Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Rab5 is necessary for the biogenesis of the endolysosomal system in vivo


An outstanding question is how cells control the number and size of membrane organelles. The small GTPase Rab5 has been proposed to be a master regulator of endosome biogenesis. Here, to test this hypothesis, we developed a mathematical model of endosome dependency on Rab5 and validated it by titrating down all three Rab5 isoforms in adult mouse liver using state-of-the-art RNA interference technology. Unexpectedly, the endocytic system was resilient to depletion of Rab5 and collapsed only when Rab5 decreased to a critical level. Loss of Rab5 below this threshold caused a marked reduction in the number of early endosomes, late endosomes and lysosomes, associated with a block of low-density lipoprotein endocytosis. Loss of endosomes caused failure to deliver apical proteins to the bile canaliculi, suggesting a requirement for polarized cargo sorting. Our results demonstrate for the first time, to our knowledge, the role of Rab5 as an endosome organizer in vivo and reveal the resilience mechanisms of the endocytic system.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Mathematical model of endosome number as function of Rab5 levels.
Figure 2: Time course of Rab5 mRNA depletion and analysis of protein expression in mouse liver in vivo.
Figure 3: Loss of EEA1 structures upon Rab5KD in vivo.
Figure 4: Loss of early endosomes, MVBs and lysosomes upon Rab5KD.
Figure 5: Block of LDL endocytosis upon Rab5KD in primary hepatocytes in vitro.
Figure 6: Rab5KD causes mis-sorting of apical proteins in hepatocytes in vivo.


  1. 1

    Mellman, I. Endocytosis and molecular sorting. Annu. Rev. Cell Dev. Biol. 12, 575–625 (1996)

    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

    Robinson, M. S. Adaptable adaptors for coated vesicles. Trends Cell Biol. 14, 167–174 (2004)

    CAS  Article  Google Scholar 

  4. 4

    Jahn, R. & Scheller, R. H. SNAREs–engines for membrane fusion. Nature Rev. Mol. Cell Biol. 7, 631–643 (2006)

    CAS  Article  Google Scholar 

  5. 5

    Pfeffer, S. R. Unsolved mysteries in membrane traffic. Annu. Rev. Biochem. 76, 629–645 (2007)

    CAS  Article  Google Scholar 

  6. 6

    Gruenberg, J. & Maxfield, F. R. Membrane transport in the endocytic pathway. Curr. Opin. Cell Biol. 7, 552–563 (1995)

    CAS  Article  Google Scholar 

  7. 7

    Rink, J., Ghigo, E., Kalaidzidis, Y. & Zerial, M. Rab conversion as a mechanism of progression from early to late endosomes. Cell 122, 735–749 (2005)

    CAS  Article  Google Scholar 

  8. 8

    Poteryaev, D., Datta, S., Ackema, K., Zerial, M. & Spang, A. Identification of the switch in early-to-late endosome transition. Cell 141, 497–508 (2010)

    CAS  Article  Google Scholar 

  9. 9

    Vonderheit, A. & Helenius, A. Rab7 associates with early endosomes to mediate sorting and transport of Semliki forest virus to late endosomes. PLoS Biol. 3, e233 (2005)

    Article  Google Scholar 

  10. 10

    Gorvel, J. P., Chavrier, P., Zerial, M. & Gruenberg, J. rab5 controls early endosome fusion in vitro . Cell 64, 915–925 (1991)

    CAS  Article  Google Scholar 

  11. 11

    Rybin, V. et al. GTPase activity of Rab5 acts as a timer for endocytic membrane fusion. Nature 383, 266–269 (1996)

    CAS  ADS  Article  Google Scholar 

  12. 12

    Skjeldal, F. M. et al. The fusion of early endosomes induces molecular motor-driven tubule formation and fission. J . Cell Science (22 February 2012)

  13. 13

    Futter, C. E., Connolly, C. N., Cutler, D. F. & Hopkins, C. R. Newly synthesized transferrin receptors can be detected in the endosome before they appear on the cell surface. J. Biol. Chem. 270, 10999–11003 (1995)

