title: The four cytoplasmically made subunits of yeast mitochondrial cytochrome c oxidase are synthesized individually and not as a polyprotein.
abstract: Subunit-specific antisera prepared against each of the four cytoplasmically made subunits (IV, V, VI, and VII) of yeast mitochondrial cytochrome c oxidase (EC 1.9.3.1) were used to precipitate immunoreactive polypeptides that were synthesized either in vitro, in a cell-free protein-synthesizing system programmed with total yeast mRNA, or in vivo, in intact cells and in spheroplasts, under conditions of pulse labeling, pulse-chase labeling, and continuous labeling. Using N-formyl-[35S]Met-rTNA as the only radioactively labeled component in the cell-free system, we demonstrated (i) that each of the four cytoplasmically made subunits is synthesized as a separate entity and not as part of a polyprotein as was claimed by others; (ii) that subunits IV, V, and VI are synthesized as precursors, larger by 1500-3000 daltons than their mature counterparts; in contrast, subunit VII is not synthesized as a larger precursor. Precursor forms of subunits IV, V, and VI identical to those synthesized in vitro were also detected in vivo by pulse-labeling of spheroplasts. The observed disappearance of these larger forms after a chase is compatible with the notion that they represent short-lived precursors that are rapidly converted to their mature counterparts during or shortly after import into mitochondria. Furthermore, using N-formyl-[35S]Met-tRNA, we provide definitive evidence that two of the cytoplasmically made subunits (beta and gamma) of another oligomeric inner mitochondrial membrane protein (F1-ATPase, EC 3.6.1.3) are not synthesized as part of a polyprotein but as individual precursors.
mesh_terms: D000251:Adenosine Triphosphatases / Q000096:biosynthesis*; D003593:Cytoplasm / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000096:biosynthesis*; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000096:biosynthesis; D008928:Mitochondria / Q000378:metabolism*; D008970:Molecular Weight; D011498:Protein Precursors / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D046911:Macromolecular Substances; D008565:Membrane Proteins; D011498:Protein Precursors; D003576:Electron Transport Complex IV; D000251:Adenosine Triphosphatases