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In vitro import into mitochondria of the precursor of mitochondrial aspartate aminotransferase
Abstract:
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).
Insights
This study demonstrates that the precursor to mitochondrial aspartate aminotransferase is imported into mitochondria post-translationally. The precursor requires passage through an energized inner mitochondrial membrane for processing by a matrix protease.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Mitochondrial proteins are synthesized in the cytosol and imported into mitochondria post-translationally.
- Mitochondrial aspartate aminotransferase (mAspAT) is a key enzyme in cellular metabolism.
- Understanding the import and processing of mAspAT is crucial for mitochondrial function.
Purpose of the Study:
- To investigate the in vitro import and processing of the mAspAT precursor.
- To elucidate the role of mitochondrial membranes and matrix factors in mAspAT maturation.
- To characterize the oligomeric state of the mAspAT precursor.
Main Methods:
- In vitro import assays using isolated chicken heart mitochondria and reticulocyte lysate-synthesized mAspAT precursor.
- Enzyme activity assays to monitor conversion to mature mAspAT.
- Inhibition studies using uncouplers and chelators to probe membrane energization and protease activity.
- Analysis of precursor and mature enzyme oligomeric states via gel electrophoresis.
Main Results:
- Reconstitution of mAspAT precursor import and maturation in vitro.
- Maturation is a time- and mitochondria-dependent process.
- Import and processing require an energized inner mitochondrial membrane and a chelator-sensitive mitochondrial matrix protease.
- The mAspAT precursor exists as dimers and high molecular weight complexes.
Conclusions:
- The mAspAT precursor undergoes post-translational import into mitochondria.
- Mitochondrial inner membrane energization is essential for precursor translocation.
- Processing occurs in the mitochondrial matrix via a protease sensitive to chelators.
- The precursor's oligomeric state differs from the mature enzyme.