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J Biol Chem, Vol. 275, Issue 4, 2745-2755, January 28, 2000

A Developmentally Regulated Aconitase Related to Iron-regulatory Protein-1 Is Localized in the Cytoplasm and in the Mitochondrion of Trypanosoma brucei^*

Joachim Saas^§, Karl Ziegelbauer^¶, Arndt von Haeseler^**, Beate Fast^§, and Michael Boshart^§

From the  Arbeitsgruppe Molekulare Zellbiologie, Institut für Molekularbiologie und Biochemie und Institut für Infektionsmedizin, Freie Universität, Berlin, the ^§ Max-Planck-Institut für Biochemie, Martinsried, the ^¶ Max-Planck-Institut für Biologie, Tübingen, and the ^** Max-Planck-Institut für Evolutionäre Anthropologie, Leipzig, Germany

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.

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^* This work was supported by Bundesministerium für Bildung, Wissenschaft, und Forschung Grant 0311092 (to M. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AF127456, AF127457, and AF190556.

Present address: Bayer Yakuhin Ltd., Research Center Kyoto, 6-5-1-3 Kunimidai, Kizu-cho, Soraku-gun, Kyoto, 619-02, Japan.

To whom correspondence should be addressed: AG Molekulare Zellbiologie, Institut für Molekularbiologie und Biochemie, FU Berlin, Hindenburgdamm 27, D-12203 Berlin, Germany. Tel.: 49-30-8445-3820; Fax: 49-30-8445-3840; E-mail: boshart@ukbf.fu-berlin.de.

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Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

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