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

Abstract

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.