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.

Synthesis and Intracellular Transport of Mitochondrial Proteins

“…Poyton and McKemmie (1979) suggested from their studies on the biosynthesis of cytochrome oxidase that the synthesis of subunits I, II, and III by mitochondria was stimulated by a precursor to subunits IV-VII. The data on which this suggestion was made is open to question as they also suggested that the precursor was a polyprotein which is not in accordance with recent findings by other groups (Schmelzer and Heinrich, 1980;Lewin et aL, 1980;Mihara and Blobel, 1980). /X.…”

In vitro synthesis, glycosylation, and membrane insertion of the four subunits of Torpedo acetylcholine receptor.

“…Calf liver tRNA was aminoacylated in the presence of [mS]methionine and formylated as described (19). The prepartion was free of ['S]MettRNAmet by the assay of Mihara and Blobel (19). Gel electrophoresis was performed throughout on NaDodSO4/7.5-15% linear gradient polyacrylamide gels as described (12), and isotopically labeled proteins were visualized by fluorography (20) of the stained dried gels.…”

“…The translocation activity of these membranes was enhanced with rough endoplasmic reticulum signal recognition protein (18) at 12 equivalents per piof membranes in a 200-,ul reaction mixture. Calf liver tRNA was aminoacylated in the presence of [mS]methionine and formylated as described (19). The prepartion was free of ['S]MettRNAmet by the assay of Mihara and Blobel (19).…”

Biogenesis of Mitochondrial Energy Transducing Complexes

“…Posttranslational transport. In this case, the fully synthesized peptide chain is released from the polysome prior to membrane association, Mihara and Blobel (1980); Lewin et al (1980); Wright et al (1984) 5,000-7,500 N.D. Mihara and Blobel (1980) …”

“…(1983c) Korb and Neupert (1978); Zimmermann et al (1979a) Matsuura et al (1981 Hatalovä and Kolarov (1983) Schmelzer and Heinrich (1980); Heinrich (1982) Mihara and Blobel (1980); Maarse et al (1984); Hurt et al (1984b) Heinrich (1982 Mihara and Blobel (1980); Lewin et al (1980); Koerner et al (1985) …”