Degradation of the precursor of mitochondrial aspartate aminotransferase in chicken embryo fibroblasts.
The precursor of mitochondrial aspartate aminotransferase accumulates in the cytosol of cultured chicken embryo fibroblasts if its import into mitochondria is inhibited by an uncoupling agent. However, its accumulation is limited by degradation with a half-life of only approximately 5 min (Jaussi, R., Sonderegger, P., Flückiger, J., and Christen, P. (1982) J. Biol. Chem. 257, 13334-13340). The aim of the present study was the characterization of the proteolytic system(s) responsible for this very rapid intracellular degradation. On depleting chicken embryo fibroblasts of ATP, the rate of degradation of the precursor was lowered by approximately 70%. Chicken embryo fibroblasts depleted of divalent metal ions showed a degradative activity of 10% of the initial value. Reconstitution of these cells with Mg2+ and Ca2+ increased the degradative activity from 10 to 107 and 24%, respectively. Thiol reagents almost completely prevented the degradation, whereas specific peptide inhibitors of cysteine proteases or inhibitors of intralysosomal proteolysis decreased the rate of degradation by only approximately 30%. Inhibitors of serine proteases had little effect. No rapid degradation of the precursor was observed in crude extracts of chicken embryo fibroblasts. The data indicate that the bulk of the precursor accumulated under conditions of import block is degraded by one or several cytosolic proteases dependent on ATP, Mg2+, and thiol groups of unknown localization, conceivably by proteolytic enzymes identical with or similar to one of the high molecular weight cytosolic proteases (Waxman, L., Fagan, J.M., Tanaka, K., and Goldberg, A. L. (1985) J. Biol. Chem. 260, 11994-12000). The rest of the precursor appears to be degraded by lysosomes.
Flückiger
J
J
Biochemisches Institut, Universität Zürich, Switzerland.
Christen
P
P
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000976:Antipain / Q000378:metabolism; D001219:Aspartate Aminotransferases / Q000378:metabolism*; D002642:Chick Embryo; D003600:Cytosol / Q000201:enzymology; D004792:Enzyme Precursors / Q000378:metabolism*; D005347:Fibroblasts / Q000648:ultrastructure; D006207:Half-Life; D007976:Leupeptins / Q000378:metabolism; D008744:Methylamines / Q000494:pharmacology; D008928:Mitochondria / Q000201:enzymology*; D008985:Monensin / Q000494:pharmacology
D004792:Enzyme Precursors; D007976:Leupeptins; D008744:Methylamines; D000976:Antipain; D000255:Adenosine Triphosphate; D008985:Monensin; C027451:methylamine; D001219:Aspartate Aminotransferases; C032854:leupeptin