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J. Biol. Chem., Vol. 281, Issue 51, 39339-39348, December 22, 2006

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A Solanesyl-diphosphate Synthase Localizes in Glycosomes of Trypanosoma cruzi^*

Marcela Ferella^¶||, Andrea Montalvetti^¶, Peter Rohloff^¶, Kildare Miranda^**, Jianmin Fang^**, Silvia Reina^¶, Makoto Kawamukai, Jacqueline Búa, Daniel Nilsson^||, Carlos Pravia, Alejandro Katzin, Maria B. Cassera, Lena Åslund, Björn Andersson^||**, Roberto Docampo^¶**1, and Esteban J. Bontempi^||2

From the Instituto Nacional de Parasitología Dr. M. Fatala Chabén, Av. Paseo Colón 568, Administración Nacional de Laboratorios e Institutos de Salud, Ministerio de Salud, Buenos Aires 1063, Argentina, the Department of Genetics and Pathology, Uppsala University, Uppsala SE751 85, Sweden, the ^¶Department of Pathobiology, University of Illinois at Urbana-Champaign, Urbana 61802, Illinois, the ^||Center for Genomics and Bioinformatics, Karolinska Institute, Stockholm SE171 77, Sweden, the ^**Center for Tropical and Emerging Global Diseases and Department of Cellular Biology, University of Georgia, Athens, Georgia 30602-2607, the Department of Applied Bioscience and Biotechnology, Faculty of Life and Environmental Science, Shimane University Matsue 690-8540, Japan, and the Departamento de Parasitologia, Instituto de Ciencias Biomédicas, Universidade de São Paulo, São Paulo 05508-900, Brazil

We report the cloning of a Trypanosoma cruzi gene encoding a solanesyl-diphosphate synthase, TcSPPS. The amino acid sequence (molecular mass 39 kDa) is homologous to polyprenyl-diphosphate synthases from different organisms, showing the seven conserved motifs and the typical hydrophobic profile. TcSPPS preferred geranylgeranyl diphosphate as the allylic substrate. The final product, as determined by TLC, had nine isoprene units. This suggests that the parasite synthesizes mainly ubiquinone-9 (UQ-9), as described for Trypanosoma brucei and Leishmania major. In fact, that was the length of the ubiquinone extracted from epimastigotes, as determined by high-performance liquid chromatography. Expression of TcSPPS was able to complement an Escherichia coli ispB mutant. A punctuated pattern in the cytoplasm of the parasite was detected by immunofluorescence analysis with a specific polyclonal antibody against TcSPPS. An overlapping fluorescence pattern was observed using an antibody directed against the glycosomal marker pyruvate phosphate dikinase, suggesting that this step of the isoprenoid biosynthetic pathway is located in the glycosomes. Co-localization in glycosomes was confirmed by immunogold electron microscopy and subcellular fractionation. Because UQ has a central role in energy production and in reoxidation of reduction equivalents, TcSPPS is promising as a new chemotherapeutic target.

Received for publication, August 4, 2006 , and in revised form, October 23, 2006.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AF282771 [GenBank] .

^* This work was supported in part by National Institutes of Health Grants AI-68647 and GM-65307 (to R. D.), the Programa de Nanociencia e Nanotecnologia, MCT/CNPq, Brazil (to K. M), the NASA/ChagaSpace network, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, Argentina), the Network for Research and Training in Parasitic Diseases at the Southern Cone of Latin America Swedish International Development Agency/Swedish Agency for Research Cooperation and Wallenberg Consortium North, and by the Instituto Nacional de Parasitología Dr. Mario Fatala Chabén, Administración Nacional de Laboratorios e Institutos de Salud, Dr. Carlos G. Malbrán. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence may be addressed: Center for Tropical and Emerging Global Diseases and Dept. of Cellular Biology, 350 Paul D. Coverdell Center, University of Georgia, Athens, GA 30602-2607. Tel.: 706-542-3310; Fax: 706-583-0181; E-mail: rdocampo{at}uga.edu. ² To whom correspondence may be addressed. Tel.: 54-11-4331-4019; Fax: 54-11-4331-7142; E-mail: ejbon{at}yahoo.com.

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