Abstract
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.
Highlights
Chem. 250, 762-76o1n)low resolution sodium dode- port and energy conservation [1,2,3], the assembly of oligocy1sulfate (SDS)polyacrylamide gels, can be partially meric membrane proteins [4,5,6], and those interactions beresolved on high resolution SDS polyacrylamide gels, and can be completely separated from one another by reversed phase high performance liquid chromatography
In orderto determine thesequences of each of these six nuclear-codedpolypeptides, we have developed new methods for the largescale purification of the holoenzymeand have tween mitochondrial and nuclear genomes which are required for mitochondrial biogenesis ( 7 ) .While most of the structural and functional data for this enzyme have been derived from studies with bovine heart cytochrome c oxidase, much of the biosynthetic data have come from studies with cytochrome c oxidase from the yeast Saccharomyces cereuisiae
Phase high performance liquid chromatography, a sin- While there are a number of similarities between bovine gle band during SDS-polyacrylamide gel electropho- heart and yeast cytochrome c oxidases, there are some resis, a single NHderminal sequence, and a unique amino acid composition and trypticpeptide map
Summary
A necessary prerequisite for the sequence analysis of the nuclear-coded subunits of yeast cytochrome c oxidase is the availability of large quantities (several hundred milligrams) of the holoenzyme as startingmaterial. The advantages afforded by the use of octyl-Sepharose chromatography as thefinal step in the purification procedure are its specificity for hydrophobic proteins and its high binding capacity These properties have allowed us to scale up the isolation of yeast cytochrome c oxidase without sacrificing yield or purity. The effectiveness of this step can be seen from SDS-PAGE of the various fractions obtained duringthe preparation of the enzyme (Fig. 1B). Despite the loss of activity which results from urea treatment, this step is included in the large scale purification procedure because it is very effective in removing residual Tween 20 which is bound to the resin and to the holoenzyme and because it facilitates the subsequent elution of the holoenzyme in a smalvl olume of ME buffer, 3%Triton X-100.
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