INTERACTIONS IN THE TONB-DEPENDENT ENERGY TRANSDUCTION COMPLEX - EXBBAND EXBD FORM HOMOMULTIMERS

The cytoplasmic membrane proteins ExbB and ExbD support TonB-dependent active transport of iron siderophores and vitamin B-12 across the ess entially unenergized outer membrane of Escherichia coil. In this study , in vivo formaldehyde cross-linking analysis was used to investigate the interactions of T7 epitope-tagged ExbB or ExbD proteins. ExbB and ExbD each formed two unique cross-linked complexes which were not depe ndent on the presence of TonB, the outer membrane receptor protein Fep A, or the other Exb protein. Cross-linking analysis of ExbB- and ExbD- derived size variants demonstrated instead that these ExbB and ExbD co mplexes were homodimers and homotrimers and suggested that ExbB also i nteracted with an unidentified protein(s). Cross-linking analysis of e pitope-tagged ExbB and ExbD proteins with TonB antisera afforded detec tion of a previously unrecognized TonB-ExbD cross-linked complex and c onfirmed the composition of the TonB-ExbB cross-linked complex. The im plications of these findings for the mechanism of TonB-dependent energ y transduction are discussed.