    CAS  Article  Google Scholar 

  14. 14

    Harsay, E. & Schekman, R. A subset of yeast vacuolar protein sorting mutants is blocked in one branch of the exocytic pathway. J. Cell Biol. 156, 271–286 (2002)

    CAS  Article  Google Scholar 

  15. 15

    Ang, A. L. et al. Recycling endosomes can serve as intermediates during transport from the Golgi to the plasma membrane of MDCK cells. J. Cell Biol. 167, 531–543 (2004)

    CAS  Article  Google Scholar 

  16. 16

    Weisz, O. A. & Rodriguez-Boulan, E. Apical trafficking in epithelial cells: signals, clusters and motors. J. Cell Sci. 122, 4253–4266 (2009)

    CAS  Article  Google Scholar 

  17. 17

    Golachowska, M. R., Hoekstra, D. & van IJzendoorn, S. C. D. Recycling endosomes in apical plasma membrane domain formation and epithelial cell polarity. Trends Cell Biol. 20, 618–626 (2010)

    CAS  Article  Google Scholar 

  18. 18

    Ihrke, G. et al. Apical plasma membrane proteins and endolyn-78 travel through a subapical compartment in polarized WIF-B hepatocytes. J. Cell Biol. 141, 115–133 (1998)

    CAS  Article  Google Scholar 

  19. 19

    Cresawn, K. O. et al. Differential involvement of endocytic compartments in the biosynthetic traffic of apical proteins. EMBO J. 26, 3737–3748 (2007)

    CAS  Article  Google Scholar 

  20. 20

    Nokes, R. L., Fields, I. C., Collins, R. N. & Folsch, H. Rab13 regulates membrane trafficking between TGN and recycling endosomes in polarized epithelial cells. J. Cell Biol. 182, 845–853 (2008)

    CAS  Article  Google Scholar 

  21. 21

    Farr, G. A., Hull, M., Mellman, I. & Caplan, M. J. Membrane proteins follow multiple pathways to the basolateral cell surface in polarized epithelial cells. J. Cell Biol. 186, 269–282 (2009)

    CAS  Article  Google Scholar 

  22. 22

    Zerial, M. & McBride, H. Rab proteins as membrane organizers. Nature Rev. Mol. Cell Biol. 2, 107–117 (2001)

    CAS  Article  Google Scholar 

  23. 23

    Ohya, T. et al. Reconstitution of Rab- and SNARE-dependent membrane fusion by synthetic endosomes. Nature 459, 1091–1097 (2009)

    CAS  ADS  Article  Google Scholar 

  24. 24

    Bucci, C. et al. Co-operative regulation of endocytosis by three Rab5 isoforms. FEBS Lett. 366, 65–71 (1995)

    CAS  Article  Google Scholar 

  25. 25

    Wucherpfennig, T., Wilsch-Brauninger, M. & Gonzalez-Gaitan, M. Role of Drosophila Rab5 during endosomal trafficking at the synapse and evoked neurotransmitter release. J. Cell Biol. 161, 609–624 (2003)

    CAS  Article  Google Scholar 

  26. 26

    Morrison, H. A. et al. Regulation of early endosomal entry by the Drosophila tumor suppressors Rabenosyn and Vps45. Mol. Biol. Cell 19, 4167–4176 (2008)

    CAS  Article  Google Scholar 

  27. 27

    Bucci, C. et al. The small GTPase rab5 functions as a regulatory factor in the early endocytic pathway. Cell 70, 715–728 (1992)

    CAS  Article  Google Scholar 

  28. 28

    Akinc, A. et al. Targeted delivery of RNAi therapeutics with endogenous and exogenous ligand-based mechanisms. Mol. Ther. 18, 1357–1364 (2010)

    CAS  Article  Google Scholar 

  29. 29

    Del Conte-Zerial, P. et al. Membrane identity and GTPase cascades regulated by toggle and cut-out switches. Mol. Syst. Biol. 4, 206 (2008)

