title: Identification and functional characterization of a novel arginine/ornithine transporter, a member of a cationic amino acid transporter subfamily in the Trypanosoma cruzi genome.
abstract: BACKGROUND
  Trypanosoma cruzi, the etiological agent of Chagas disease, is auxotrophic for arginine. It obtains this amino acid from the host through transporters expressed on the plasma membrane and on the membranes of intracellular compartments. A few cationic amino acid transporters have been characterized at the molecular level, such as the novel intracellular arginine/ornithine transporter, TcCAT1.1, a member of the TcCAT subfamily that is composed of four almost identical open reading frames in the T. cruzi genome.
  
  
  METHODS
  The functional characterization of the TcCAT1.1 isoform was performed in two heterologous expression systems. TcCAT subfamily expression was evaluated by real-time PCR in polysomal RNA fractions, and the cellular localization of TcCAT1.1 fused to EGFP was performed by confocal and immunoelectron microscopy.
  
  
  RESULTS
  In the S. cerevisiae expression system, TcCAT1.1 showed high affinity for arginine (K m  = 0.085 ± 0.04 mM) and low affinity for ornithine (K m  = 1.7 ± 0.2 mM). Xenopus laevis oocytes expressing TcCAT1.1 showed a 7-fold increase in arginine uptake when they were pre-loaded with arginine, indicating that transport is enhanced by substrates on the trans side of the membrane (trans-stimulation). Oocytes that were pre-loaded with [(3)H]-arginine displayed a 16-fold higher efflux of [(3)H]-arginine compared with that of the control. Analysis of polysomal RNA fractions demonstrated that the expression of members of the arginine transporter TcCAT subfamily is upregulated under nutritional stress and that this upregulation precedes metacyclogenesis. To investigate the cellular localization of the transporter, EGFP was fused to TcCAT1.1, and fluorescence microscopy and immunocytochemistry revealed the intracellular labeling of vesicles in the anterior region, in a network of tubules and vesicles.
  
  
  CONCLUSIONS
  TcCAT1.1 is a novel arginine/ornithine transporter, an exchanger expressed in intracellular compartments that is physiologically involved in arginine homeostasis throughout the T. cruzi life cycle. The properties and estimated kinetic parameters of TcCAT1.1 can be extended to other members of the TcCAT subfamily.
authors.affiliation: Fundação Oswaldo Cruz, Fiocruz-Mato Grosso do Sul, Rua Gabriel Abrão 92-Jardim das Nações, Campo Grande, MS, 89081-746, Brazil. henriques@fiocruz.br.|Instituto de Biofísica Carlos Chagas Filho-UFRJ, CCS-Bloco G-Laboratório de Ultraestrutura Celular Hertha Meyer, Rio de Janeiro, RJ, 21949-900, Brazil. henriques@fiocruz.br.|Nucleo de Biologia Estrutural e Biomagens, Universidade Federal do Rio de Janeiro-CENABIO, Rio de Janeiro, RJ, Brazil. henriques@fiocruz.br.
authors.forename: Cristina
authors.initials: C
authors.lastname: Henriques
authors.affiliation: Department of Neurobiology, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15260, USA. mpl8@pitt.edu.
authors.forename: Megan P
authors.initials: MP
authors.lastname: Miller
authors.affiliation: Fiocruz, Instituto Oswaldo Cruz, Laboratório de Genômica Funcional e Bioinformática, Av. Brasil 4365, Manguinhos, 21040-900, Rio de Janeiro, RJ, Brazil. mcatanho@fiocruz.br.
authors.forename: Marcos
authors.initials: M
authors.lastname: Catanho
authors.affiliation: Instituto de Biofísica Carlos Chagas Filho-UFRJ, CCS-Bloco G-Laboratório de Ultraestrutura Celular Hertha Meyer, Rio de Janeiro, RJ, 21949-900, Brazil. tecia.ulisses@gmail.com.
authors.forename: Técia Maria Ulisses
authors.initials: TM
authors.lastname: de Carvalho
authors.affiliation: Instituto Carlos Chagas-ICC-FIOCRUZ, Curitiba, PR, 81350-010, Brazil. mkrieger@fiocruz.br.
authors.forename: Marco Aurélio
authors.initials: MA
authors.lastname: Krieger
authors.affiliation: Instituto Carlos Chagas-ICC-FIOCRUZ, Curitiba, PR, 81350-010, Brazil. cprobst@tecpar.br.
authors.forename: Christian M
authors.initials: CM
authors.lastname: Probst
authors.affiliation: Instituto de Biofísica Carlos Chagas Filho-UFRJ, CCS-Bloco G-Laboratório de Ultraestrutura Celular Hertha Meyer, Rio de Janeiro, RJ, 21949-900, Brazil. wsouza@biof.ufrj.br.|Nucleo de Biologia Estrutural e Biomagens, Universidade Federal do Rio de Janeiro-CENABIO, Rio de Janeiro, RJ, Brazil. wsouza@biof.ufrj.br.|Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Biomagens-INBEB, Rio de Janeiro, Brazil. wsouza@biof.ufrj.br.
authors.forename: Wanderley
authors.initials: W
authors.lastname: de Souza
authors.affiliation: Fiocruz, Instituto Oswaldo Cruz, Laboratório de Genômica Funcional e Bioinformática, Av. Brasil 4365, Manguinhos, 21040-900, Rio de Janeiro, RJ, Brazil. wdegrave@fiocruz.br.
authors.forename: Wim
authors.initials: W
authors.lastname: Degrave
authors.affiliation: National Institute of Mental Health, NIH Building 10 Center Driver, Room 4N222, MSC 1381, Bethesda, MD, 20892-1381, USA. amarasg@mail.nih.gov.|Department of Neurobiology, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15260, USA. amarasg@mail.nih.gov.
authors.forename: Susan Gaye
authors.initials: SG
authors.lastname: Amara
mesh_terms: D000595:Amino Acid Sequence; D026923:Amino Acid Transport Systems, Basic / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001120:Arginine / Q000378:metabolism*; D014355:Chagas Disease / Q000469:parasitology*; D018503:Genome, Protozoan; D006801:Humans; D008297:Male; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D005810:Multigene Family; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D014349:Trypanosoma cruzi / Q000235:genetics* / Q000378:metabolism*
chemical_list: D026923:Amino Acid Transport Systems, Basic; D015800:Protozoan Proteins; C041356:ornithine transporter; D001120:Arginine