Transport of carbamyl phosphate synthetase I and ornithine transcarbamylase into mitochondria. Inhibition by rhodamine 123 and accumulation of enzyme precursors in isolated hepatocytes.

Carbamoyl-phosphate synthetase (ammonia) (EC 6.3.4.16) and ornithine carbamoyltransferase (EC 2.1.3.3) 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, a laser dye which specifically stains mitochondria, resulted both in a strong inhibition of the processing of the enzyme precursors and in accumulation. Rhodamine 123 did not specifically inhibit the synthesis of the synthetase and the transcarbamylase precursors in a reticulocyte lysate cell-free system programmed with rat liver free polysomes. The dye strongly inhibited the uptake and processing of the ornithine transcarbamylase precursor by isolated rat liver mitochondria. When the mitochondria and the medium were separated by centrifugation, the unprocessed precursor was recovered almost exclusively in the medium. These results indicate that rhodamine 123 inhibits either the binding of the enzyme precursors to the mitochondria or their transport into the organelle.