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BIOENERGETICS AND PHOTOSYNTHESIS

Mitochondrial and Nuclear Localization of a Novel Pea Thioredoxin: Identification of Its Mitochondrial Target Proteins^1,[W]

Department of Stress Biology and Plant Pathology, Centro de Edafología y Biología Aplicada del Segura, Consejo Superior de Investigaciones Científicas, E–30100 Murcia, Spain (M.C.M., E.O., F.S., A.J.); Laboratory of Structural Proteomics, Instituto de Biomedicina, Consejo Superior de Investigaciones Científicas, E–46010 Valencia, Spain (J.J.C.); Department of Biotechnology, Escuela Técnica Superior de Ingenieros Agrónomos-Universidad Politécnica de Madrid, E–28040 Madrid, Spain (I.D.); Department of Biochemistry, Cellular and Molecular Biology of Plants, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, E–18080 Granada, Spain (S.B.-M., J.J.L.); and Australian Research Council Centre of Excellence in Plant Energy Biology, University of Western Australia, Crawley 6009, Western Australia, Australia (J.W.)

Plants contain several genes encoding thioredoxins (Trxs), small proteins involved in the regulation of the activity of many enzymes through dithiol-disulfide exchange. In addition to chloroplastic and cytoplasmic Trx systems, plant mitochondria contain a reduced nicotinamide adenine dinucleotide phosphate-dependent Trx reductase and a specific Trx o, and to date, there have been no reports of a gene encoding a plant nuclear Trx. We report here the presence in pea (Pisum sativum) mitochondria and nuclei of a Trx isoform (PsTrxo1) that seems to belong to the Trx o group, although it differs from this Trx type by its absence of introns in the genomic sequence. Western-blot analysis with isolated mitochondria and nuclei, immunogold labeling, and green fluorescent protein fusion constructs all indicated that PsTrxo1 is present in both cell compartments. Moreover, the identification by tandem mass spectrometry of the native mitochondrial Trx after gel filtration using the fast-protein liquid chromatography system of highly purified mitochondria and the in vitro uptake assay into isolated mitochondria also corroborated a mitochondrial location for this protein. The recombinant PsTrxo1 protein has been shown to be reduced more effectively by the Saccharomyces cerevisiae mitochondrial Trx reductase Trr2 than by the wheat (Triticum aestivum) cytoplasmic reduced nicotinamide adenine dinucleotide phosphate-dependent Trx reductase. PsTrxo1 was able to activate alternative oxidase, and it was shown to interact with a number of mitochondrial proteins, including peroxiredoxin and enzymes mainly involved in the photorespiratory process.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Ana Jiménez (ajimenez{at}cebas.csic.es).

^[W] The online version of this article contains Web-only data.

www.plantphysiol.org/cgi/doi/10.1104/pp.109.138073

^* Corresponding author; e-mail ajimenez{at}cebas.csic.es.

Received March 4, 2009; accepted April 6, 2009; published April 10, 2009.