Biosynthesis and topogenesis of aspartate aminotransferase isoenzymes in chicken embryo fibroblasts. The precursor of the mitochondrial isoenzyme is either imported into mitochondria or degraded in the cytosol.
In chicken embryo fibroblasts pulsed wih [35S]methionine, a precursor of mitochondrial aspartate aminotransferase with higher molecular weight (delta Mr approximately 3000) was detected by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Peptide mapping of the precursor and the mature enzyme confirmed their precursor-product relationship. No precursor of the homologous cytosolic isoenzyme was found. The precursor of the mitochondrial isoenzyme is synthesized on membrane-free polysomes in the cytosol (Sonderegger, P., Jaussi, R., Christen, P., and Gehring, H. (1982) J. Biol. Chem. 257, 3339-3345); its half-life is 30 to 60 s. The pronounced susceptibility of the precursor toward exogenous proteases contrasts the stability of the mature enzyme and thus indicates that the conformation or the quarternary structure of the protein must change concomitantly with its import into mitochondria. Administration of the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) to the cell cultures blocks the import of many matrix and inner membrane proteins into mitochondria. The precursor of mitochondrial aspartate aminotransferase is found to be accumulated in the cytosol. However, its steady state concentration in CCCP-treated cells exceeds the concentration in untreated cells by not more than 1 order of magnitude. During a chase, the radioactive precursor disappears with a half-life of approximately 5 with min without formation of mature enzyme. Thus, in CCCP-treated cells, a degradative process is limiting the accumulation of the precursor in the cytosol. When the chase is performed in the presence of cysteamine, an antagonist of CCCP, the precursor is processed to the mature enzyme. Newly synthesized cytosolic aspartate aminotransferase is not degraded.
D000818:Animals; D001219:Aspartate Aminotransferases / Q000096:biosynthesis*; D001692:Biological Transport; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002478:Cells, Cultured; D002642:Chick Embryo; D003600:Cytosol / Q000201:enzymology; D005347:Fibroblasts / Q000201:enzymology; D007425:Intracellular Membranes / Q000378:metabolism; D007527:Isoenzymes / Q000096:biosynthesis; D008565:Membrane Proteins / Q000096:biosynthesis; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology*; D010446:Peptide Fragments / Q000032:analysis
D007527:Isoenzymes; D008565:Membrane Proteins; D010446:Peptide Fragments; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D001219:Aspartate Aminotransferases