Histochemistry and Cell Biology

, Volume 121, Issue 6, pp 429–439 | Cite as

Endosomal ricin transport: involvement of Rab4- and Rab5-positive compartments

  • Mihail Moisenovich
  • Alex Tonevitsky
  • Natalia Maljuchenko
  • Natalia Kozlovskaya
  • Igor Agapov
  • Walter Volknandt
  • Jürgen Bereiter-HahnEmail author
Original Paper


Transport of the ribosome-inactivating protein ricin through endosomes was studied in A431 cells expressing Rab5-, Rab4-, and Rab11-GFP. It was shown that Rab5- and Rab4-positive functional domains of early endosomes are involved in ricin transport. Ricin enters cells by both clathrin-dependent and clathrin-independent mechanisms. The main pool of internalized toxin accumulates in early endosomes and remains associated with them for a long time. In contrast to earlier observations, current observations indicate that the majority of ricin avoids transport to lysosomes. The low level of ricin association with Rab11 as well as with transferrin accumulated in the pericentriolar recycling compartment shows that the compartment is not responsible for keeping ricin away from degradation in lysosomes. Escape from degradation in lysosomes is assumed to result from the potentiality of ricin to form assemblies within compartments.


Endocytosis Ricin Transferrin Recycling Rab proteins Lysosome 



We would like to thank Dr. Zerial for kindly providing the Rab constructs. This work was supported by the Russian Foundation for Basic Research grant 03-04-49278, Internationales Büro des BMBF Rus 01-239.


