A leader peptide is sufficient to direct mitochondrial import of a chimeric protein.
Most mitochondrial proteins are encoded in the nucleus and synthesized in the cytoplasm as larger precursors containing NH2-terminal 'leader' peptides. To test whether a leader peptide is sufficient to direct mitochondrial import, we fused the cloned nucleotide sequence encoding the leader peptide of the mitochondrial matrix enzyme ornithine transcarbamylase (OTC) with the sequence encoding the cytosolic enzyme dihydrofolate reductase (DHFR). The fused sequence, joined with SV40 regulatory elements, was introduced along with a selectable marker into a mutant CHO cell line devoid of endogenous DHFR. In stable transformants, the predicted 26-K chimeric precursor protein and two additional proteins, 22 K and 20 K, were detected by immunoprecipitation with anti-DHFR antiserum. In the presence of rhodamine 6G, an inhibitor of mitochondrial import, only the chimeric precursor was detected. Immunofluorescent staining of stably transformed cells with anti-DHFR antiserum produced a pattern characteristic of mitochondrial localization of immunoreactive material. When the chimeric precursor was synthesized in a cell-free system and incubated post-translationally with isolated rat liver mitochondria, it was imported and converted to a major product of 20 K that associated with mitochondria and was resistant to proteolytic digestion by externally added trypsin. Thus, both in intact cells and in vitro, a leader sequence is sufficient to direct the post-translational import of a chimeric precursor protein by mitochondria.
D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D002678:Chimera; D006224:Cricetinae; D003412:Cricetulus; D005455:Fluorescent Antibody Technique; D066298:In Vitro Techniques; D008928:Mitochondria / Q000378:metabolism*; D009954:Ornithine Carbamoyltransferase / Q000235:genetics; D010957:Plasmids; D014176:Protein Biosynthesis; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D014170:Transformation, Genetic
D013762:Tetrahydrofolate Dehydrogenase; D009954:Ornithine Carbamoyltransferase