We have isolated the yeast ATP2 gene encoding the beta-subunit of mitochondrial ATP synthase and determined its nucleotide sequence. A fusion between the N-terminal 15 amino acid residues of beta-subunit and the mouse cytosolic protein dihydrofolate reductase (DHFR) was transcribed and translated in vitro and found to be transported into isolated yeast mitochondria. A fusion with the first 35 amino acid residues of beta-subunit attached to DHFR was not only transported but also proteolytically processed by a mitochondrial protease. Amino acid substitutions were introduced into the N-terminal presequence of the beta-subunit by bisulphite mutagenesis of the corresponding DNA. The effects of these mutations on mitochondrial targeting were assessed by transport experiments in vitro using DHFR fusion proteins. All of the mutants, harbourin from one to six amino acid substitutions in the first 14 residues of the presequence, were transported into mitochondria, though at least one of them (I8) was transported and proteolytically processed at a much reduced rate. The I8 mutant beta-subunit also exhibited poor transport and processing in vivo, and expression of this mutant polypeptide failed to complement the glycerol- phenotype of a yeast ATP2 mutant. More remarkably, the expression of I8 beta-subunit induced a more general growth defect in yeast, possibly due to interference with the transport of other, essential, mitochondrial proteins.
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February 1990
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Research Article|
February 15 1990
Transport of the yeast ATP synthase β-subunit into mitochondria. Effects of amino acid substitutions on targeting
M E Walker;
M E Walker
1Department of Microbiology, University of Edinburgh, West Mains Road, Edinburgh EH9 3JG, U.K.
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E Valentin;
E Valentin
1Department of Microbiology, University of Edinburgh, West Mains Road, Edinburgh EH9 3JG, U.K.
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G A Reid
G A Reid
1Department of Microbiology, University of Edinburgh, West Mains Road, Edinburgh EH9 3JG, U.K.
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Biochem J (1990) 266 (1): 227–234.
Citation
M E Walker, E Valentin, G A Reid; Transport of the yeast ATP synthase β-subunit into mitochondria. Effects of amino acid substitutions on targeting. Biochem J 15 February 1990; 266 (1): 227–234. doi: https://doi.org/10.1042/bj2660227
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