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J Biol Chem, Vol. 275, Issue 4, 2745-2755, January 28, 2000
§,
,
§, and
§
From the 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 Km
(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.
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
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/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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