Membrane and cytosolic components affecting transport of the precursor for ornithine carbamyltransferase into mitochondria.

C Argan,C J Lusty,G C Shore

doi:10.1016/s0021-9258(18)32263-4

Abstract

A higher molecular weight precursor (Mr = 39,000) to the liver mitochondrial matrix enzyme, ornithine carbamyltransferase (Mr = 36,000), is imported and processed by heart mitochondria in vitro in a manner similar to liver mitochondria. In both systems, however, an additional 37-kDa ornithine carbamyltransferase polypeptide appears, but this arises from nonspecific events and, therefore, does not represent a bona fide intermediate in the overall processing sequence. Our experiments demonstrate that the outer mitochondrial membrane of mitochondria contains a protease-sensitive (5 micrograms of trypsin or chymotrypsin/ml, 15 min at 2 degrees C), salt-resistant (1.0 M KCl) protein which is required to maintain import functions. In addition, functional post-translational import requires a component of the reticulocyte lysate (i.e. cytosol) that is used for initially synthesizing precursor enzyme. The component is retained by Sephadex G-25. Import of Sephadex G-25-excluded precursor is restored by fresh reticulocyte lysate but not by a combination of other additives, including Mg2+, K+, ATP, ADP, Pi, succinate, and total translation mixture (minus lysate).

Highlights

University, Mclntyre Medical Sciences Building, Montreal, Quebec, Canada H3G 1Y6 and §The Public Health Research Institute of the City of New York, Inc., New York, New York 10016
We suggest that the37K product arises in vitro due to leakage from mitochondria of the soluble matrix endoprotease described by Mori et al [4]; this protease was shown to convert precursor ornithine car
The precursor to ornithine carbamyltransferase is one of a limited number of mammalian mitochondrial proteins which have proven amenable to studies of import and processing by isolated mitochondria in vitro [4, 5, 13, 21,22,23,24, 26]

Summary

Introduction

I n Vitro Import andProcessing by Heart Mitochondria-Translation of rat liver mRNA was performed in a reticulocyte cell-free system [17]for 60 min at 30 "C, after which the reaction mixture was adjusted to 0.25 M sucrose and 10 pg/ml of cycloheximide.

Results

Conclusion