Molecular and Cellular Biochemistry

, Volume 292, Issue 1–2, pp 107–117 | Cite as

Rab5b localization to early endosomes in the protozoan human pathogen Leishmania donovani

  • Diane E. Marotta
  • Noel Gerald
  • Dennis M. Dwyer


Leishmania donovani is a primitive trypanosomatid pathogen of humans. This protozoan is apically polarized such that the flagellar reservoir, the exclusive site of endocytosis and exocytosis, is situated at the anterior end. Recent evidence for the existence of an endocytic pathway in Leishmania has prompted us to investigate candidate temporal markers for endocytosis. In this study we identify the L. donovani Rab5b gene, and demonstrate the localization of a Rab5b chimera to early endosomes. A full-length Rab5b protein was fused to green fluorescent protein (GFP) to generate a chimeric protein GFP::Rab5b. Transfected L. donovani promastigotes carrying this chimeric construct displayed GFP::Rab5b localization. Additionally, incubation of transfected promastigotes with the fluid-phase marker Texas Red dextran demonstrated anterior co-localization of GFP::Rab5b and dye. This suggests Rab5b may act as a marker for early endosomes in L. donovani.


endocytosis GFP chimera Leishmania Rab5b trafficking 


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  1. 1.
    Burchmore RJS, Barrett MP: Life in vacuoles-nutrient acquisition by Leishmania amastigotes. Int J Parasitol 31: 1311–1320, 2001PubMedCrossRefGoogle Scholar
  2. 2.
    Webster P, Russell DG: The flagellar pocket of trypanosomatids. Parasitol Today 9: 201–206, 1993PubMedCrossRefGoogle Scholar
  3. 3.
    Ghedin E, Debrabant A, Engel JC, Dwyer, DM: Secretory and endocytic pathways converge in a dynamic endosomal system in a primitive protozoan. Traffic 2: 175–188, 2001PubMedCrossRefGoogle Scholar
  4. 4.
    McLauchlan H, Newell J, Morrice N, Osborne A, West M, Smythe E: A novel role for Rab5-GDI in ligand sequestration into clathrin-coated pits. Curr Biol 8: 34–45, 1998PubMedCrossRefGoogle Scholar
  5. 5.
    Christoforidis S, McBride HM, Burgoyne RD, Zerial M: The Rab effector EEA1 is a core component of endosome docking. Nature 397: 621–625, 1999PubMedCrossRefGoogle Scholar
  6. 6.
    Seabra MC, Mules EH, Hume AN: Rab GTPases, intracellular traffic and disease. Trends Mol Med 8: 23–30, 2002PubMedCrossRefGoogle Scholar
  7. 7.
    Duclos S, Diez R, Garin J, Papadopoulou B, Descoteau A, Stenmark H, Desjardins M: Rab5 regulates the kiss and run fusion between phagosomes and endosomes and the acquisition of phagosome leishmanicidal properties in RAW 264.7 macrophages. J Cell Sci 113: 3531–3541, 2000Google Scholar
  8. 8.
    Singh SB, Tandon R, Krishnamurthy G, Vikram R, Sharma N, Basu SK, Mukhopadhyay A: Rab5-mediated endosome-endosome fusion regulates hemoglobin endocytosis in Leishmania donovani. EMBO J 22: 5712–5722, 2003PubMedCrossRefGoogle Scholar
  9. 9.
    Kamau SW, Grimm F, Hehl AB: Expression of green fluorescent protein as a marker for effects of antileishmanial compounds in vitro. Antimicrob Agents Chemother 45: 3654–3656, 2001PubMedCrossRefGoogle Scholar
  10. 10.
    Bates PA, Hermes I, Dwyer DM: Golgi-mediated post-translational processing of secretory acid phosphatase by Leishmania donovani promastigotes. Mol Biochem Parasitol 392: 247–256, 1990Google Scholar
  11. 11.
    Shakarian AM, Dwyer DM: The Ld Cht 1 gene encodes the secretory chitinases of the human pathogen Leishmania donovani. Gene 208: 315–322, 1998PubMedCrossRefGoogle Scholar
  12. 12.
    Clayton C, Adams M, Almeida R, et al.: Genetic nomenclature for Trypanosoma and Leishmania. Mol Biochem Parasitol 97: 221–224, 1998PubMedCrossRefGoogle Scholar
