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.
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 carbonylcyanidechlorophenyl-hydrazone (CCCP), ionophores (valinomycin, gramicidin), and inhibitors of the respiratory chain (myxothiazol, KCN), which decrease the TMP, also diminish the APMC accumulation and labelling. And conversely, drugs, which hyperpolarize the inner membrane (nigericin, atractyloside), favour APMC labelling. Another driving force of APMC accumulation is the dye's lipophilicity, which facilitates dye accumulation by hydrophobic interaction with the very lipophilic proteins of the inner mitochondrial membranes. This was shown by competitive double staining experiments. Thiamine strongly inhibits APMC labelling. Obviously, the transport of the APMC cations is facilitated by the thiamine carrier, and thiamine competes for the same binding sites, which are occupied by the dye cations. Chloramphenicol is an inhibitor of the mitochondrial protein synthesis without affecting the TMP. On preincubation, chloramphenicol completely quenches the signal of the 40 kDa protein. Therefore, this protein must be encoded on the mtDNA. The only 40 kDa protein with adequate properties is the subunit I of cytochrome c oxidase. Obviously, it is the preferred target of the APMC cations on photoaffinity labelling. This assignment agrees with the strong hydrophobicity of the labelled 40 kDa protein, which was tested with various detergents. It also agrees with the solvatochromism of the protein-bound APMC label, and finally with the paralellism of the labelled protein with cytochrome c oxidase on fractional ammonium sulfate precipitation.
Haass-Männle
H
H
Institut für Physikalische Chemie der Universität Freiburg, Freiburg, Germany.
Zimmermann
H W
HW
D002232:Carbocyanines / Q000737:chemistry / Q000378:metabolism*; D002701:Chloramphenicol / Q000494:pharmacology; D003576:Electron Transport Complex IV / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism; D019920:Photoaffinity Labels; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000378:metabolism*
D002232:Carbocyanines; D005456:Fluorescent Dyes; D005656:Fungal Proteins; D019920:Photoaffinity Labels; C089003:4,4-azo-n-pentyl-1'-methyl-3,3,3',3'-tetramethylindocarbocyanine; D002701:Chloramphenicol; D003576:Electron Transport Complex IV