Inhibition of pyruvate carboxylase degradation and total protein breakdown by lysosomotropic agents in 3T3-L1 cells

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Abstract
Exposure to [3H]biotin during the differentiation of [mouse embryo] 3T3-L1 cells to adipocytes selectively labeled pyruvate carboxylase. A subsequent incubation of labeled cells permitted the measurement of the degradation rate constant of this mitochondrial enzyme. In medium without serum, pyruvate carboxylase was degraded with a half-life of 64 h, considerably longer than that found for average cell protein. The long half-life is commensurate with the enzyme being catabolized when whole mitochondria are destroyed. The breakdown of pyruvate carboxylase was inhibited to a greater extent than the breakdown of total cell protein by insulin, NH4Cl and inhibitors of lysosomal proteinases [leupeptin, chymostatin, pepstatin], suggesting that the enzyme is degraded by the autophagic lysosomal system of the cell. The above evidence implies that whole mitochondria are degraded in lysosomes, a conclusion that agrees with earlier EM evidence showing mitochondria within autophagic vacuoles. A second degradative pathway must be invoked to account for the breakdown of mitochondrial proteins of short half-life.