Products of the Mitochondrial Protein-Synthesizing System

The products of the mitochondrial protein-synthesizing system are all found in the inner mitochondrial membrane and are hydrophobic in nature. This hydrophobic nature makes it particularly difficult to identify mitochondrial products as part of a functional entity, since on extraction they are dissociated from other proteins of the entity and are often denatured. A more productive approach has been to eliminate the mitochondrial contribution by the administration of antibiotics or by mutation of the mitochondrial DNA, and assay the function in situ for altered properties. To date, possibly four inner membrane-associated functions have been implicated as containing products of the mitochondrial synthetic systems. In all cases they are multicomponent complexes containing both mitochon-drially and cytoplasmically synthesized components. These are the cytochrome oxidase complex, the oligomycin-sensitive ATPase, the cytochrome b complex, and the mitochondrial ribosomes. All of the other inner membrane functions which have so far been studied are synthesized entirely in the cytoplasm. 

Yeast cytochrome oxidase is composed of probably seven subunits shown by gel electrophoresis to have molecular weights of about 40,000 27,300 25,000 13,800 13,000 10,200 and 9500. Studies with protein synthesis inhibitors have demonstrated that both the cytoplasmic and mitochondrial systems contribute to the complex.Immunological studies by Kraml and Mahler have shown that cytoplasmic petite mutant cells contain the copper-associated protein of the complex, demonstrating the cytoplasmic origin of this component. Studies on the mitochondrial products have shown that the three proteins of highest molecular weight in the complex are mitochondrially synthesized and account for about half of the protein, by weight, of the cytochrome oxidase complex. 

Although the biogenesis of cytochrome b has not been closely examined in yeast, studies on Neurospora crassa have shown that in this organism, both the mitochondrial and cytoplasmic protein-synthesizing systems contribute to the cytochrome b complex. Cytochrome b probably contains two polypeptide types of molecular weight 30,000. Inhibition studies have shown that only one of these is translated on mitochondrial ribosomes (see Chapter 5). 

The mitochondrial ribosomes are associated with the inner mitochondrial membrane and are synthesized cooperatively in a fashion analogous to that of the cooperatively synthesized protein complexes of the inner membrane. It has been shown by inhibition studies that the ribosomal proteins are synthesized by the cytoplasmic protein-synthesizing system, while the ribosomal RNA is transcribed in the mitochondria from mitochondrial DNA, as mentioned above. 

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In all our studies on this diverse range of systems, we have been led back at some stage to the properties and function of the mitochondrial membrane. Seen in retrospect, this is not surprising, as the products of the mitochondrial protein-synthesizing system are all components of the inner mitochondrial membrane, and the mitochondrial replication, transcription, and translation systems are all inner-membrane-associated to some extent. 

Mitochondrial proteins arise both from the cytoplasmic protein-synthesizing system and the mitochondrial protein-synthesizing system. The proteins of the outer membrane are apparently synthesized entirely by the former, while the inner membrane is composed of products of both protein-synthesizing systems (for review Linnane et The differential effects of antibiotics on the protein-synthesizing systems have been used to estimate the total contribution of the mitochondrial system to the organelle. It has been shown that in catabolite-derepressed cells, the mitochondria synthesize about 10% of the total mitochondrial protein, while in ca-tabolite-repressed cells, the mitochondrial contribution is reduced to 4% of the total mitochondrial protein. 

We first became aware of the possibility that continued translation by the intramitochondrial system of protein synthesis might be contingent on the operations of the cytoribosomal system by the discovery that effective synthesis of cytochrome oxidase could be inhibited, not only by CAP, but also by CHX. This particular effect we now know to be due to the fact that this enzyme complex actually contains two classes of polypeptides synthesized by the two systems (see Sections 2.1.1 and 2.3.1). However, upon exploring the use of formate as a tag for mitochondrial translational products (Section 2.2.1), we met with very similar, and equally puzzling, observations. Now it was the incorporation of labeled formate into membrane proteins that appeared to be equally sensitive to both these in-... 

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