  1. Agapov II, Tonevitsky AG, Shamshiev AT, Pohl E, Pohl P, Palmer RA, Kirpichnikov MP (1997) The role of structural domains in RIP II toxin model membrane binding. FEBS Lett 402:91–93CrossRefPubMedGoogle Scholar
  2. Babst M, Odorizzi G, Estepa EJ, Emr SD (2000) Mammalian tumor susceptibility gene 101 (TSG101) and the yeast homologue,Vps23p, both function in late endosomal trafficking. Traffic 1:248–258CrossRefPubMedGoogle Scholar
  3. Bao J, Alroy I, Waterman H, Schejter ED, Brodie C, Gruenberg J, Yarden Y (2000) Threonine phosphorylation diverts internalized epidermal growth factor receptors from a degradative pathway to the recycling endosome. J Biol Chem 275:26178–26186CrossRefPubMedGoogle Scholar
  4. Barbieri L, Battelli MG, Stirpe F (1993) Ribosome-inactivating proteins from plants. Biochim Biophys Acta 1154:237–282PubMedGoogle Scholar
  5. Benlimame N, Le PU, Nabi IR (1998) Localization of autocrine motility factor receptor to caveolae and clathrin-independent internalization of its ligand to smooth endoplasmic reticulum. Mol Biol Cell 9:1773–1786PubMedGoogle Scholar
  6. Ben-Zeev O, Mao HZ, Doolittle MH (2002) Maturation of lipoprotein lipase in the endoplasmic reticulum. Concurrent formation of functional dimers and inactive aggregates. J Biol Chem 277:10727–10738PubMedGoogle Scholar
  7. Birkeli KA, Llorente A, Torgersen ML, Keryer G, Tasken K, Sandvig K (2003) Endosome-to-Golgi transport is regulated by protein kinase A type II alpha. J Biol Chem 278:1991–1997CrossRefPubMedGoogle Scholar
  8. Christoforidis S, McBride HM, Burgoyne RD, Zerial M (1999) The Rab5 effector EEA1 is a core component of endosome docking. Nature 397:621–625CrossRefPubMedGoogle Scholar
  9. Day PJ, Owens SR, Wesche J, Olsnes S, Roberts LM, Lord JM (2001) An interaction between ricin and calreticulin that may have implications for toxin trafficking. J Biol Chem 276:7202–7208CrossRefPubMedGoogle Scholar
  10. Day PJ, Pinheiro TJ, Roberts LM, Lord JM (2002) Binding of ricin A-chain to negatively charged phospholipid vesicles leads to protein structural changes and destabilizes the lipid bilayer. Biochemistry 41:2836–2843CrossRefPubMedGoogle Scholar
  11. Gibson A, Futter CE, Maxwell S, Allchin EH, Shipman M, Kraehenbuhl JP, Domingo D, Odorizzi G, Trowbridge IS, Hopkins CR (1998) Sorting mechanisms regulating membrane protein traffic in the apical transcytotic pathway of polarized MDCK cells. J Cell Biol 143:81–94Google Scholar
  12. Gournier H, Stenmark H, Rybin V, Lippe R, Zerial M (1998) Two distinct effectors of the small GTPase Rab5 cooperate in endocytic membrane fusion. EMBO J 17:1930–1940CrossRefPubMedGoogle Scholar
  13. Grimmer S, Iversen TG, van Deurs B, Sandvig K (2000) Endosome to Golgi transport of ricin is regulated by cholesterol. Mol Biol Cell 11:4205–4216PubMedGoogle Scholar
  14. Hazes B, Read RJ (1997) Accumulating evidence suggests that several AB-toxins subvert the endoplasmic reticulum-associated protein degradation pathway to enter target cells. Biochemistry 36:11051–11054CrossRefPubMedGoogle Scholar
  15. Iversen TG, Skretting G, Llorente A, Nicoziani P, Deurs B, Sandvig K (2001) Endosome to Golgi transport of ricin is independent of clathrin and of the Rab9- and Rab11-GTPases. Mol Biol Cell 12:2099–2107PubMedGoogle Scholar
  16. Lauvrak SU, Llorente A, Iversen TG, Sandvig K (2002) Selective regulation of the Rab9-independent transport of ricin to the Golgi apparatus by calcium. J Cell Sci 115:3449–3456Google Scholar
  17. Llorente A, Rapak A, Schmid SL, van Deurs B, Sandvig K (1998) Expression of mutant dynamin inhibits toxicity and transport of endocytosed ricin to the Golgi apparatus. J Cell Biol 140:553–563Google Scholar
  18. Lombard S, Helmy ME, Pieroni G (2001) Lipolytic activity of ricin from Ricinus sanguineus and Ricinus communis on neutral lipids. Biochem J 358:773–781CrossRefPubMedGoogle Scholar
  19. Lord JM, Roberts LM (1998) Toxin entry: retrograde transport through the secretory pathway. J Cell Biol 140:733–736CrossRefPubMedGoogle Scholar
  20. McCaffrey MW, Bielli A, Cantalupo G, Mora S, Roberti V, Santillo M, Drummond F, Bucci C (2001) Rab4 affects both recycling and degradative endosomal trafficking. FEBS Lett 495:21–30PubMedGoogle Scholar
  21. Mellman I (1996) Endocytosis and molecular sorting. Annu Rev Cell Dev Biol 12:575–625Google Scholar
