Characterization of Loops of the Yeast Mitochondrial ADP/ATP Carrier Facing the Cytosol by Site-Directed Mutagenesis

• Published in: Biochemical and biophysical research communications Vol. 286; no. 5; pp. 936 - 942
• Main Authors: Hatanaka, Takashi, Kihira, Yoshitaka, Shinohara, Yasuo, Majima, Eiji, Terada, Hiroshi
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 07.09.2001
• Subjects: Adenosine Diphosphate - metabolism; ADP/ATP carrier; Alanine - chemistry; Anti-Bacterial Agents - pharmacology; Blotting, Western; Bongkrekic Acid - pharmacology; Cell Division; chemical modification; Cys-less mutant; Cysteine - chemistry; cysteine residue; Cytosol - chemistry; DNA - metabolism; DNA Primers - metabolism; Electrophoresis, Polyacrylamide Gel; eosin-5-maleimide; Eosine Yellowish-(YS) - analogs & derivatives; Eosine Yellowish-(YS) - pharmacology; Fluorescent Dyes - pharmacology; mitochondria; Mitochondria - metabolism; Mitochondrial ADP, ATP Translocases - chemistry; Mitochondrial ADP, ATP Translocases - genetics; Models, Biological; Mutagenesis, Site-Directed; Mutation; oxidative phosphorylation; Protein Structure, Tertiary; Protein Transport; Saccharomyces cerevisiae; Saccharomyces cerevisiae - chemistry; Saccharomyces cerevisiae Proteins; Serine - chemistry; SH reagent; transporter; yeast; mitochondria; cysteine residue; transporter; SH reagent; Cys-less mutant; chemical modification; eosin-5-maleimide; oxidative phosphorylation; yeast; ADP/ATP carrier
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1006/bbrc.2001.5498

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 Ser21 in the putative N-terminal region, Ala124 and Ser222 in the first and second loops facing cytosol, respectively, and Leu312 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.