    Article  Google Scholar 

  30. 30

    Heinrich, R. & Rapoport, T. A. Generation of nonidentical compartments in vesicular transport systems. J. Cell Biol. 168, 271–280 (2005)

    CAS  Article  Google Scholar 

  31. 31

    Serio, G. et al. Small GTPase Rab5 participates in chromosome congression and regulates localization of the centromere-associated protein CENP-F to kinetochores. Proc. Natl Acad. Sci. USA 108, 17337–17342 (2011)

    CAS  ADS  Article  Google Scholar 

  32. 32

    Akinc, A. et al. A combinatorial library of lipid-like materials for delivery of RNAi therapeutics. Nature Biotechnol. 26, 561–569 (2008)

    CAS  Article  Google Scholar 

  33. 33

    Love, K. T. et al. Lipid-like materials for low-dose, in vivo gene silencing. Proc. Natl Acad. Sci. USA 107, 1864–1869 (2010)

    CAS  ADS  Article  Google Scholar 

  34. 34

    Christoforidis, S., McBride, H. M., Burgoyne, R. D. & Zerial, M. The Rab5 effector EEA1 is a core component of endosome docking. Nature 397, 621–625 (1999)

    CAS  ADS  Article  Google Scholar 

  35. 35

    Collinet, C. et al. Systems survey of endocytosis by multiparametric image analysis. Nature 464, 243–249 (2010)

    CAS  ADS  Article  Google Scholar 

  36. 36

    Chen, P. I., Kong, C., Su, X. & Stahl, P. D. Rab5 isoforms differentially regulate the trafficking and degradation of epidermal growth factor receptors. J. Biol. Chem. 284, 30328–30338 (2009)

    CAS  Article  Google Scholar 

  37. 37

    Mukherjee, S., Ghosh, R. N. & Maxfield, F. R. Endocytosis. Physiol. Rev. 77, 759–803 (1997)

    CAS  Article  Google Scholar 

  38. 38

    Schroeder, B. M. M. in The Liver: Biology and Pathobiology 5th edn (eds Arias, I. et al.) Ch. 7 107–123 (Wiley-Blackwell, 2009)

    Book  Google Scholar 

  39. 39

    Wolfsdorf, J. I. & Weinstein, D. A. Glycogen storage diseases. Rev. Endocr. Metab. Disord. 4, 95–102 (2003)

    CAS  Article  Google Scholar 

  40. 40

    van der Bliek, A. M. et al. Mutations in human dynamin block an intermediate stage in coated vesicle formation. J. Cell Biol. 122, 553–563 (1993)

    CAS  Article  Google Scholar 

  41. 41

    Shen, H. et al. Constitutive activated Cdc42-associated kinase (Ack) phosphorylation at arrested endocytic clathrin-coated pits of cells that lack dynamin. Mol. Biol. Cell 22, 493–502 (2011)

    CAS  Article  Google Scholar 

  42. 42

    Kipp, H., Pichetshote, N. & Arias, I. M. Transporters on demand: intrahepatic pools of canalicular ATP binding cassette transporters in rat liver. J. Biol. Chem. 276, 7218–7224 (2001)

    CAS  Article  Google Scholar 

  43. 43

    Wang, L. & Boyer, J. L. The maintenance and generation of membrane polarity in hepatocytes. Hepatology 39, 892–899 (2004)

    Article  Google Scholar 

  44. 44

    Wakabayashi, Y., Dutt, P., Lippincott-Schwartz, J. & Arias, I. M. Rab11a and myosin Vb are required for bile canalicular formation in WIF-B9 cells. Proc. Natl Acad. Sci. USA 102, 15087–15092 (2005)

    CAS  ADS  Article  Google Scholar 

  45. 45

    Lawe, D. C. et al. Sequential roles for phosphatidylinositol 3-phosphate and Rab5 in tethering and fusion of early endosomes via their interaction with EEA1. J. Biol. Chem. 277, 8611–8617 (2002)