  22. Miaczynska M, Zerial M (2002) Mosaic organization of the endocytic pathway. Exp Cell Res 272:8–14CrossRefPubMedGoogle Scholar
  23. Mohrmann K, van der Sluijs P (1999) Regulation of membrane transport through the endocytic pathway by rabGTPases. Mol Membr Biol 16:81–87CrossRefPubMedGoogle Scholar
  24. Mohrmann K, Gerez L, Oorschot V, Klumperman J, Sluijs P (2002) Rab4 function in membrane recycling from early endosomes depends on a membrane to cytoplasm cycle. J Biol Chem 277:32029–32035CrossRefPubMedGoogle Scholar
  25. Moisenovich M, Tonevitsky A, Agapov I, Niwa H, Schewe H, Bereiter-Hahn J (2002) Differences in endocytosis and intracellular sorting of ricin and viscumin in 3T3 cells. Eur J Cell Biol 81:529–538Google Scholar
  26. Moisenovich M, Agapov I, Marx U, Bereiter-Hahn J, Tonevitsky A (2003) Intracellular transport of plant toxins ricin and viscumin from different plasma membrane sites. Arzneimittelforschung/Drug Research 53:393–476Google Scholar
  27. Morlon-Guyot J, Helmy M, Lombard-Frasca S, Pignol D, Pieroni G, Beaumelle B (2003) Identification of the ricin lipase site and implication in cytotoxicity. J Biol Chem 278:17006–17011CrossRefPubMedGoogle Scholar
  28. Mukherjee S, Ghosh RN, Maxfield FR (1997) Endocytosis. Physiol Rev 77:759–803PubMedGoogle Scholar
  29. Nichols BJ, Kenworthy AK, Polishchuk RS, Lodge R, Roberts TH, Hirschberg K, Phair RD, Lippincott-Schwartz J (2001) Rapid cycling of lipid raft markers between the cell surface and Golgi complex. J Cell Biol 153:529–541PubMedGoogle Scholar
  30. Nielsen E, Severin F, Backer JM, Hyman AA, Zerial M (1999) Rab5 regulates motility of early endosomes on microtubules. Nat Cell Biol 1:376–382CrossRefPubMedGoogle Scholar
  31. Pelkmans L, Helenius A (2002) Endocytosis via caveolae. Traffic 3:311–320CrossRefPubMedGoogle Scholar
  32. Poussin C, Foti M, Carpentier JL, Pugin J (1998) CD14-dependent endotoxin internalization via a macropinocytic pathway. J Biol Chem 273:20285–20291CrossRefPubMedGoogle Scholar
  33. Ramalingam TS, Das PK, Podder SK (1994) Ricin-membrane interaction: membrane penetration depth by fluorescence quenching and resonance energy transfer. Biochemistry 33:12247–12254PubMedGoogle Scholar
  34. Rapak A, Falnes PO, Olsnes S (1997) Retrograde transport of mutant ricin to the endoplasmic reticulum with subsequent translocation to cytosol. Proc Natl Acad Sci U S A 94:3783–3788Google Scholar
  35. Ren M, Xu G, Zeng J, Lemos-Chiarandini C, Adesnik M, Sabatini D (1998) Hydrolysis of GTP on rab11 is required for the direct delivery of transferrin from the pericentriolar recycling compartment to the cell surface but not from sorting endosomes. Proc Natl Acad Sci U S A 95:6187–6192CrossRefPubMedGoogle Scholar
  36. Renzis S, Sonnichsen B, Zerial M (2002) Divalent Rab effectors regulate the sub-compartmental organization and sorting of early endosomes. Nat Cell Biol 4:124–133PubMedGoogle Scholar
  37. Rodman J, Wandinger-Ness A (2000) Rab GTPases coordinate endocytosis. J Cell Sci 113:183–192Google Scholar
  38. Sachse M, Urbe S, Oorschot V, Strous GJ, Klumperman J (2002) Bilayered clathrin coats on endosomal vacuoles are involved in protein sorting toward lysosomes. Mol Biol Cell 13:1313–1328CrossRefPubMedGoogle Scholar
  39. Sandvig K, van Deurs B (1994) Endocytosis and intracellular sorting of ricin and Shiga toxin. FEBS Lett 346:99–102CrossRefPubMedGoogle Scholar
  40. Sandvig K, van Deurs B (1996) Endocytosis, intracellular transport, and cytotoxic action of Shiga toxin and ricin. Physiol Rev 76:949–966PubMedGoogle Scholar
  41. Sandvig K, van Deurs B (2000) Entry ricin and Shiga toxin into cells: molecular mechanisms and medical perspectives. EMBO J 19:5943–5950CrossRefPubMedGoogle Scholar
  42. Sandvig K, Prydz K, Hansen SH, van Deurs B (1991) Ricin transport in brefeldin A-treated cells: correlation between Golgi structure and toxic effect. J Cell Biol 115:971–981PubMedGoogle Scholar
  43. Sandvig K, Grimmer S, Lauvrak SU, Torgersen ML, Skretting G, van Deurs B, Iversen TG (2002) Pathways followed by ricin and Shiga toxin into cells. Histochem Cell Biol 117:131–141CrossRefPubMedGoogle Scholar
  44. Sheff DR, Daro EA, Hull M, Mellman I (1999) The receptor recycling pathway contains two distinct populations of early endosomes with different sorting functions. J Cell Biol 145:123–139PubMedGoogle Scholar
