title: A solanesyl-diphosphate synthase localizes in glycosomes of Trypanosoma cruzi.
abstract: We report the cloning of a Trypanosoma cruzi gene encoding a solanesyl-diphosphate synthase, TcSPPS. The amino acid sequence (molecular mass approximately 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.
authors.affiliation: 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.
authors.forename: Marcela
authors.initials: M
authors.lastname: Ferella
authors.forename: Andrea
authors.initials: A
authors.lastname: Montalvetti
authors.forename: Peter
authors.initials: P
authors.lastname: Rohloff
authors.forename: Kildare
authors.initials: K
authors.lastname: Miranda
authors.forename: Jianmin
authors.initials: J
authors.lastname: Fang
authors.forename: Silvia
authors.initials: S
authors.lastname: Reina
authors.forename: Makoto
authors.initials: M
authors.lastname: Kawamukai
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Búa
authors.forename: Daniel
authors.initials: D
authors.lastname: Nilsson
authors.forename: Carlos
authors.initials: C
authors.lastname: Pravia
authors.forename: Alejandro
authors.initials: A
authors.lastname: Katzin
authors.forename: Maria B
authors.initials: MB
authors.lastname: Cassera
authors.forename: Lena
authors.initials: L
authors.lastname: Aslund
authors.forename: Björn
authors.initials: B
authors.lastname: Andersson
authors.forename: Roberto
authors.initials: R
authors.lastname: Docampo
authors.forename: Esteban J
authors.initials: EJ
authors.lastname: Bontempi
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000096:biosynthesis* / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D003360:Cosmids; D004926:Escherichia coli / Q000378:metabolism; D005816:Genetic Complementation Test; D008830:Microbodies / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014349:Trypanosoma cruzi / Q000378:metabolism*; D014451:Ubiquinone / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D014451:Ubiquinone; D019883:Alkyl and Aryl Transferases; C022644:trans-octaprenyltranstransferase; C030571:ubiquinone 9