Immunoprecipitation of a Cytoplasmic Precursor of Rat-Liver Cytochrome Oxidase

Abstract

A simple method for the isolation of rat liver cells is described. The cells are shown, by an isotope dilution method, to maintain a constant rate of protein synthesis for 8 h of incubation. Antibodies to purified rat liver cytochrome oxidase were raised in rabbits and used to investigate the labeling of cytochrome oxidase in isolated rat liver cells and in vivo. The data demonstrate the occurrence of a precursor of the subunits of cytochrome oxidase that are synthesized in the cytoplasm.1. Dodecylsulfate gel electrophoresis of the immunoprecipitates from isolated rat liver cells that had been labeled with [35S]methionine for 1 h showed a single radioactive peak with a molecular weight of 50000.2. Judged by the effects of cycloheximide and chloramphenicol the labeled protein is synthesized on cytoplasmic ribosomes.3. After labeling for 1 h in vivo with [3H]leucine the labeled protein appears to be exclusively associated with the hepatic niicrosomal fraction.4. Ouchterlony double-diffusion analysis demonstrated immunological relationship between the precipitates from microsomes and cytochrome oxidase. In addition to the precipitates derived from mitochondria and microsomes immunoprecipitates were also obtained from the cytosol in comparable amounts ; these again were immunologically related.The occurrence of large amounts of precursor(s) (or degradation products) of cytochrome oxidase in rat liver fractions is interpreted in terms of a regulatory pool for amino acid homeostasis in the organism.Cytochrome oxidase, the terminal oxygen acceptor of the respiratory chain in the inner mitochondrial membrane, represents a hydrophobic enzyme complex that is composed of seven protein subunits, two heme a groups, two copper atoms and tightly bound lipids (for reviews see [l-31). The biosynthesis of the enzyme is a complex process involving the assembly of four small subunits that are coded for in the nucleus and synthesized on cytoplasmic ribosomes, and three larger subunits that are coded for and synthesized within the mitochondria (for reviews see . Although these results are mainly based on studies with yeast similar data on subunit composition [9, of the cytoplasmically synthesized subunits and on the mechanism of assembling the enzyme complex. Poyton et al. described the extramitochondrial occurrence of a precursor protein for the four small subunits of yeast cytochrome oxidase which stimulates the synthesis of the three large subunits in mitochondria and which is immunoprecipitated from a postpolysomal supernatant fraction by antibodies to the holoenzyme 129,301. This protein has a molecular weight of 55000 and is thought to contain a small peptide in addition to the sequences of the four cytoplasmically synthesized subunits of cytochrome oxidase. Rucker et al. also presented evidence for the occurrence of precursor subunits VI and VII in the postribosomal cytoplasmic supernatant fraction of Neurospora crassa cells [31]. An intermediate in the assembly of mitochondria1 and cytoplasmically synthesized subunits of cy...

Mechanisms for the incorporation of proteins in membranes and organelles.

“…With the exception of cytochrome c and the ADP-ATP translocators of the inner mitochondrial membrane (133,251,252), for which high molecular weight precursors have not been detected, studies with other mitochondrial polypeptides of cytoplasmic origin, such as subunit V of cytochrome bCL (39), cytochrome c peroxidase (129), citrate synthase (85), subunits of cytochrome oxidase (171), and ATPase (120,128,192), as well as polypeptides of the mitochondrial matrix such as carbamylphosphate synthetase (141,203) and ornithine transcarbamylase (38), have shown that these polypeptides are released from cytoplasmic polysomes, as precursors that are proteolytically processed upon entrance into the organelle . A similar mechanism appears to operate in chloroplasts.…”

Regulation of mitochondrial biogenesis. Occurrence of non-functioning components of the mitochondrial respiratory chain in Saccharomyces cerevisiae grown in the presence of proteinase inhibitors: evidence for proteolytic control over assembly of the respiratory chain

“…This can hardly be explained by any side effects of anti-proteolytic agents, such as suppression or distortion of the synthesis and/or assembly process. An intriguing issue arising in connection with the conceivable sequelae of proteinase inhibitors is that several mitochondrial proteins, including some respiratorychain components, have been reported to be synthesized as larger precursors (see, e.g., Ries et al, 1978;Schatz, 1979;Poyton & McKemmie, 1979a,b). An implication of these findings is that mitochondrial assembly involves a proteolytic processing step that might be affected by inhibitor treatment.…”

Hydrophobic interaction between the monomer of mitochondrial malate dehydrogenase and phospholipid membranes

“…At present there is no direct evidence for transport pores, vectorial discharge or post-transcriptional modification concerned with the transport of the soluble matrix proteins (Godinot & Lardy, 1973;Hallermayer et al, 1977;Harmey et al, 1977;Marra et al, 1978). This is in contrast with some intrinsic membrane proteins, which have been shown to be synthesized in precursor forms (Ries et al, 1978;Maccecchini et al, 1979;Cote et al, 1979). By using mitochondrial malate dehydrogenase (EC 1.1.1.37) from porcine heart and model phospholipid membranes our studies have been directed towards determining how transport of this enzyme into the matrix occurs (Webster et al, 1979).…”