The uncoupling protein dimer can form a disulfide cross‐link between the mobile C‐terminal SH groups

Abstract

Isolated uncoupling protein (UCP) can be cross‐linked, by various disulfide‐forming reagents, to dimers. The best cross‐linking is achieved with Cu2+‐phenanthroline oxidation. Because cross‐linking is independent of UCP concentration and prevented by SDS addition, a disulfide bridge must be formed between the two subunits of the native dimer. Cross‐linking is prevented by SH reagent and reversed by SH‐reducing reagents. In mitochondria, cross‐linking of UCP with disulfide‐forming agents is even more efficient than in isolated state. It proves that UCP is a dimer in mitochondria, before isolation. Disulfide‐bridge formation does not inhibit GTP‐binding to UCP. Cross‐linked UCP re‐incorporated in proteoliposomes either before or after cross‐linking fully retains the H+‐transport function. Rapid cross‐linking by membrane impermeant reagents indicates a surface localization of the C‐terminus in soluble UCP and projection to the outer surface in mitochondria. Intermolecular disulfide‐bridge formation in a dimer requires juxtaposition of identical cysteines at the twofold symmetry axis. A rigid juxtaposition of cysteines is unlikely, unless intended for a native disulfide bridge. The absence of such a bridge in UCP suggests that juxtaposition of cysteines is generated by high mobility. In order to localize the cysteine involved, cross‐linked UCP was cleaved by BrCN. The CB‐7 C‐terminal peptide, which contains cysteines at positions 287 and 304, disappears. Limited trypsinolytic cleavage, previously shown to occur at Lys‐292, removed cross‐linking in UCP both in the solubilized and mitochondrially bound state. The cleaved C‐terminal peptide of 11 residues contains only cystein‐304 which, thus, should be the only one (out of 7 cysteines in UCP) involved in the S—S bridge formation. Obviously, the C‐terminal location of the cysteine, because of its high mobility, permits juxtapositioning for cross‐linking. This agrees with predictions from hydrophobicity analysis that the last 14 residues in UCP protrude from the membrane. Copyright © 1989, Wiley Blackwell. All rights reserved