ROLE OF THE TONB AMINO-TERMINUS IN ENERGY TRANSDUCTION BETWEEN MEMBRANES

Escherichia coli TonB protein is an energy transducer, coupling cytopl asmic membrane energy to active transport of vitamin B-12 and iron-sid erophores across the outer membrane. TonB is anchored in the cytoplasm ic membrane by its hydrophobic amino terminus, with the remainder occu pying the periplasmic space. In this report we establish several funct ions for the hydrophobic amino terminus of TonB. A G-26-->D substituti on in the amino terminus prevents export of TonB, suggesting that the amino terminus contains an export signal for proper localization of To nB within the cell envelope. Substitution of the first membrane-spanni ng domain of the cytoplasmic membrane protein TetA for the TonB amino terminus eliminates TonB activity without altering TonB export, sugges ting that the amino terminus contains sequence-specific information. D etectable TonB cross-linking to ExbB is also prevented, suggesting tha t the two proteins interact primarily through their transmembrane doma ins. In vivo cleavage of the amino terminus of TonB carrying an engine ered leader peptidase cleavage site eliminates (i) TonB activity, (ii) detectable interaction with a membrane fraction having a density inte rmediate to those of the cytoplasmic and outer membranes, and (iii) cr oss-linking to ExbB. In contrast, the amino terminus is not required f or cross-linking to other proteins with which TonB can form complexes, including FepA. Additionally, although the amino terminus clearly is a membrane anchor, it is not the only means by which TonB associates w ith the cytoplasmic membrane. TonB lacking its amino-terminal membrane anchor still remains largely associated with the cytoplasmic membrane .