  45. Sheff D, Pelletier L, O’Connell CB, Warren G, Mellman I (2002) Transferrin receptor recycling in the absence of perinuclear recycling endosomes. J Cell Biol 156:797–804Google Scholar
  46. Sönnichsen B, Renzis S, Nielsen E, Rietdorf J, Zerial M (2000) Distinct membrane domains on endosomes in the recycling pathway visualized by multicolor imaging of rab4, rab5, and rab11. J Cell Biol 149:901–914PubMedGoogle Scholar
  47. Steeves RM, Denton ME, Barnard FC, Henry A, Lambert JM (1999) Identification of three oligosaccharide binding sites in ricin. Biochemistry 38:11677–11685CrossRefPubMedGoogle Scholar
  48. Sweeney EC, Tonevitsky AG, Palmer RA, Niwa H, Pfueller U, Eck J, Lentzen H, Agapov II, Kirpichnikov MP (1998) Mistletoe lectin I forms a double trefoil structure. FEBS Lett 431:367–370CrossRefPubMedGoogle Scholar
  49. Tonevitsky AG, Zhukova OS, Mirimanova NV, Omelyanenko VG, Timofeeva NV, Bergelson LD (1990) Effect of gangliosides on binding, internalization and cytotoxic activity of ricin. FEBS Lett 264:249–252CrossRefPubMedGoogle Scholar
  50. Tonevitsky AG, Marx U, Agapov I, Moisenovich M (2002) Detection of isolated mistletoe lectin chains in plant extracts. Arzneimittelforschung/Drug Research 52:67–71Google Scholar
  51. Trischler M, Stoorvogel W, Ullrich O (1999) Biochemical analysis of distinct Rab5- and Rab11-positive endosomes along the transferrin pathway J Cell Sci 112:4773–4783Google Scholar
  52. Ullrich O, Reinsch S, Urbe S, Zerial M, Parton RG (1996) Rab11 regulates recycling through the pericentriolar recycling endosome. J Cell Biol 135:913–924PubMedGoogle Scholar
  53. van Dam EM, Stoorvogel W (2002) Dynamin-dependent transferrin receptor recycling by endosome-derived clathrin-coated vesicles. Mol Biol Cell 13:169–182CrossRefPubMedGoogle Scholar
  54. van Dam E, Broeke T, Jansen K, Spijkers P, Stoorvogel W (2002) Endocytosed transferrin receptors recycle via distinct dynamin and phosphatidylinositol 3-kinase-dependent pathways. J Biol Chem 277:48876–48883CrossRefPubMedGoogle Scholar
  55. van Deurs B, Tonnessen TI, Petersen OW, Sandvig K, Olsnes S (1986) Routing of internalized ricin and ricin conjugates to the Golgi complex. J Cell Biol 102:37–47Google Scholar
  56. van Deurs B, Sandvig K, Petersen OW, Olsnes S, Simons K, Griffiths G (1988) Estimation of the amount of internalized ricin that reaches the trans-Golgi network. J Cell Biol 106:253–267Google Scholar
  57. van Deurs B, Hansen SH, Olsnes S, Sandvig K (1993) Protein uptake and cytoplasmic access in animal cells. In: Audus KL, Raub TJ (eds) Pharmaceutical Biotechnology, vol 4. Biological barriers to protein delivery. Plenum, New York, pp 71–104Google Scholar
  58. Venkatesh YP, Lambert JM (1997) Galactose-induced dimerization of blocked ricin at acidic pH: evidence for a third galactose-binding site in ricin B-chain. Glycobiology 7:329–335PubMedGoogle Scholar
  59. Volknandt W, Küster F, Wilhelm A, Obermüller E, Steinmann A, Zhang L, Zimmermann H (2002) Expression and allocation of proteins of the exo-endocytotic machinery in U373 glioma cells: similarities to long-term cultured astrocytes. Cell Mol Neurobiol 22:153–169CrossRefPubMedGoogle Scholar
  60. Wesche J (2002) Retrograde transport of ricin. Int J Med Microbiol 291:517–521PubMedGoogle Scholar
  61. Wesche J, Rapak A, Olsnes S (1999) Dependence of ricin toxicity on translocation of the toxin A-chain from the endoplasmic reticulum to the cytosol. J Biol Chem 274:34443–34449CrossRefPubMedGoogle Scholar
  62. Wit H, Lichtenstein Y, Kelly RB, Geuze HJ, Klumperman J, Sluijs P (2001) Rab4 regulates formation of synaptic-like microvesicles from early endosomes in PC12 Cells. Mol Biol Cell 12:3703–3715PubMedGoogle Scholar
  63. Zerial M, McBride H (2001) Rab proteins as membrane organizers. Nat Rev Mol Cell Biol 2:107–117PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • Mihail Moisenovich
    • 2
  • Alex Tonevitsky
    • 2
  • Natalia Maljuchenko
    • 2
  • Natalia Kozlovskaya
    • 2
  • Igor Agapov
    • 2
  • Walter Volknandt
    • 1
  • Jürgen Bereiter-Hahn
    • 1
    Email author
  1. 1.Institute of Zoology, BiocentreJohann Wolfgang Goethe UniversityFrankfurt am MainGermany
  2. 2.Biological FacultyLomonosov Moscow State UniversityMoscowRussia

Personalised recommendations