Functionally important conserved length of C-terminal regions of yeast and bovine ADP/ATP carriers, identified by deletion mutants studies, and water accessibility of the amino acids at the C-terminal region of the yeast carrier.
Comparison of the amino acid sequence of yeast type 2 ADP/ATP carrier (yAAC2) with that of bovine type 1 AAC (bAAC1) revealed that the N- and C-terminus of yAAC2 are 15- and 6-amino acids longer, respectively, than those of bAAC1. In the present study, we focused on the difference in the C-terminal region between yAAC2 and bAAC1. Deletion of first six residues of C-terminus of yAAC did not markedly affect the function of yAAC2; however, further deletion of 1 amino acid (7th amino acid from the C-terminus) destroyed its function. On the contrary, deletion of the first amino acid residue of the C-terminus of bAAC1 caused failure of its functional expression in yeast mitochondria. Based on these results, we concluded that the 6-amino acid residue extension of the C-terminus of yAAC2 was not necessary for the function of this carrier and that the remainder of the C-terminal region of yAAC2, having a length conserved with that of bAAC1, is important for the transport function of AACs. We next prepared various single-Cys mutants in which each of 32 residues in the C-terminus of yAAC2 was replaced by a Cys residue. Since all mutants were successfully expressed in yeast mitochondria, we examined the reactivity of these cysteine residues with the membrane-impermeable sulfhydryl reagent eosin 5-maleimide (EMA). As a result, all cysteine residues that replaced the 9 continuous amino acids in Met310-Lys318 showed high reactivity with EMA regardless of the presence of carboxyatractyloside or bongkrekic acid; and so this region was concluded to be exposed to the water-accessible environment. Furthermore, based on the reactivities of cysteine residues that replaced amino acids in the sixth transmembrane segment, the probable structural features of the C-terminal region of this carrier in the presence of bongkrekic acid were discussed.
Iwahashi
Akihiro
A
Institute for Genome Research, University of Tokushima, Kuramotocho-3, Tokushima 770-8503, Japan.
Ishii
Aoi
A
Yamazaki
Naoshi
N
Hashimoto
Mitsuru
M
Ohkura
Kazuto
K
Kataoka
Masatoshi
M
Majima
Eiji
E
Terada
Hiroshi
H
Shinohara
Yasuo
Y
D000595:Amino Acid Sequence; D000818:Animals; D001865:Bongkrekic Acid / Q000494:pharmacology; D002417:Cattle; D004801:Eosine Yellowish-(YS) / Q000031:analogs & derivatives / Q000378:metabolism; D017353:Gene Deletion; D000226:Mitochondrial ADP, ATP Translocases / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D011487:Protein Conformation; D029701:Saccharomyces cerevisiae Proteins / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D014170:Transformation, Genetic
C112225:PET9 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D001865:Bongkrekic Acid; C033531:eosin maleimide; D000226:Mitochondrial ADP, ATP Translocases; D004801:Eosine Yellowish-(YS)