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Molecular and Cellular Biology, September 1999, p. 6253-6259, Vol. 19, No. 9
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
In Vitro Import of a Nuclearly Encoded tRNA into
the Mitochondrion of Trypanosoma brucei
Audra E.
Yermovsky-Kammerer and
Stephen L.
Hajduk*
Department of Biochemistry and Molecular
Genetics, Schools of Medicine and Dentistry, University of Alabama
at Birmingham, Birmingham, Alabama 35294
Received 3 March 1999/Returned for modification 14 April
1999/Accepted 12 June 1999
All of the mitochondrial tRNAs of Trypanosoma brucei
have been shown to be encoded in the nucleus and must be imported into the mitochondrion. The import of nuclearly encoded tRNAs into the
mitochondrion has been demonstrated in a variety of organisms and is
essential for proper function in the mitochondrion. An in vitro import
assay has been developed to study the pathway of tRNA import in
T. brucei. The in vitro system utilizes crude isolated
trypanosome mitochondria and synthetic RNAs transcribed from a cloned
nucleus-encoded tRNA gene cluster. The substrate, composed of
tRNASer and tRNALeu, is transcribed in tandem
with a 59-nucleotide intergenic region. The tandem tRNA substrate is
imported rapidly, while the mature-size tRNALeu fails to be
imported in this system. These results suggest that the preferred
substrate for tRNA import into trypanosome mitochondria is a precursor
molecule composed of tandemly linked tRNAs. Import of the tandem tRNA
substrate requires (i) a protein component that is associated with the
surface of the mitochondrion, (ii) ATP pools both outside and within
the mitochondrion, and (iii) a membrane potential. Dissipation of the
proton gradient across the inner mitochondrial membrane by treatment
with an uncoupling agent inhibits import of the tandem tRNA substrate.
Characterization of the import requirements indicates that
mitochondrial RNA import proceeds by a pathway including a protein
component associated with the outer mitochondrial membrane,
ATP-dependent steps, and a mitochondrial membrane potential.
*
Corresponding author. Mailing address: Department of
Biochemistry and Molecular Genetics, Schools of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, AL 35294. Phone: (205)
934-6033. Fax: (205) 975-2547. E-mail:
shajduk{at}bmg.bhs.uab.edu.
Molecular and Cellular Biology, September 1999, p. 6253-6259, Vol. 19, No. 9
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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