Transport of the yeast ATP synthase beta-subunit into mitochondria. Effects of amino acid substitutions on targeting.
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.
Walker
M E
ME
Department of Microbiology, University of Edinburgh, U.K.
Valentin
E
E
Reid
G A
GA
D000595:Amino Acid Sequence; D001483:Base Sequence; D001692:Biological Transport; D004926:Escherichia coli / Q000235:genetics; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D009154:Mutation; D011498:Protein Precursors / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D013329:Structure-Activity Relationship; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D014170:Transformation, Genetic
D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D013762:Tetrahydrofolate Dehydrogenase; D006180:Proton-Translocating ATPases