Posttranslational uptake and processing of in vitro synthesized ornithine transcarbamoylase precursor by isolated rat liver mitochondria.

Abstract

The mitochondrial matrix enzyme ornithine transcarbamoylase (OTCase; ornithine carbamoyltransferase; carbamoylphosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3) is encoded by a nuclear gene on the X chromosome, synthesized on cytoplasmic ribosomes and translocated across both mitochondrial membranes. Using specific immunoprecipitation evidence was presented previously that the primary in vitro translation product of OTCase in rat liver is a polypeptide about 4000 daltons larger than the mature OTCase subunit purified from homologous mitochondria. In this report the immunological identification of this cell-free translation product (pOTCase is augmented) with structural information. By electrophoresis of proteolysis products pOTCase is shown to be structurally similar to mitochondrial OTCase. When pOTCase is incubated posttranslatinally with isolated rat liver mitochondria, it is converted to the size of mature OTCae and is sequestered within the mitochondria in such a way that it becomes resistant to externally added proteases. Such posttranslatinal processing is catalyzed specifically by the mitochondrial fraction of rat liver cells and is dependent both on the duration of incubation with mitochondria and on the amount of mitochondrial protein added. Apparently, pOTCase is indeed the bona fide precursor of mitochondrial OTCase and that use of this simplified cell-free system will fascilitate analysis of OTCase biogenesis at both the cellular and the molecular level.