V-ATPase interacts with ARNO and Arf6 in early endosomes and regulates the protein degradative pathway


The recruitment of the small GTPase Arf6 and ARNO from cytosol to endosomal membranes is driven by V-ATPase-dependent intra-endosomal acidification. The molecular mechanism that mediates this pH-sensitive recruitment and its role are unknown. Here, we demonstrate that Arf6 interacts with the c-subunit, and ARNO with the a2-isoform of V-ATPase. The a2-isoform is targeted to early endosomes, interacts with ARNO in an intra-endosomal acidification-dependent manner, and disruption of this interaction results in reversible inhibition of endocytosis. Inhibition of endosomal acidification abrogates protein trafficking between early and late endosomal compartments. These data demonstrate the crucial role of early endosomal acidification and V-ATPase/ARNO/Arf6 interactions in the regulation of the endocytic degradative pathway. They also indicate that V-ATPase could modulate membrane trafficking by recruiting and interacting with ARNO and Arf6; characteristics that are consistent with the role of V-ATPase as an essential component of the endosomal pH-sensing machinery.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Expression, distribution and targeting of a-isoforms of V-ATPase in kidney proximal tubule epithelial cells in situ.
Figure 2: The a2-isoform of V-ATPase is targeted to early endosomes.
Figure 3: Endosomal V-ATPase directly interacts with cytosolic ARNO and Arf6.
Figure 4: Vesicular trafficking in proximal tubule cells is an acidification-dependent process that correlates with intra-endosomal acidification-dependent V-ATPase–ARNO interaction.
Figure 5: Overexpression of soluble cytosolic tail of the a2-isoform (a2N) inhibits receptor-mediated endocytosis in MTC cells.
Figure 6: An interaction-competent mutant of ARNO rescues MTC cells from the inhibitory effect of a2N on receptor-mediated protein endocytosis.
Figure 7: Differential role of V-ATPase-driven endosomal acidification on vesicular trafficking via recycling and degradative pathways during endocytosis.
Figure 8: Model of the novel role of V-ATPase as an essential component of the endosomal pH-sensing machinery.


  1. 1

    Sorkin, A. & Von Zastrow, M. Signal transduction and endocytosis: close encounters of many kinds. Nature Rev. Mol. Cell. Biol. 3, 600–614 (2002).

    CAS  Article  Google Scholar 

  2. 2

    Bonifacino, J.S. & Glick, B.S. The mechanisms of vesicle budding and fusion. Cell 116, 153–166 (2004).

    CAS  Article  Google Scholar 

  3. 3

    Kirchhausen, T. Three ways to make a vesicle. Nature Rev. Mol. Cell. Biol. 1, 187–198 (2000).

    CAS  Article  Google Scholar 

  4. 4

    Donaldson, J. G. Multiple roles for Arf6: Sorting, structuring, and signaling plasma membrane. J. Biol. Chem. 278, 41573–41576 (2003).

    CAS  Article  Google Scholar 

  5. 5

    D'Souza-Schorey, C., Li, G., Colombo, M.I. & Stahl, P.D. A regulatory role for ARF6 in receptor-mediated endocytosis. Science 267, 1175–1178 (1995).

    CAS  Article  Google Scholar 

  6. 6

    Paleotti, O. et al. The small G-protein Arf6GTP recruits the AP-2 adaptor complex to membranes. J. Biol. Chem. 280, 21661–21665 (2005).

    CAS  Article  Google Scholar 

  7. 7

    Peters, P.J. et al. Characterization of coated vesicles that participate in endocytic recycling. Traffic 2, 885–895 (2001).

    CAS  Article  Google Scholar 

  8. 8

    Radhakrishna, H., Klausner, R.D. & Donaldson, J.G. Aluminumfluoride stimulates surface protrusions in cells overexpressing the ARF6 GTPase. J. Cell. Biol. 134, 935–947 (1996).

    CAS  Article  Google Scholar 

  9. 9

    Frank, S.R., Hatfield, J.C. & Casanova, J.E. Remodeling of the actin cytoskeleton is coordinately regulated by protein kinase C and the ADP-ribosylation factor nucleotide exchange factor ARNO. Mol. Biol. Cell. 9, 3133–3146 (1998).

