PMID: 9440317Jan 24, 1998Paper

Selective photoaffinity labelling of one mitochondrial protein in living cells of Saccharomyces cerevisiae with the fluorescent probe APMC. Identification of the target protein as subunit I of cytochrome c oxidase

Journal of Photochemistry and Photobiology. B, Biology
H Haass-Männle, H W Zimmermann

Abstract

The lipophilic, cationic fluorochrome azopentylmethylindocarbocyanine (APMC) specifically stains the mitochondria in living yeast cells (Saccharomyces cerevisiae WT X 2180). It contains a photosensitive diazirine ring and is suitable for photoaffinity labelling. By combining photoaffinity labelling, micro-gel electrophoresis (SDS-PAGE), and detection of the APMC fluorescence with a microfluorimeter, we established a highly sensitive procedure for determining the apparent molecular weight of the APMC-labelled proteins in yeast cells. On vital staining at 0.1 microM APMC for 30 min, only one mitochondrial protein with an apparent molecular weight of 40 kDa is labelled with high intensity. At increased dye concentrations proteins of 47 and 49 kDa are labelled too, however not until all binding sites of the 40 kDa protein are occupied. Obviously, the APMC cations have a pronounced affinity for this protein. It was shown by fractional centrifugation that the labelled 40 kDa protein is a constituent of the inner mitochondrial membrane. One driving force for the accumulation of the APMC cations is the trans-membrane potential (TMP) across the inner mitochondrial membrane. Consequently, uncouplers like dinitrophenol (DNP) and carbonylc...Continue Reading

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