi: 10.1016/0092-8674(87)90499-5. [DOI] [PubMed] [Google Scholar]
  6. Fuller S. D., Simons K. Transferrin receptor polarity and recycling accuracy in "tight" and "leaky" strains of Madin-Darby canine kidney cells. J Cell Biol. 1986 Nov;103(5):1767–1779. doi: 10.1083/jcb.103.5.1767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Griffiths G., McDowall A., Back R., Dubochet J. On the preparation of cryosections for immunocytochemistry. J Ultrastruct Res. 1984 Oct;89(1):65–78. doi: 10.1016/s0022-5320(84)80024-6. [DOI] [PubMed] [Google Scholar]
  8. Griffiths G., Pfeiffer S., Simons K., Matlin K. Exit of newly synthesized membrane proteins from the trans cisterna of the Golgi complex to the plasma membrane. J Cell Biol. 1985 Sep;101(3):949–964. doi: 10.1083/jcb.101.3.949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Griffiths G., Simons K. The trans Golgi network: sorting at the exit site of the Golgi complex. Science. 1986 Oct 24;234(4775):438–443. doi: 10.1126/science.2945253. [DOI] [PubMed] [Google Scholar]
  10. Griffiths G., Simons K., Warren G., Tokuyasu K. T. Immunoelectron microscopy using thin, frozen sections: application to studies of the intracellular transport of Semliki Forest virus spike glycoproteins. Methods Enzymol. 1983;96:466–485. doi: 10.1016/s0076-6879(83)96041-x. [DOI] [PubMed] [Google Scholar]
  11. Hemmilä I., Dakubu S., Mukkala V. M., Siitari H., Lövgren T. Europium as a label in time-resolved immunofluorometric assays. Anal Biochem. 1984 Mar;137(2):335–343. doi: 10.1016/0003-2697(84)90095-2. [DOI] [PubMed] [Google Scholar]
  12. Hopkins C. R., Trowbridge I. S. Internalization and processing of transferrin and the transferrin receptor in human carcinoma A431 cells. J Cell Biol. 1983 Aug;97(2):508–521. doi: 10.1083/jcb.97.2.508. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kaila K., Voipio J. Postsynaptic fall in intracellular pH induced by GABA-activated bicarbonate conductance. Nature. 1987 Nov 12;330(6144):163–165. doi: 10.1038/330163a0. [DOI] [PubMed] [Google Scholar]
  14. Klausner R. D., Ashwell G., van Renswoude J., Harford J. B., Bridges K. R. Binding of apotransferrin to K562 cells: explanation of the transferrin cycle. Proc Natl Acad Sci U S A. 1983 Apr;80(8):2263–2266. doi: 10.1073/pnas.80.8.2263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. L'Allemain G., Paris S., Pouysségur J. Growth factor action and intracellular pH regulation in fibroblasts. Evidence for a major role of the Na+/H+ antiport. J Biol Chem. 1984 May 10;259(9):5809–5815. [PubMed] [Google Scholar]
  16. L'Allemain G., Paris S., Pouysségur J. Role of a Na+-dependent Cl-/HCO3- exchange in regulation of intracellular pH in fibroblasts. J Biol Chem. 1985 Apr 25;260(8):4877–4883. [PubMed] [Google Scholar]
  17. Lemansky P., Hasilik A., von Figura K., Helmy S., Fishman J., Fine R. E., Kedersha N. L., Rome L. H. Lysosomal enzyme precursors in coated vesicles derived from the exocytic and endocytic pathways. J Cell Biol. 1987 Jun;104(6):1743–1748. doi: 10.1083/jcb.104.6.1743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Melançon P., Glick B. S., Malhotra V., Weidman P. J., Serafini T., Gleason M. L., Orci L., Rothman J. E. Involvement of GTP-binding "G" proteins in transport through the Golgi stack. Cell. 1987 Dec 24;51(6):1053–1062. doi: 10.1016/0092-8674(87)90591-5. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Moolenaar W. H., Tsien R. Y., van der Saag P. T., de Laat S. W. Na+/H+ exchange and cytoplasmic pH in the action of growth factors in human fibroblasts. Nature. 1983 Aug 18;304(5927):645–648. doi: 10.1038/304645a0. [DOI] [PubMed] [Google Scholar]