    CAS  Article  Google Scholar 

  10. 10

    Massenburg, D. et al. Activation of rat brain phospholipase D by ADP-ribosylation factors 1,5, and 6: separation of ADP-ribosylation factor-dependent and oleate-dependent enzymes. Proc. Natl Acad. Sci. USA 91, 11718–11722 (1994).

    CAS  Article  Google Scholar 

  11. 11

    Santy, L.C. & Casanova, J.E. Activation of ARF6 by ARNO stimulates epithelial cell migration through downstream activation of both Rac1 and phospholipase D. J. Cell. Biol. 154, 599–610 (2001).

    CAS  Article  Google Scholar 

  12. 12

    Mellman, I. The importance of being acid: the role of acidification in intracellular membrane traffic. J. Exp. Biol. 172, 39–45 (1992).

    CAS  PubMed  Google Scholar 

  13. 13

    Mellman, I., Fuchs, R. & Helenius, A. Acidification of the endocytic and exocytic pathways. Annu. Rev. Biochem. 55, 663–700 (1986).

    CAS  Article  Google Scholar 

  14. 14

    Trombetta, E. S., Ebersold, M., Garrett, W., Pypaert, M. & Mellman, I. Activation of lysosomal function during dendritic cell maturation. Science 299, 1400–1403 (2003).

    CAS  Article  Google Scholar 

  15. 15

    Nishi, T. & Forgac, M. The vacuolar (H+)-ATPases-nature's most versatile proton pumps. Nature Rev. Mol. Cell. Biol. 3, 94–103 (2002).

    CAS  Article  Google Scholar 

  16. 16

    Sun-Wada, G.H., Wada Y. & Futai, M. Lysosome and lysosome-related organelles responsible for specialized functions in higher organisms, with special emphasis on vacuolar-type proton ATPase. Cell. Struct. Funct. 28, 455–463 (2003).

    CAS  Article  Google Scholar 

  17. 17

    Sun-Wada, G.H., Wada Y. & Futai, M. Diverse and essential roles of mammalian vacuolar-type proton pump ATPase: toward the physiological understanding of inside acidic compartments. Biochim. Biophys. Acta 1658, 106–114 (2004).

    CAS  Article  Google Scholar 

  18. 18

    Toyomura, T., Oka, T., Yamaguchi, C., Wada, Y. & Futai, M. Three subunit a isoforms of mouse vacuolar H(+)-ATPase. Preferential expression of the a3 isoform during osteoclast differentiation. J. Biol. Chem. 275, 8760–8765 (2000).

    CAS  Article  Google Scholar 

  19. 19

    Oka, T. et al. a4, a unique kidney-specific isoform of mouse vacuolar H+ATPase subunit a. J. Biol. Chem. 276, 40050–40054 (2001).

    CAS  Article  Google Scholar 

  20. 20

    Toyomura, T. et al. From lysosomes to the plasma membrane: localization of vacuolar-type H+ATPase with the a3 isoform during osteoclast differentiation. J. Biol. Chem. 278, 22023–22030 (2003).

    CAS  Article  Google Scholar 

  21. 21

    Marshansky, V., Fleser, A., Noel, J., Bourgoin, S. & Vinay, P. Isolation of heavy endosomes from dog proximal tubules in suspension. J. Membr. Biol. 153, 59–73 (1996).

    CAS  Article  Google Scholar 

  22. 22

    Marshansky, V. & Vinay, P. Proton gradient formation in early endosomes from proximal tubules. Biochim. Biophys. Acta 1284, 171–180 (1996).

    Article  Google Scholar 

  23. 23

    Marshansky, V. et al. Identification of ADP-ribosylation factor-6 in brush-border membrane and early endosomes of human kidney proximal tubules. Electrophoresis 18, 538–547 (1997).

