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IDENTIFICATION AND FUNCTIONAL CHARACTERIZATION OF A NOVEL ARGININE/ORNITHINE TRANSPORTER, A MEMBER OF A CATIONIC AMINO ACID TRANSPORTER SUBFAMILY IN THE TRYPANOSOMA CRUZI GENOME
Author
Affilliation
Fundação Oswaldo Cruz. Campo Grande, MS, Brasil / Universidade Federal do Rio de Janeiro. Centro de Ciências da Saúde. Instituto de Biofísica Carlos Chagas Filho. Laboratório de Ultraestrutura Celular Hertha Meyer. Rio de Janeiro, RJ, Brasil / Universidade Federal do Rio de Janeiro. CENABIO. Núcleo de Biologia Estrutural e Bioimagem. Rio de Janeiro, RJ, Brasil.
University of Pittsburgh. School of Medicine. Department of Neurobiology. Pittsburgh, PA, USA.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Genômica Funcional e Bioinformática. Rio de Janeiro, RJ, Brasil.
Universidade Federal do Rio de Janeiro. Centro de Ciências da Saúde. Instituto de Biofísica Carlos Chagas Filho. Laboratório de Ultraestrutura Celular Hertha Meyer. Rio de Janeiro, RJ, Brasil
Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.
Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.
Universidade Federal do Rio de Janeiro. Centro de Ciências da Saúde. Instituto de Biofísica Carlos Chagas Filho. Laboratório de Ultraestrutura Celular Hertha Meyer. Rio de Janeiro, RJ, Brasil / Universidade Federal do Rio de Janeiro. CENABIO. Núcleo de Biologia Estrutural e Bioimagem. Rio de Janeiro, RJ, Brasil / Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Biomagens - INBEB. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Genômica Funcional e Bioinformática. Rio de Janeiro, RJ, Brasil.
National Institute of Mental Health. Bethesda, MD, USA / University of Pittsburgh. School of Medicine. Departmento of Neurobiology. Pittsburg, PA, USA.
University of Pittsburgh. School of Medicine. Department of Neurobiology. Pittsburgh, PA, USA.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Genômica Funcional e Bioinformática. Rio de Janeiro, RJ, Brasil.
Universidade Federal do Rio de Janeiro. Centro de Ciências da Saúde. Instituto de Biofísica Carlos Chagas Filho. Laboratório de Ultraestrutura Celular Hertha Meyer. Rio de Janeiro, RJ, Brasil
Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.
Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.
Universidade Federal do Rio de Janeiro. Centro de Ciências da Saúde. Instituto de Biofísica Carlos Chagas Filho. Laboratório de Ultraestrutura Celular Hertha Meyer. Rio de Janeiro, RJ, Brasil / Universidade Federal do Rio de Janeiro. CENABIO. Núcleo de Biologia Estrutural e Bioimagem. Rio de Janeiro, RJ, Brasil / Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Biomagens - INBEB. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Genômica Funcional e Bioinformática. Rio de Janeiro, RJ, Brasil.
National Institute of Mental Health. Bethesda, MD, USA / University of Pittsburgh. School of Medicine. Departmento of Neurobiology. Pittsburg, PA, USA.
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 (Km = 0.085 ± 0.04 mM)
and low affinity for ornithine (Km = 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.
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