Cell-Free Synthesis of a Larger-Molecular-Weight Precursor of Cytochrome c Oxidase Subunit V from Rat Liver and the Distribution of Its mRNA between Free and Membrane-Bound Polysomes

Abstract

Poly(A)-rich RNA from phenol-extracted rat liver polysomes was translated in a heterologous cell-free system derived from wheat germ. The labeled translation products were incubated with an antiserum against cytochrome c oxidase subunit V. After immunoprecipitation and affinity chromatography with protein-A-Sepahrose, the isolated antigen-Ig complexes were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoersis and fluorography. Only 1 protein with an apparent MW of 15,500 was visualized. In immunocompetition experiments with unlabeled individual cytochrome c oxidase subunits IV, V, VI or VII only subunit V could compete with the 15,500-MW protein synthesized in vitro. Two-dimensional fingerprints of cytochrome c oxidase subunit V and the polypeptide synthesized in vitro showed a high degree of similarity. The cytochrome c oxidase subunit V is synthesized as a precursor with an amino-terminal extension of about 25 amino acids. It was possible to convert the precursor of cytochrome C oxidase subunit V synthesized in vitro to its mature form by intact mitochondria as well as by submitochondrial particles. A chain length of 830 .+-. 70 nucletoides was estimated for the poly(A)-rich mRNA of the higher-molecular-weight precursor of rat liver cytochrome c oxidase subunit V. Assuming a MW of 15,500 for the precursor a non-coding region of about 300 nucleotides must exist. In experiments on the site of synthesis the poly(A)-rich RNA for the higher-molecular-weight precursor of cytochrome c oxidase subunit V is found in free, loosely and tightly membrane-bound polyribosomes.