Translocation of Loops Regulates Transport Activity of Mitochondrial ADP/ATP Carrier Deduced from Formation of a Specific Intermolecular Disulfide Bridge Catalyzed by Copper-o-Phenanthroline

Eiji Majima,Kazuro Ikawa,Masashi Takeda,Mitsuru Hashimoto,Yasuo Shinohara,Hiroshi Terada

doi:10.1074/jbc.270.49.29548

Eiji Majima, Kazuro Ikawa + Show 4 more

Open Access

https://doi.org/10.1074/jbc.270.49.29548

Copy DOI

Abstract

The cross-linking reagent copper-o-phenanthroline complex (Cu(OP)2) specifically caused a decrease in the amount of the 30-kDa ADP/ATP carrier in bovine submitochondrial particles associated predominantly with formation of a 60-kDa protein consisting of a cross-linked dimer of the carrier. However, Cu(OP)2 had no effect on mitochondria. The transport of ADP via the carrier through submitochondrial particle membranes was found to be inhibited in parallel with the progress of intermolecular cross-linking. Analysis of the cross-linked site showed that a disulfide bridge was formed only between two Cys56 residues in a pair of the first loops facing the matrix space. The transport inhibitor bongkrekic acid, which locks the m-state conformation of the carrier, had no effect on disulfide bridge formation catalyzed by Cu(OP)2, but carboxyatractyloside, which locks the c-state conformation by acting from the cytosolic side, completely inhibited the cross-linking. These results show that the ADP/ATP carrier functions as a dimer form, and a pair of the first loops protrudes into the matrix space in the m-state, but possibly intrudes into the membrane in the c-state. Thus, it is suggested that a pair of the first loops acts as a gate and that its opening and closing are regulated by their translocation.

Highlights

The 30-kDa ADP/ATP carrier present in the inner mitochondrial membrane mediates the transport of ADP and ATP from both the cytosolic and matrix sides of the mitochondrial inner membrane, and its structural properties including its primary structures in preparations from various sources have been well studied [1,2,3]
Joshi and Torok reported that an ADP/ATP carrier contaminating the preparation of ATPase was the protein modified by Cu(OP)2 and that Cu(OP)2 caused intermolecular and intramolecular disulfide bridges in electron transport particles from bovine heart mitochondria [20], but it seemed to induce only intramolecular cross-linking of the carrier present in the preparation of Hϩ-ATPase [21]
Submitochondrial particles were incubated with various concentrations of Cu(OP)2 at 0 °C for 10 min; the reaction was terminated by addition of EDTA to trap free and complexed Cu2ϩ; and NEM was added to modify the remaining free cysteine residues

Summary

Introduction

The 30-kDa ADP/ATP carrier present in the inner mitochondrial membrane mediates the transport of ADP and ATP from both the cytosolic and matrix sides of the mitochondrial inner membrane, and its structural properties including its primary structures in preparations from various sources have been well studied [1,2,3]. Joshi and Torok reported that an ADP/ATP carrier contaminating the preparation of ATPase was the protein modified by Cu(OP) and that Cu(OP) caused intermolecular and intramolecular disulfide bridges in electron transport particles from bovine heart mitochondria [20], but it seemed to induce only intramolecular cross-linking of the carrier present in the preparation of Hϩ-ATPase [21]. They suggested that Cys159 and Cys256 were associated with the intramolecular cross-linking. Based on our present results, we discuss changes in configuration of the loops in the ADP/ATP carrier in relation to its transport function

Methods

Results

Conclusion