    CAS  Article  Google Scholar 

  46. 46

    Renaud, G., Hamilton, R. L. & Havel, R. J. Hepatic metabolism of colloidal gold-low-density lipoprotein complexes in the rat: evidence for bulk excretion of lysosomal contents into bile. Hepatology 9, 380–392 (1989)

    CAS  Article  Google Scholar 

  47. 47

    Damke, H., Baba, T., van der Bliek, A. M. & Schmid, S. L. Clathrin-independent pinocytosis is induced in cells overexpressing a temperature-sensitive mutant of dynamin. J. Cell Biol. 131, 69–80 (1995)

    CAS  Article  Google Scholar 

  48. 48

    Koenig, J. H. & Ikeda, K. Transformational process of the endosomal compartment in nephrocytes of Drosophila melanogaster . Cell Tissue Res. 262, 233–244 (1990)

    CAS  Article  Google Scholar 

  49. 49

    Wakabayashi, Y., Lippincott-Schwartz, J. & Arias, I. M. Intracellular trafficking of bile salt export pump (ABCB11) in polarized hepatic cells: constitutive cycling between the canalicular membrane and rab11-positive endosomes. Mol. Biol. Cell 15, 3485–3496 (2004)

    CAS  Article  Google Scholar 

Download references


We acknowledge K. Simons, E. Knust, C. Fetzer, T. Galvez, G. O’Sullivan and M. P. McShane for discussions and comments on the manuscript. We thank W. John and A. Muench-Wuttke from the Biomedical Services Facility for mouse care and injections. We acknowledge J. Peychl for the management of the Light Microscopy Facility and K. Manygoats as well as J.-M. Verbavatz for their support. We thank B. Bettencourt and J. Hettinger for siRNA design and serum biochemistry assays, respectively. This work was financially supported by the Virtual Liver initiative (, funded by the German Federal Ministry of Research and Education (BMBF), the Max Planck Society (MPG) and the DFG. A.Z. was supported by a grant from Marie Curie Action, Intra-European Fellowship (fp7-people-ief-2008) and J.G. from an EMBO long-term fellowship.

Author information




M.Z. and V.K. conceived the project and M.Z. directed it. A.Z. designed and directed the animal injection experiments, and performed the stainings and imaging of liver sections. J.G. and A.Z. co-developed the staining procedures for the tissue sections. J.G.H. helped A.Z. to establish the hepatocyte isolation technique. A.Z. established the primary culture, developed the endocytosis assays, staining protocols and the knockdown technique with LNPs in primary hepatocytes. J.G. performed the electron microscopy analysis and quantifications, and the sectioning of liver tissue. R.L.B. selected siRNAs and validated their efficacy and specificity in vitro and in vivo, and designed, performed and analysed some in vivo experiments. H.E.-B. prepared the siRNAs into lipidoid-based formulations and analysed them. S.K. and C.G.P. performed the synthesis and analysis of siRNAs. V.M.R. designed, performed and analysed the 5′RACE assay. H.N. performed the RT–PCR in primary hepatocytes. S.S. under the supervision of A.Z. and J.G. performed the western blot analysis and performed the hepatocyte isolation and primary culturing under the supervision of A.Z.; G.M. under the supervision of Y.K. adapted the QMPIA for primary hepatocytes and liver tissue and performed the image analysis. Y.K. and P.dC.-Z. developed the mathematical model. M.Z., Y.K., A.Z. and J.G. wrote the manuscript, R.L.B. and V.K. participated in the editing.

Corresponding author

Correspondence to Marino Zerial.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-11, Supplementary Methods, Supplementary Model Descriptions, a Supplementary Discussion and Supplementary References. (PDF 2944 kb)

Supplementary Data 1

This zipped fie contains Supplementary Data. (ZIP 12403 kb)

Supplementary Data 2

This zipped fie contains Supplementary Data. (ZIP 38 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Zeigerer, A., Gilleron, J., Bogorad, R. et al. Rab5 is necessary for the biogenesis of the endolysosomal system in vivo. Nature 485, 465–470 (2012).

Download citation

Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


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