Chapter 6 Mass spectrometric characterization of the thirteen subunits of bovine respiratory complexes that are encoded in mitochondrial DNA.
The genomes of mammalian mitochondria encode 13 hydrophobic membrane proteins. All of them are subunits of the respiratory complexes found in the inner membranes of the organelle. Although the sequences of human and bovine mitochondrial DNA were described in 1981 and 1982, respectively, and the encoded proteins were identified at the same time or soon after, because of their hydrophobic properties, the chemical compositions of some of these proteins have never been characterized. Therefore, we have developed procedures to extract them with organic solvents from the inner membranes of bovine mitochondria and from purified respiratory complexes and to fractionate the extracts, allowing the precise molecular masses of all 13 proteins to be measured by electrospray ionization mass spectrometry. It was found that, with one exception, the proteins retain their translational initiator formyl-methionine residues, and the only posttranslational modification detected was the removal of the formyl group or the formyl-methionine from the Cox III protein. These procedures can be adapted for analyzing the proteins encoded in mitochondrial DNAs in other species, for analyzing the subunit compositions of their respiratory complexes, and for establishing accurate and comprehensive proteomes of other cellular membranes. Because many membrane proteins have few proteolytic enzyme cleavage sites, identifying them by mass spectrometric sequencing of proteolytic peptides can be difficult. Therefore, we have studied the tandem mass spectra of fragment ions from a range of membrane proteins from mitochondria, including 10 of the 13 proteins encoded in mitochondrial DNA. In contrast to the highly complex spectra produced in this way by globular proteins, the spectra of membrane proteins are simple and easy to interpret, and so they provide sequence tags for the identification of membrane proteins.
Medical Research Council Mitochondrial Biology Unit, Cambridge, United Kingdom.
John E
JE
Walker
Joe
J
Carroll
Matthew C
MC
Altman
Ian M
IM
Fearnley
D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid; D004272:DNA, Mitochondrial / Q000378:metabolism*; D004579:Electron Transport; D004591:Electrophoresis, Polyacrylamide Gel; D008929:Mitochondria, Heart / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013056:Spectrophotometry, Ultraviolet; D053719:Tandem Mass Spectrometry / Q000379:methods*
D004272:DNA, Mitochondrial