Advances in Animal Science, Theriogenology, Genetics and Breeding

The molecular and cellular characterisation of the ADP - ribosylation factor of African trypanosomes

Abstract


Aaron Kingsley*, Roger Pell├â┬ę, Francis Chuma, Clive Wells and Noel B. Murphy

Small GTP-binding proteins of the ADP-ribosylation factor (Arf) family are essential for vesicle formation, intracellular and membrane trafficking and signal transduction. We have cloned and characterized the homologue of Arf1 from Trypanosoma b. brucei, Trypanosoma evansi, Trypanosoma congolense and Trypanosoma vivax. The African trypanosome Arf1 cDNA contains an open reading frame of 546 base pairs, corresponding to a 181 amino acid residue polypeptide. The protein carries conserved motifs in the GTP-binding domains for Arf1 proteins and a myristoylation site in addition to domains specific to trypanosomes. The TbArf1 gene generates a transcript of approximately 0.9 kb that is developmentally regulated. Recombinant TbArf1 expressed in Escherichia coli binds GTP. GDP and GTP were efficient competitors of [a32P]- GTP binding to the protein but not ATP or CTP. Polyclonal antibodies raised against the recombinant TbArf1 revealed a major protein band of about 21 kDa in T. b. brucei and T. congolense lysates and additional weaker binding to proteins of higher molecular weight in both lysates. The protein localizes in endosomal compartments between the nucleus and kinetoplast but in addition it also displays a flagellar localisation. The TbArf1 protein is detected in supernatant of in vitro cultivated parasites.

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