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Molecular and Cellular Biology, June 2003, p. 4000-4012, Vol. 23, No. 11
0270-7306/03/$08.00+0     DOI: 10.1128/MCB.23.11.4000-4012.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

In Vitro Import of a Nuclearly Encoded tRNA into Mitochondria of Solanum tuberosum

Ludovic Delage,{dagger} André Dietrich, Anne Cosset, and Laurence Maréchal-Drouard*

Institut de Biologie Moléculaire des Plantes, UPR 2357 CNRS, Université Louis Pasteur, 67084 Strasbourg Cedex, France

Received 13 January 2003/ Accepted 14 March 2003

Some of the mitochondrial tRNAs of higher plants are nuclearly encoded and imported into mitochondria. The import of tRNAs encoded in the nucleus has been shown to be essential for proper protein translation within mitochondria of a variety of organisms. Here, we report the development of an in vitro assay for import of nuclearly encoded tRNAs into plant mitochondria. This in vitro system utilizes isolated mitochondria from Solanum tuberosum and synthetic tRNAs transcribed from cloned nuclear tRNA genes. Although incubation of radioactively labeled in vitro-transcribed tRNAAla, tRNAPhe, and tRNAMet-e with isolated potato mitochondria resulted in importation, as measured by nuclease protection, the amount of tRNA transcripts protected at saturation was at least five times higher for tRNAAla than for the two other tRNAs. This difference in in vitro saturation levels of import is consistent with the in vivo localization of these tRNAs, since cytosolic tRNAAla is naturally imported into potato mitochondria whereas tRNAPhe and tRNAMet-e are not. Characterization of in vitro tRNA import requirements indicates that mitochondrial tRNA import proceeds in the absence of any added cytosolic protein fraction, involves at least one protein component on the surface of mitochondria, and requires ATP-dependent step(s) and a membrane potential.

* Corresponding author. Mailing address: Institut de Biologie Moléculaire des Plantes du CNRS, 12 rue du Général Zimmer, 67084 Strasbourg Cedex, France. Phone: (33) 3 88 41 72 44. Fax: (33) 3 88 61 44 42. E-mail: laurence.drouard{at}ibmp-ulp.u-strasbg.fr.

{dagger} Present address: Station Biologique, UMR 1931 CNRS, 29680 Roscoff Cedex, France.

Molecular and Cellular Biology, June 2003, p. 4000-4012, Vol. 23, No. 11
0022-538X/03/$08.00+0     DOI: 10.1128/MCB.23.11.4000-4012.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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