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Eukaryotic Cell, October 2008, p. 1750-1757, Vol. 7, No. 10
1535-9778/08/$08.00+0     doi:10.1128/EC.00206-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Protein Import into Hydrogenosomes of Trichomonas vaginalis Involves both N-Terminal and Internal Targeting Signals: a Case Study of Thioredoxin Reductases ^,

Marek Mentel, Verena Zimorski, Patrick Haferkamp, William Martin, and Katrin Henze^*

Institute of Botany III, University of Duesseldorf, D-40225 Duesseldorf, Germany

Received 23 June 2008/ Accepted 21 July 2008

The parabasalian flagellate Trichomonas vaginalis harbors mitochondrion-related and H₂-producing organelles of anaerobic ATP synthesis, called hydrogenosomes, which harbor oxygen-sensitive enzymes essential to its pyruvate metabolism. In the human urogenital tract, however, T. vaginalis is regularly exposed to low oxygen concentrations and therefore must possess antioxidant systems protecting the organellar environment against the detrimental effects of molecular oxygen and reactive oxygen species. We have identified two closely related hydrogenosomal thioredoxin reductases (TrxRs), the hitherto-missing component of a thioredoxin-linked hydrogenosomal antioxidant system. One of the two hydrogenosomal TrxR isoforms, TrxRh1, carried an N-terminal extension resembling known hydrogenosomal targeting signals. Expression of hemagglutinin-tagged TrxRh1 in transfected T. vaginalis cells revealed that its N-terminal extension was necessary to import the protein into the organelles. The second hydrogenosomal TrxR isoform, TrxRh2, had no N-terminal targeting signal but was nonetheless efficiently targeted to hydrogenosomes. N-terminal presequences from hydrogenosomal proteins with known processing sites, i.e., the alpha subunit of succinyl coenzyme A synthetase (SCS) and pyruvate:ferredoxin oxidoreductase A, were investigated for their ability to direct mature TrxRh1 to hydrogenosomes. Neither presequence directed TrxRh1 to hydrogenosomes, indicating that neither extension is, by itself, sufficient for hydrogenosomal targeting. Moreover, SCS lacking its N-terminal extension was efficiently imported into hydrogenosomes, indicating that this extension is not required for import of this major hydrogenosomal protein. The finding that some hydrogenosomal enzymes require N-terminal signals for import but that in others the N-terminal extension is not necessary for targeting indicates the presence of additional targeting signals within the mature subunits of several hydrogenosome-localized proteins.

Published ahead of print on 1 August 2008.

Supplemental material for this article may be found at http://ec.asm.org/.

Present address: Department of Biochemistry, Faculty of Natural Sciences, Comenius University, Mlynská dolina CH-1, 842 15 Bratislava, Slovakia.

Present address: Molekulare Enzymtechnologie, Universität Duisburg-Essen, Campus Duisburg, Geibelstr. 41, 47057 Duisburg, Germany.