  21. Orci L., Glick B. S., Rothman J. E. A new type of coated vesicular carrier that appears not to contain clathrin: its possible role in protein transport within the Golgi stack. Cell. 1986 Jul 18;46(2):171–184. doi: 10.1016/0092-8674(86)90734-8. [DOI] [PubMed] [Google Scholar]
  22. Orci L., Halban P., Amherdt M., Ravazzola M., Vassalli J. D., Perrelet A. A clathrin-coated, Golgi-related compartment of the insulin secreting cell accumulates proinsulin in the presence of monensin. Cell. 1984 Nov;39(1):39–47. doi: 10.1016/0092-8674(84)90189-2. [DOI] [PubMed] [Google Scholar]
  23. Orci L., Ravazzola M., Amherdt M., Louvard D., Perrelet A. Clathrin-immunoreactive sites in the Golgi apparatus are concentrated at the trans pole in polypeptide hormone-secreting cells. Proc Natl Acad Sci U S A. 1985 Aug;82(16):5385–5389. doi: 10.1073/pnas.82.16.5385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Paris S., Pouysségur J. Growth factors activate the Na+/H+ antiporter in quiescent fibroblasts by increasing its affinity for intracellular H+. J Biol Chem. 1984 Sep 10;259(17):10989–10994. [PubMed] [Google Scholar]
  25. Pfeffer S. R., Rothman J. E. Biosynthetic protein transport and sorting by the endoplasmic reticulum and Golgi. Annu Rev Biochem. 1987;56:829–852. doi: 10.1146/annurev.bi.56.070187.004145. [DOI] [PubMed] [Google Scholar]
  26. Pouysségur J., Sardet C., Franchi A., L'Allemain G., Paris S. A specific mutation abolishing Na+/H+ antiport activity in hamster fibroblasts precludes growth at neutral and acidic pH. Proc Natl Acad Sci U S A. 1984 Aug;81(15):4833–4837. doi: 10.1073/pnas.81.15.4833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Roos A., Boron W. F. Intracellular pH. Physiol Rev. 1981 Apr;61(2):296–434. doi: 10.1152/physrev.1981.61.2.296. [DOI] [PubMed] [Google Scholar]
  28. Rozengurt E. Early signals in the mitogenic response. Science. 1986 Oct 10;234(4773):161–166. doi: 10.1126/science.3018928. [DOI] [PubMed] [Google Scholar]
  29. Sandvig K., Olsnes S., Petersen O. W., van Deurs B. Acidification of the cytosol inhibits endocytosis from coated pits. J Cell Biol. 1987 Aug;105(2):679–689. doi: 10.1083/jcb.105.2.679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sandvig K., Olsnes S., Petersen O. W., van Deurs B. Inhibition of endocytosis from coated pits by acidification of the cytosol. J Cell Biochem. 1988 Jan;36(1):73–81. doi: 10.1002/jcb.240360108. [DOI] [PubMed] [Google Scholar]
  31. Steinman R. M., Silver J. M., Cohn Z. A. Pinocytosis in fibroblasts. Quantitative studies in vitro. J Cell Biol. 1974 Dec;63(3):949–969. doi: 10.1083/jcb.63.3.949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Swanson J. A., Yirinec B. D., Silverstein S. C. Phorbol esters and horseradish peroxidase stimulate pinocytosis and redirect the flow of pinocytosed fluid in macrophages. J Cell Biol. 1985 Mar;100(3):851–859. doi: 10.1083/jcb.100.3.851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Tooze J., Tooze S. A. Clathrin-coated vesicular transport of secretory proteins during the formation of ACTH-containing secretory granules in AtT20 cells. J Cell Biol. 1986 Sep;103(3):839–850. doi: 10.1083/jcb.103.3.839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. de Curtis I., Howell K. E., Simons K. Isolation of a fraction enriched in the trans-Golgi network from baby hamster kidney cells. Exp Cell Res. 1988 Apr;175(2):248–265. doi: 10.1016/0014-4827(88)90190-5. [DOI] [PubMed] [Google Scholar]
  35. van Adelsberg J., Al-Awqati Q. Regulation of cell pH by Ca+2-mediated exocytotic insertion of H+-ATPases. J Cell Biol. 1986 May;102(5):1638–1645. doi: 10.1083/jcb.102.5.1638. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES