Transport and processing of the precursors of mitochondrial carbamoyl phosphate synthetase I and ornithine transcarbamoylase.

The mitochondrial matrix of ureotelic animal liver contains CPS I* and OTCase* in amounts of the order of 20% and 0.5% respectively of the matrix proteins. CPS I exists as a dimer consisting of two identical subunits (MI approx 160000 each) and OTCase exists as a trimer consisting of three identical subunits (MI approx. 36000 each). mRNA molecules for both enzymes are transcribed from nuclear genes and can be translated in a cell-free protein-synthesizing system by free cytoplasmic polyribosomes. The translation product of the mRNA for CPS I has MI approx. 165000 and is a precursor (pCPS 1)of CPS I. The translation product of the mRNA for OTCase has M, approx. 39500 (pOTCase) and is a precursor of OTCase (for reviews, see Mori et al., 1982, 1983). pCPS I synthesized from mRNA preparations of rat liver (Shore et al., 1979; Raymond & Shore, 1979; Mori et al., 1979)exists in an aggregation state larger than that of CPS I (Miura et al., 1981) and has a half-life in the cytoplasm of about 2min (Mori et al., 1981a; Raymond & Shore, 1981). The uptake and processing of pCPS I by isolated mitochondria has been reported to be inhibited by paminobenzamidine, with degradation of the precursor (Raymond & Shore, 1981). Treatment of isolated rat hepatocytes with Rhodamine 123 strongly inhibits processing of both pCPS I and pOTCase, with accumulation of the precursors (Morita et al., 1982~). Import of pCPS I into mitochondria from foetalrat liver has been demonstrated by Campbell et a/ . (1982). Studies by Conboy et al. (1979) and Mori et al. (1980a) provided initial evidence for the synthesis of pOTCase from mRNA preparations of rat liver. Subsequent studies revealed that pOTCase : (1) has an N-terminal peptide not present in OTCase (Mori et al., 1980~; Kraus et al., 1981); (2) is synthesized on free polyribosomes (Morita et al., 1981); (3) exists in an aggregated form and in a conformation different from that of OTCase (Miura et ol., 1981) and (4) has a higher isoelectric point that OTCase (Miura et al., 19826). pOTCase has a half-life in the cytoplasm of 1-2min (Mori et al., 1981~). Transport of pOTCase into mitochondria and processing of pOTCase to form OTCase have been studied in a system consisting of isolated intact mitochondria and preparations of isotopically labelled pOTCase. Mori et al. (1982) have reviewed the optimal requirements for this system. Evidence that the import process is dependent on an energy source and a membrane potential is indicated by the inhibitory effect of uncoupling agents (Mori etal., 1981~; Morita et al., 1982b; Kolansky et a / . , 1982). Additional requirements for optimal activity are K + ( l 2 0 m ~ ) and Mg2+ (approx. 1 . 6 m ~ ) (Miura et al., 1983) plus a reticulocyte cytosolic protein(s) (Mori et al., 1982; Miura et al., 1983; Argan et d., 1983). A similar, if not identical, factor is present in dialysed supernatant of rabbit erythrocyte lysate (Mori et al., 1982; Miura et al., 1983). Import of pOTCase is not specific for rat liver mitochondria, even though these organelles (other than mitochondria of the intestinal mucosa; see Cohen, 1981a) are the only ones containing OTCase in a ureotelic animal such as the