The first twelve amino acids of a yeast mitochondrial outer membrane protein can direct a nuclear-coded cytochrome oxidase subunit to the mitochondrial inner membrane.
We have used an in vivo complementation assay to test whether a given polypeptide sequence can direct an attached protein to the mitochondrial inner membrane. The host is a previously described yeast deletion mutant that lacks cytochrome oxidase subunit IV (an imported protein) and, thus neither assembles cytochrome oxidase in its mitochondrial inner membrane nor grows on the non-fermentable carbon source, glycerol. Growth on glycerol and cytochrome oxidase assembly are restored to the mutant if it is transformed with the gene encoding authentic subunit IV precursor, a protein carrying a 25-residue transient pre-sequence. No restoration is seen with a plasmid encoding a subunit IV precursor whose pre-sequence has been shortened to seven residues. Partial, but significant restoration is achieved by an artificial subunit IV precursor in which the authentic pre-sequence has been replaced by the first 12 amino acids of a 70-kd protein of the mitochondrial outer membrane. If this dodecapeptide is fused to the amino terminus of mouse dihydrofolate reductase (a cytosolic protein), the resulting fusion protein is imported into the matrix of yeast mitochondria in vitro and in vivo. Import in vitro requires an energized inner membrane. We conclude that the extreme amino terminus of the 70-kd outer membrane protein can direct an attached protein across the mitochondrial inner membrane.
D001692:Biological Transport; D002451:Cell Compartmentation; D002467:Cell Nucleus / Q000502:physiology; D004274:DNA, Recombinant; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000235:genetics / Q000378:metabolism*; D005816:Genetic Complementation Test; D007425:Intracellular Membranes / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism*; D010101:Oxygen Consumption; D011498:Protein Precursors / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism
D004274:DNA, Recombinant; D011498:Protein Precursors; D013762:Tetrahydrofolate Dehydrogenase; D003576:Electron Transport Complex IV