Trypanosoma cruzi: TcRAB7 protein is localized at the Golgi apparatus in epimastigotes

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Abstract

In mammalian cells, the Rab7 protein is a key element of late endocytic membrane traffic. Several results suggest that it is involved in the transport from early to late endosome or from late endosome to lysosome. We have previously characterized a Rab7 gene homologue (TcRAB7) in Trypanosoma cruzi. Now, using an affinity-purified antibody specific to TcRAB7 protein we have determined that it is localized at the Golgi apparatus of the parasite. Our results indicate that the T. cruzi Rab7 homologue may function in a different route than its counterparts in mammalian cells.

Section snippets

Materials and methods

Parasites. T. cruzi clones, CL Brener, and Dm28c, were used. Epimastigotes were cultivated at 28 °C in liver infusion tryptose (LIT) liquid medium supplemented with 10% fetal calf serum [18]. Stably transformed epimastigote clones, transfected with either control plasmid pTEX [19] or with pTAG, a plasmid that leads to over-expression of a hemagglutinin (HA) epitope-tagged TcRAB7 protein [17], were maintained in the presence of 500 μg/ml of G418 (Geneticin, Gibco, Grand Island, NY).

Nucleic acid

Characterization of an immune serum against TcRAB7 protein of T. cruzi

The immune serum was prepared against the synthetic peptide corresponding to the non-conserved carboxy-terminal 14-amino acid sequence of TcRAB7. Immunoblotting experiments were performed using the recombinant TcRAB7 protein as antigen. Total immune serum recognized a protein with molecular weight corresponding to GST-fused TcRAB7 protein (around 56 kDa, Fig. 1A) on Western blots of the fusion protein from total extract of E. coli. The total immune serum recognized neither bacterial proteins nor

Acknowledgments

We thank Rosane Silva and Turán P. Ürményi for critical reading of the manuscript and César Félix Schmidt, Cláudio Nunes Pereira, and Sebastisião Cruz for technical assistance. This work was supported by grants from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação Coordenação de Aperfeiçoamento de Pessoal de Nı́vel Superior (CAPES), Financiadora de Estudos e Projetos (FINEP), Fundação José Bonifácio (FUJB), Fundação Carlos Chagas Filho de Apoio à Pesquisa no

References (29)

  • G.W. Morgan et al.

    The kinetoplastida endocytic apparatus. Part I: a dynamic system for nutrition and evasion of host defenses

    Trends Parasitol.

    (2002)
  • Y. Feng et al.

    Rab7: an important regulator of late endocytic membrane traffic

    J. Cell Biol.

    (1995)
  • C. Bucci et al.

    Rab7: a key to lysosome biogenesis

    Mol. Biol. Cell

    (2000)
  • W. De Souza

    Basic cell biology of Trypanosoma cruzi

    Curr. Pharm. Des.

    (2002)
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    Present address: The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA.

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