Difference between yeast and bovine mitochondrial ADP/ATP carriers in terms of conformational properties of the first matrix loop as deduced by use of copper-o-phenanthroline.
The mitochondrial ADP/ATP carrier (AAC) has 6 transmembrane regions and 3 matrix loops. Our previous mutational study on the Cys residue in the LM1s of chimeric bovine type 1 AAC (yN-bAAC1), in which the N-terminal 11 amino acids of bovine type 1 AAC are substituted with the corresponding 26 amino acids of yeast type 2 AAC (yAAC2), and yAAC2 in the yeast expression system suggested the possibility of a different structural feature between their LM1s. In the present study, we compared the effects of the SH cross-linking reagent copper-o-phenanthroline (Cu(OP)(2)) on yN-bAAC1 and yAAC2 in order to study the difference between these LM1s of the 2 carriers. Cu(OP)(2) is known to cross-link 2 AAC molecules in a functional dimer via a Cys residue in each first matrix loop (LM1). yN-bAAC1 exhibited intra- and inter-molecular cross-linking, in agreement with the results of a previous study on the native bovine carrier and suggesting that yN-bAAC1 had the same structure as the native carrier. yAAC2 also showed intra- and inter-molecular cross-linking. However, the speed of formation of the inter-molecular cross-linking of yN-bAAC1 was faster than that of yAAC2, suggesting that the conformational state of the LM1 was different between the 2 carriers. In addition, we also studied the effects of AAC-specific inhibitors and solubilization with Triton X-100 on the cross-linking.
Kihira
Yoshitaka
Y
Faculty of Pharmaceutical Sciences, University of Toyama, Toyama, Japan. kihira@pha.u-toyama.ac.jp
Ueno
Masaharu
M
Terada
Hiroshi
H
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D003600:Cytosol / Q000201:enzymology; D008928:Mitochondria / Q000201:enzymology*; D000226:Mitochondrial ADP, ATP Translocases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010618:Phenanthrolines / Q000494:pharmacology*; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D003432:Cross-Linking Reagents; D010618:Phenanthrolines; D029701:Saccharomyces cerevisiae Proteins; D000226:Mitochondrial ADP, ATP Translocases; C025205:1,10-phenanthroline