Characterization of loops of the yeast mitochondrial ADP/ATP carrier facing the cytosol by site-directed mutagenesis.
To characterize structural features of the regions of the yeast type 2 ADP/ATP carrier (yAAC2) facing the cytosol, we prepared its Cys-less mutant, in which all four cysteine residues were replaced by alanine residues. The Cys-less mutant functioned like native yAAC2, showing that the cysteine residues are not essential. We then prepared cysteine mutants by substituting Ser(21) in the putative N-terminal region, Ala(124) and Ser(222) in the first and second loops facing cytosol, respectively, and Leu(312) in the C-terminal region of the Cys-less mutant for cysteine and examined the labeling of the substituted cysteine residues of the mutants with the membrane-impermeable SH reagent eosin-5-maleimide (EMA) from the cytosol. EMA labeled all the mutants, showing that all regions containing mutated residues faced the cytosolic side. The effects of transport inhibitors on EMA labeling were also examined. From the results, the location and conformation of the region around mutated residues were discussed.
Hatanaka
T
T
Faculty of Pharmaceutical Sciences, University of Tokushima, Shomachi-1, Tokushima, 770-8505, Japan.
Kihira
Y
Y
Shinohara
Y
Y
Majima
E
E
Terada
H
H
D000244:Adenosine Diphosphate / Q000378:metabolism; D000409:Alanine / Q000737:chemistry; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D015153:Blotting, Western; D001865:Bongkrekic Acid / Q000494:pharmacology; D002455:Cell Division; D003545:Cysteine / Q000737:chemistry; D003600:Cytosol / Q000737:chemistry*; D004247:DNA / Q000378:metabolism; D017931:DNA Primers / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004801:Eosine Yellowish-(YS) / Q000031:analogs & derivatives / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000494:pharmacology; D008928:Mitochondria / Q000378:metabolism; D000226:Mitochondrial ADP, ATP Translocases / Q000737:chemistry* / Q000235:genetics; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins; D012694:Serine / Q000737:chemistry
D000900:Anti-Bacterial Agents; D017931:DNA Primers; D005456:Fluorescent Dyes; C112225:PET9 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D001865:Bongkrekic Acid; D012694:Serine; D000244:Adenosine Diphosphate; C033531:eosin maleimide; D004247:DNA; D000226:Mitochondrial ADP, ATP Translocases; D003545:Cysteine; D000409:Alanine; D004801:Eosine Yellowish-(YS)