The structure of the second cytosolic loop of the yeast mitochondrial ADP/ATP carrier AAC2 is dependent on the conformational state.
To detect structural changes in the second cytosolic loop of the mitochondrial ADP/ATP carrier of Saccharomyces cerevisiae AAC2, we prepared 20 single cysteine mutants by replacing each amino acid in the S213 to L232 region. All single cysteine mutants were fully functional, because they could restore growth on glycerol of a yeast strain lacking functional ADP/ATP carriers. First, these single-Cys mutants were treated with carboxyatractyloside to lock the carrier in the cytosolic state or with bongkrekic acid to generate the matrix state, and then with the membrane-impermeable SH reagent eosin-5-maleimide (EMA) to probe accessibility. The amino acid residues S213C, L214C, F231C and L232C were not labeled, indicating that these 4 residues must have been buried in the membrane, whereas the region between residues K215 and S230 is accessible to labeling and must, therefore, have protruded into the aqueous phase. Residue L218C showed strong resistance against EMA labeling regardless of the state of the carrier, but the reason for such behavior is unclear. On the contrary, the labeling of the residues between F227C and S230C was strongly dependent on the state of the carrier. Thus, the C-terminal region of the second cytosolic loop in AAC2 changes its environment when the carrier cycles between the matrix and cytosolic state.
Iwahashi
Akihiro
A
Institute for Genome Research, University of Tokushima, Kuramotocho-3, Tokushima 770-8503, Japan.
Kihira
Yoshitaka
Y
Majima
Eiji
E
Terada
Hiroshi
H
Yamazaki
Naoshi
N
Kataoka
Masatoshi
M
Shinohara
Yasuo
Y
D019943:Amino Acid Substitution; D003545:Cysteine / Q000378:metabolism; D003600:Cytosol / Q000737:chemistry / Q000201:enzymology; D004801:Eosine Yellowish-(YS) / Q000031:analogs & derivatives; D008928:Mitochondria / Q000737:chemistry / Q000201:enzymology*; D000226:Mitochondrial ADP, ATP Translocases / Q000737:chemistry*; D008969:Molecular Sequence Data; D009154:Mutation / Q000187:drug effects; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*
C112225:PET9 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C033531:eosin maleimide; D000226:Mitochondrial ADP, ATP Translocases; D003545:Cysteine; D004801:Eosine Yellowish-(YS)