Previously, cytochrome c oxidase from the yeast Saccharomyces cerevisiae has been thought to be composed of seven different polypeptide subunits. Four of these are small polypeptides (4,000-15,000 daltons), subunits IV-VII, which are encoded by nuclear DNA. Studies described here reveal the presence of two new polypeptides in this size range. These polypeptides, designated as subunits VIIa and VIII, co-migrate with subunit VII (R.O. Poyton and G. Schatz (1975) J. Biol. Chem. 250, 752-761) on low resolution sodium dodecyl sulfate (SDS) polyacrylamide gels, can be partially resolved on high resolution SDS polyacrylamide gels, and can be completely separated from one another by reversed phase high performance liquid chromatography. In order to determine the sequences of each of these six nuclear-coded polypeptides (subunits IV, V, VI, VII, VIIa, and VIII), we have developed new methods for the large scale purification of the holoenzyme and have employed a new strategy for the isolation of each polypeptide. By using octyl-Sepharose chromatography to isolate holocytochrome c oxidase and by extracting the holoenzyme with aprotic organic solvents and fractionating these extracts by reversed phase high performance liquid chromatography, it is possible to isolate several milligrams of each of these subunits. Each subunit preparation gives a single peak during reversed phase high performance liquid chromatography, a single band during SDS-polyacrylamide gel electrophoresis, a single NH2-terminal sequence, and a unique amino acid composition and tryptic peptide map. Since each purified subunit preparation gives close to a 100% yield of its NH2-terminal amino acid during quantitative Edman degradation, we conclude that no subunit has a blocked NH2 terminus and that no subunit preparation contains either blocked or unblocked contaminating polypeptides. Thus, each consists of a single unique polypeptide species. Together, these results demonstrate that yeast cytochrome c oxidase contains six, rather than four, small subunit polypeptides. Thus, it appears that these polypeptides, in combination with the three polypeptides encoded by mitochondrial DNA, constitute a holoenzyme which contains nine subunits, instead of seven as proposed earlier.

• PMID: 6327684
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Reference Type

Journal Article | Research Support, Non-U.S. Gov't | Research Support, U.S. Gov't, P.H.S.

Authors

Power SD, Lochrie MA, Sevarino KA, Patterson TE, Poyton RO

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