In vitro import into mitochondria of the precursor of mitochondrial aspartate aminotransferase.
The import of the precursor of mitochondrial aspartate aminotransferase was reconstituted in vitro with isolated mitochondria thus corroborating the earlier conclusion of a post-translational uptake. The higher Mr precursor was synthesized in a reticulocyte lysate programmed with free polysomes from chicken liver. After incubation with intact mitochondria from chicken heart about 50% of the precursor was converted to the mature form in a time-dependent process, its rate being a function of the amount of mitochondria added. The same amount of precursor was processed to the mature form on addition of a mitochondrial extract. No conversion to the mature enzyme took place when the precursor was incubated with intact mitochondria in the presence of the uncoupling agent carbonyl cyanide m-chlorophenylhydrazone or of the chelator o-phenanthroline which penetrates the mitochondrial inner membrane. In contrast, the chelator bathophenanthroline disulfonate which does not diffuse into the mitochondrial matrix did not inhibit the appearance of the mature form. The results indicate that that precursor must pass through an energized inner mitochondrial membrane before it is processed by a chelator-sensitive protease in the mitochondrial matrix. Excess mature mitochondrial aspartate aminotransferase did not compete with the precursor for its uptake into mitochondria. Mature mitochondrial aspartate aminotransferase is an alpha 2-dimer with Mr = 2 X 45,000. Both the precursor synthesized in a rabbit reticulocyte lysate and the precursor accumulated in the cytosol of carbonyl cyanide m-chlorophenylhydrazone-treated chicken embryo fibroblasts were found to exist as homodimer or hetero-oligomer and high Mr complexes (Mr greater than 300,000).
D000818:Animals; D001219:Aspartate Aminotransferases / Q000096:biosynthesis*; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002499:Centrifugation, Density Gradient; D002642:Chick Embryo; D002645:Chickens; D002850:Chromatography, Gel; D003600:Cytosol / Q000201:enzymology; D004792:Enzyme Precursors / Q000378:metabolism*; D007527:Isoenzymes / Q000096:biosynthesis; D008297:Male; D008928:Mitochondria / Q000201:enzymology*; D008929:Mitochondria, Heart / Q000201:enzymology; D008930:Mitochondria, Liver / Q000201:enzymology; D008970:Molecular Weight; D010618:Phenanthrolines / Q000494:pharmacology; D011132:Polyribosomes / Q000032:analysis
D004792:Enzyme Precursors; D007527:Isoenzymes; D010618:Phenanthrolines; C017049:bathophenanthroline disulfonic acid; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D001219:Aspartate Aminotransferases; C025205:1,10-phenanthroline