Synthesis, intracellular transport and processing of mitochondrial urea cycle enzymes.

Carbamyl phosphate synthetase I and ornithine transcarbamylase are matrix enzymes synthesized outside the mitochondria in the form of larger precursors and are transported rapidly into mitochondria, in association with post-translational proteolytic processing to the mature enzymes. Treatment of isolated rat hepatocytes with 40 micrograms/ml of rhodamine 123 resulted both in a potent inhibition of the processing of the enzyme precursors and in accumulation of the precursors. In pulse-chase experiments, the labeled precursor disappeared much more slowly in the presence of the dye. Rhodamine 123 strongly inhibited the uptake and processing of the ornithine transcarbamylase precursor by isolated rat liver mitochondria. Other positively charged rhodamines such as rhodamines 6G and 6GX were also strongly inhibitory. On the other hand, rhodamine B which has no net charge was much less inhibitory. These results suggest that the positively charged rhodamines inhibit the binding of the positively charged enzyme precursors to a negatively charged protein(s) or to phospholipids of the mitochondrial outer membrane. Potassium and magnesium ions, and probably a cytosolic protein(s), were required for the maximal uptake and processing of the ornithine transcarbamylase precursor by the isolated mitochondria. The concentrations of potassium and magnesium ions required for the maximal transport and processing were about 120 mM and 0.8-1.6 mM, respectively. Dialyzed postribosomal supernatant of rabbit reticulocyte lysate (36-72 mg protein/ml), in combination with potassium and magnesium ions, stimulated the precursor transport and processing 3- to 4-fold. The stimulatory activity of the dialyzed lysate was inactivated by trypsin treatment or heat treatment. No significant amount of the enzyme precursor was associated with the mitochondria when incubation was performed in the absence of these compounds. All these results indicate that potassium and magnesium ions, and probably a cytosolic protein(s), are required for the binding of the ornithine transcarbamylase precursor to the mitochondria or its transport into the organelle.