Synthesis, intracellular transport, and processing of the precursors for mitochondrial ornithine transcarbamylase and carbamoyl-phosphate synthetase I in isolated hepatocytes.

Abstract

The synthesis and intracellular transport of the mitochondrial matrix enzymes ornithine transcarbamylase (carbamoylphosphate: L-ornithine carbamoyltransferase, EC 2.1.3.3.) and carbamoyl-phosphate synthetase (ammonia) I [carbon-dioxide:ammonia ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.4.16] were studied in isolated rat hepatocytes. In pulse experiments at 37C, the larger precursors of the two enzymes appeared in the cytosol ofthe liver cells, where radioactivity levels of the precursors reached a plateau in 10-20 min after the pulse. The pulse-labeled mature enzymes appeared in the particulate fraction (containing mitochondria) after a time lag and increased almost linearly with time up to 40 min. The specific radioactivities of the precursors in the cytosol were much higher than those of the mature enzymes in the particulate fraction. In pulse-chase experiments, the labeled precursors disappeared from the cytosol with estimated half-lives of about 1-2 min. These results indicate that ornithine transcarbamylase and carbamoyl-phosphate synthetase I are initially synthesized as larger precursors and exist in a cytosolic pool from which they are transported into mitochondria and processed there to the mature enzymes concomitandy with or immediately after transport. Although the rates of synthesis, transport, and processing were decreased about 3-fold at 25'C (as compared to incubation at 37C), the pool size of the precursors in the cytosol were somewhat larger at this temperature.

Expression analysis of two mutant human ornithine transcarbamylases in COS-7 cells

“…On the other hand, processed, mature OTC appeared with a lag time of about 4 min and then increased rapidly. Processing of pOTC reflects its import into the mitochondria, since it occurs in the mitochondrial matrix during or immediately after the mitochondrial import (Mori et al 1981;Conboy and Rosenberg 1981). When the cells expressing A152V pOTC were labeled, labeled mutant pOTC was synthesized as rapidly as wild-type pOTC, but its increase was less than that of the wild-type.…”

“…Mitochondrial protein import and processing are very rapid (Mori et al 1981) and cannot be analyzed by routine pulse-chase experiments lasting several hours. In the present study, we report the first analysis of an OTC mutation by using a rapid pulse-chase procedure lasting for a few minutes.…”

“…OTC is initially synthesized as a larger precursor with the NH 2 -terminal presequence (pOTC), and is then imported into the mitochondrial matrix and processed to the mature form (Mori et al 1981;Conboy and Rosenberg 1981;Horwich et al 1984;Takiguchi et al 1984).…”

Selection of a cDNA clone which contains the complete coding sequence for the mature form of ornithine transcarbamylase from rat liver: expression of the cloned protein in Escherichia coli Molecular cloning of rat ornithine transcarbamylase. Molecular cloning of rat ornithine transcarbamylase

“…The precursor (pOTCase) is translocated across the mitochondrial membranes by an energy-dependent process [4] and proteolytically cleaved to a mature size of 36000 Da [5]. Pulse-labelling experiments show the pOTCase is rapidly removed from the cytoplasm with a half-life of 1 -2 min [5].…”

“…The enzyme is encoded by a nuclear gene and synthesized on membrane-free polysomes as a precursor which has a 3 400 -4000-Da N-terminal extension [2,3]. The precursor (pOTCase) is translocated across the mitochondrial membranes by an energy-dependent process [4] and proteolytically cleaved to a mature size of 36000 Da [5]. Pulse-labelling experiments show the pOTCase is rapidly removed from the cytoplasm with a half-life of 1 -2 min [5].…”