    CAS  Article  Google Scholar 

  24. 24

    Marshansky, V. et al. Receptor-mediated endocytosis in kidney proximal tubules: recent advances and hypothesis. Electrophoresis 18, 2661–2676 (1997).

    CAS  Article  Google Scholar 

  25. 25

    Maranda, B. et al. Intra-endosomal pH-sensitive recruitment of the Arf-nucleotide exchange factor ARNO and Arf6 from cytoplasm to proximal tubule endosomes. J. Biol. Chem. 276, 18540–18550 (2001).

    CAS  Article  Google Scholar 

  26. 26

    El Annan, J. et al. Differential expression and targeting of endogenous Arf1 and Arf6 small GTPases in kidney epithelial cell in situ. Am. J. Physiol. (Cell Physiol) 286, C768–C778 (2004).

    CAS  Article  Google Scholar 

  27. 27

    Marshansky, V., Ausiello, D. A. & Brown, D. Physiological importance of endosomal acidification: potential role in proximal tubulopathies. Curr. Opin. Nephrol. Hypertens. 11, 527–537 (2002).

    Article  Google Scholar 

  28. 28

    Brown, D., & Marshansky, V. in Handbook of ATPases. (eds Futai, M., Wada, Y. & Kaplan, J.H.) 413–442 (Wiley-VCH, New York, 2004).

    Google Scholar 

  29. 29

    Zeuzem, S., Zimmermann, P. & Schulz, I. Association of a 19- and a 21-kDa GTP-binding protein to pancreatic microsomal vesicles is regulated by the intravesicular pH established by a vacuolar-type H(+)-ATPase. J. Membr. Biol. 125, 231–241 (1992).

    CAS  Article  Google Scholar 

  30. 30

    Zeuzem, S. et al. Intravesicular acidification correlates with binding of ADP-ribosylation factor to microsomal membranes. Proc. Natl Acad. Sci. USA 89, 6619–6623 (1992).

    CAS  Article  Google Scholar 

  31. 31

    Aniento, F., Gu, F., Parton, R. G. & Gruenberg, J. An endosomal beta COP is involved in the pH-dependent formation of transport vesicles destined for late endosomes. J. Cell Biol. 133, 29–41 (1996).

    CAS  Article  Google Scholar 

  32. 32

    Gu, F. & Gruenberg, J. Biogenesis of transport intermediates in the endocytic pathway. FEBS Lett. 452, 61–66 (1999).

    CAS  Article  Google Scholar 

  33. 33

    Gu, F. & Gruenberg, J. ARF1 regulates pH-dependent COP functions in the early endocytic pathway. J. Biol. Chem. 275, 8154–8160 (2000).

    CAS  Article  Google Scholar 

  34. 34

    Chardin, P. et al. A human exchange factor for ARF contains Sec7- and pleckstrin-homology domains. Nature 384, 481–484 (1996).

    CAS  Article  Google Scholar 

  35. 35

    Mansour, M., Lee, S. Y. & Pohajdak, B. The N-terminal coiled coil domain of the cytohesin/ARNO family of guanine nucleotide exchange factors interacts with the scaffolding protein CASP. J. Biol. Chem. 277, 32302–32309 (2002).

    CAS  Article  Google Scholar 

  36. 36

    Santy, L. C., Frank, S. R., Hatfield, J. C. & Casanova, J. E. Regulation of ARNO nucleotide exchange by a PH domain electrostatic switch. Curr. Biol. 9, 1173–1176 (1999).

    CAS  Article  Google Scholar 

  37. 37

    Sun-Wada, G.H. et al. A proton pump ATPase with testis-specific E1-subunit isoform required for acrosome acidification. J. Biol. Chem. 277, 18098–18105 (2002).

    CAS  Article  Google Scholar 

  38. 38

    D'Souza-Schorey, C. & Stahl, P.D. Myristoylation is required for the intracellular localization and endocytic function of ARF6. Exp. Cell. Res. 221, 153–159 (1995).

    CAS  Article  Google Scholar 

  39. 39

    Christensen, E. I. & Birn, H. Megalin and cubilin: multifunctional endocytotic receptors. Nature Rev. Mol. Cell. Biol. 3, 258–268 (2002).

