Transport of the Precursor for Rat‐Liver Ornithine Carbamoyltransferase into Mitochondria in vitro

1 Transport of the precursor form of rat liver ornithine carbanioyltransferase was studied in a cell-free reconstitution system. The precursor. synthesized in vitro. was converted to an apparently mature form of the enzyme by incubation with a mitochondria1 preparation of rat liver. On further purification of the preparation by isopycnic sucrose gradient centrifugation, the processing activity cosedimented with mitochondria. The processed pro-duct was no longer susceptible to externally added proteases. 2 The activity responsible for the conversion of the precursor to the mature form disappeared when the mitochondria were disrupted by digitonin treatment. sonication or freeze-thawing. The inactivation apparently pardleled the leakage of the marker enzyme of the intermembrane space from the mitochondria. 3 Processing of the precursor by intact mitocliondria was strongly inhibited by both 0.1 mM dinitroplienol and 1 μM carbonylcyanide p-trifluoromethoxyphenylhydrazone. However. neither KCN, NaN3. antimycin A nor oligomycin had any marked effect on the processing. 4 Mitochondria from rat spleen and ascites hepatonin cells, which lack ornithine carbamoyltransferase. were able to take up the enzyme precursor and to process it to the mature form of the enzyme. Mouse liver kind kidney mitochondria also took up and processed the rat precursor. 5 In pulse and pulse-chase experiments with isolated rat hepatocytes. the labeled precursor first appeared in the cytosol and then disappeared with a half-life of less than 3 min. Dinitrophenol and carbonylcyanide p-trifluoromethoxyphenylhydrazone failed to inhibit the disappearance of the precursor from the cytosol. The results indicate that the ornithine carbamoyltransferase precursor is transported into isolated mitochondria in association with the proteolytic processing to the mature enzyme. The transport-processing system requires mitochondria of high integrity. It was sensitive to uncouplers but not much to respiratory inhibitors. and may be common to more than one mitochondria1 enzyme precursor.