A developmentally regulated aconitase related to iron-regulatory protein-1 is localized in the cytoplasm and in the mitochondrion of Trypanosoma brucei.
Mitochondrial energy metabolism and Krebs cycle activities are developmentally regulated in the life cycle of the protozoan parasite Trypanosoma brucei. Here we report cloning of a T. brucei aconitase gene that is closely related to mammalian iron-regulatory protein 1 (IRP-1) and plant aconitases. Kinetic analysis of purified recombinant TbACO expressed in Escherichia coli resulted in a K(m) (isocitrate) of 3 +/- 0.4 mM, similar to aconitases of other organisms. This was unexpected since an arginine conserved in the aconitase 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 TbACO was shown to be lowest in the slender bloodstream stage of the parasite, 8-fold elevated in the stumpy stage, and increased a further 4-fold in the procyclic stage. The differential expression of TbACO protein contrasted with only minor changes in TbACO mRNA, indicating translational or post-translational mechanisms of regulation. Whereas animal cells express two distinct compartmentalized aconitases, mitochondrial aconitase and cytoplasmic aconitase/IRP-1, TbACO accounts for total aconitase activity in trypanosomes. By cell fractionation and immunofluorescence microscopy, we show that native as well as a transfected epitope-tagged TbACO localizes in both the mitochondrion (30%) and in the cytoplasm (70%). Together with phylogenetic reconstructions of the aconitase family, this suggests that animal IRPs have evolved from a multicompartmentalized ancestral aconitase. The possible functions of a cytoplasmic aconitase in trypanosomes are discussed.
Saas
J
J
Arbeitsgruppe Molekulare Zellbiologie, Institut für Molekularbiologie und Biochemie und Institut für Infektionsmedizin, Freie Universität, Berlin, Germany.
Ziegelbauer
K
K
von Haeseler
A
A
Fast
B
B
Boshart
M
M
D000154:Aconitate Hydratase / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000201:enzymology*; D018076:DNA, Complementary; D015971:Gene Expression Regulation, Enzymologic; D035941:Iron Regulatory Protein 1; D035925:Iron-Regulatory Proteins; D007506:Iron-Sulfur Proteins / Q000235:genetics*; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010802:Phylogeny; D016601:RNA-Binding Proteins / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014346:Trypanosoma brucei brucei / Q000201:enzymology*
D018076:DNA, Complementary; D035925:Iron-Regulatory Proteins; D007506:Iron-Sulfur Proteins; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D000154:Aconitate Hydratase; D035941:Iron Regulatory Protein 1