    Article  Google Scholar 

  40. 40

    Wagner, C. A. et al. Renal vacuolar H+-ATPase. Physiol. Rev. 84, 1263–1314 (2004).

    CAS  Article  Google Scholar 

  41. 41

    Jentsch, T. J., Hubner, C. A. & Fuhrmann, J. C. Ion channels: Function unraveled by dysfunction. Nature Cell. Biol. 6, 1039–1047 (2004).

    CAS  Article  Google Scholar 

  42. 42

    Baravalle, G. et al. Transferrin recycling and dextran transport to lysosomes is differentially affected by bafilomycin, nocodazole, and low temperature. Cell. Tissue Res. 320, 99–113 (2005).

    CAS  Article  Google Scholar 

  43. 43

    Donaldson, J.G., Honda, A. & Weigert, R. Multiple activities for Arf1 at the Golgi complex. Biochim. Biophys. Acta 1744, 364–373 (2005).

    CAS  Article  Google Scholar 

  44. 44

    Shao, E. & Forgac, M. Involvement of the nonhomologous region of subunit A of the yeast V-ATPase in coupling and in vivo dissociation. J. Biol. Chem. 279, 48663–48670 (2004).

    CAS  Article  Google Scholar 

  45. 45

    Radhakrishna, H. & Donaldson, J.G. ADP-ribosylation factor 6 regulates a novel plasma membrane recycling pathway. J. Cell. Biol. 139, 49–61 (1997).

    CAS  Article  Google Scholar 

  46. 46

    Skinner, M.A. & Wildeman, A.G. Suppression of tumor-related glycosylation of cell surface receptors by the 16-kDa membrane subunit of vacuolar H+ATPase. J. Biol. Chem. 276, 48451–48457 (2001).

    CAS  Article  Google Scholar 

  47. 47

    Vinay, P., Gougoux, A. & Lemieux, G. Isolation of a pure suspension of rat proximal tubules. Am. J. Physiol. 241, F403–F411 (1981).

    CAS  PubMed  Google Scholar 

  48. 48

    Sun-Wada, G. H. et al. Mouse proton pump ATPase C subunit isoforms (C2-a and C2-b) specifically expressed in kidney and lung. J. Biol. Chem. 278, 44843–44851 (2003).

    CAS  Article  Google Scholar 

  49. 49

    Zhai X.Y. et al. Cubilin- and megalin-mediated uptake of albumin in cultured proximal tubule cells of opossum kidney. Kidney Int. 58, 1523–1533 (2000).

    CAS  Article  Google Scholar 

  50. 50

    Hryciw, D.H. et al. Cofilin interacts with ClC-5 and regulates albumin uptake in proximal tubule cell lines. J. Biol. Chem. 278, 40169–40176 (2003).

    CAS  Article  Google Scholar 

Download references


We would like to thank C. Reinecker for generously providing Rab7–EGFP and Rab11–EGFP constructs and for advice on Volocity software. We would like to thank J. Donaldson for generously providing the Arf6–HA construct and M.A. Billeter for kindly providing the HEK cell line expressing T7-polymerase. We also wish to thank H. Huang for expert technical assistance on FACS analysis. We are grateful to V. Hsu for critical discussion and reading of the manuscript. M.F. and G.-H.S.-W. were supported by CREST, the Japan Science and Technology Agency. This work was supported by a National Institutes of Health grants (DK38452 and DK42956). The Microscopy Core facility of the MGH Program in Membrane Biology receives additional support from the Boston Area Diabetes and Endocrinology Research Center (DK57521) and the Center for the Study of Inflammatory Bowel Disease (DK43341).

Author information



Corresponding author

Correspondence to Vladimir Marshansky.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Hurtado-Lorenzo, A., Skinner, M., Annan, J. et al. V-ATPase interacts with ARNO and Arf6 in early endosomes and regulates the protein degradative pathway. Nat Cell Biol 8, 124–136 (2006). https://doi.org/10.1038/ncb1348

Download citation

Further reading


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing