A developmentally regulated aconitase related to iron-regulatory protein-1is localized in the cytoplasm and in the mitochondrion of Trypanosoma brucei

Mitochondrial energy metabolism and Krebs cycle activities are development- ally regulated in the life cycle of the protozoan parasite Typanosoma bruce i. Here we report cloning of a T. brucei aconitase gene that is closely rel ated to mammalian iron-regulatory protein 1 (IRP-1) and plant aconitases. K inetic analysis of purified recombinant TbACO expressed in Escherichia coli resulted in a K-m (isocitrate) of 3 +/- 0.4 mM, similar to aconitases of o ther organisms. This was unexpected since an arginine conserved in the acon itase protein family and crucial for substrate positioning in the catalytic center and for activity of pig mitochondrial aconitase (Zheng, L., Kennedy , M. C., Beinert, H., and Zalkin, H. (1992) J. Biol. Chem. 267, 7895-7903) is substituted by leucine in the TbACO sequence. Expression of the 98-kDa T bACO was shown to be lowest in the slender bloodstream stage of the parasit e, 8-fold elevated in the stumpy stage, and increased a further 4-fold in t he procyclic stage. The differential expression of TbACO protein contrasted with only minor changes in TbACO mRNA, indicating translational or post-tr anslational mechanisms of regulation, Whereas animal cells express two dist inct compartmentalized aconitases, mitochondrial aconitase and cytoplasmic aconitase/IRP-1, TbACO accounts for total aconitase activity in trypanosome s, By cell fractionation and immunofluorescence microscopy, we show that na tive as well as a transfected epitope-tagged TbACO localizes in both the mi tochondrion (30%) and in the cytoplasm (70%), Together with phylogenetic re constructions of the aconitase family, this suggests that animal IRPs have evolved from a multicompartmentalized ancestral aconitase, The possible fun ctions of a cytoplasmic aconitase in trypanosomes are discussed.