  13. 13.
    McCombie WR, Heiner C, Kelley JM, Fitzgerald MG, Gocayne JD: Rapid and reliable fluorescent cycle sequencing of double-stranded templates. DNA Seq. 2: 289–296, 1992Google Scholar
  14. 14.
    Shakarian AM, Ellis SL, Mallinson DJ, Olafson RW, Dwyer DM: Two tandemly arrayed genes encode the (histidine) secretory acid phosphatase of Leishmania donovani. Gene 196: 127–137, 1997PubMedCrossRefGoogle Scholar
  15. 15.
    Swofford DL. PAUP. Phylogenetic Analysis Using Parsimony (and Other Methods). Version 4. Sunderland, Massachusetts: Sinauer Associates 2002.Google Scholar
  16. 16.
    Zhang WW, Charest H, Ghedin E, Matlashewshi G: Identification and overexpression of the A2 amastigote-specific protein in Leishmania donovani. Mol Biochem Parasitol 78: 79–90, 1996Google Scholar
  17. 17.
    Debrabant A, Ghedin E, Dwyer DM: Dissection of the functional domains of the Leishmania surface membrane 3′-nucleotidase/nuclease, a unique member of the Class I nuclease family. J Biol Chem 275: 16366–16372, 2000CrossRefGoogle Scholar
  18. 18.
    Overath P, Stierhof YD, Wiese M: Endocytosis and secretion in trypanosomatid parasites-tumultuous trafficking in a pocket. Trends Cell Biol 7: 27–33, 1997CrossRefGoogle Scholar
  19. 19.
    Zerial M, McBride H: Rab proteins as membrane organizers. Nat Rev Mol Cell Bio l2: 107–117, 2001CrossRefGoogle Scholar
  20. 20.
    Pfeffer SR: Rab GTPases: Specifying and deciphering organelle identity and function. Trends Cell Biol 11: 487–491, 2001PubMedCrossRefGoogle Scholar
  21. 21.
    Pereira-Leal JB, Seabra MC: The mammalian Rab family of small GTPases: definition of family and subfamily sequence motifs suggests a mechanism for functional specificity in the Ras superfamily. J Mol Biol 301: 1077–1087, 2000PubMedCrossRefGoogle Scholar
  22. 22.
    Pereira-Leal JB, Strom M, Godfrey RF, Seabra, MC. Structural determinants of Rab and Rab escort protein interaction: Rab family motifs define a conserved binding surface. Biochem Biophys Res Commun 301: 92–97, 2003Google Scholar
  23. 23.
    Feng Y, Press B, Wardinger-Ness A: Rab7: an important regulator of the late endocytic membrane traffic. J Biol Chem 131: 1435–1452, 1995Google Scholar
  24. 24.
    Mullin KA, Foth BJ, Ilgoutz SC, Callaghan JM, Zawadzki JL, McFadden GI, McConville MJ: Regulated degradation of an endoplasmic reticulum membrane protein in a tubular lysosome in Leishmania mexicana. Mol Biol Cell 12: 2364–2377, 2001PubMedGoogle Scholar
  25. 25.
    Bucci C, Parton RG, Mather IH, Stunnenberg H, Simons K, Hoflack B, Zerial M. The small GTPase Rab5b functions as a regulatory factor in the early endocytic pathway. Cell 70: 715–728, 1992Google Scholar
  26. 26.
    Carter RE, Sorkin, A: Endocytosis of functional epidermal growth factor receptor-green fluorescent protein chimera. J Biol Chem 273: 35000–35007, 1998PubMedCrossRefGoogle Scholar
  27. 27.
    Chiariello M, Bruni C, Brucci, C: The small GTPases Rab5a, Rab5b and Rab5c are differentially phosphorylated in vitro. FEBS Lett 453: 20–24, 1999PubMedCrossRefGoogle Scholar
  28. 28.
    Callaghan J, Nixon S, Bucci C, Ban-Hock T, Stenmark H: Direct interaction of EEA1 with Rab5b. Eur J Biochem 265: 361–366, 1999PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, Inc. 2006

Authors and Affiliations

  • Diane E. Marotta
    • 1
    • 2
  • Noel Gerald
    • 1
  • Dennis M. Dwyer
    • 1
  1. 1.Cell Biology Section, Laboratory of Parasitic Diseases, NIAIDNational Institutes of HealthBethesdaUSA
  2. 2.University of PennsylvaniaPhiladelphiaUSA

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