THE TONB AND TOLA TRANSMEMBRANE DOMAINS: CONTRIBUTIONS OF NON-ESSENTIAL SIDE-CHAINS TO ENERGY TRANSFER SPECIFICITY
Abstract (Summary)The gram-negative bacterium Escherichia coli has two systems that transduce energy from the cytoplasmic membrane to the outer membrane, the TonB system (TonB, ExbB, ExbD) and the TolA system (TolA, TolQ, TolR). The energy-harvesting complexes of the two systems are ExbB/ExbD and TolQ/TolR, with the energy-transducers being TonB and TolA, respectively. Sequence homologies between the transmembrane domains of ExbB/TolQ and ExbD/TolR proteins are high. TonB and TolA share only a conserved Ser-X(3)-His-X(6)-Leu-X(3)-Ser (SHLS) motif in their transmembrane domain, with the first Ser and the His residues of this motif being essential for efficient energization by the energy-harvesting complexes in E. coli. In cells lacking the energy-harvesting complex from one system, the energy-transducer is able to be energized (less efficiently) by the energy-harvesting complex from the opposite system, a phenomenon called crosstalk. Protein capture and bacteria two-hybrid assays were unable to identify interactions between proteins in the TolA system; therefore, evaluation of crosstalk interactions between the TolA and TonB systems required another approach. Crosstalk is inefficient, the result of a system specificity possibly defined by side group differences between the transmembrane domain residues of TonB and TolA. The relative contributions of such side groups were examined by systematically removing the differences between the energy-transducers via multiple alanyl substitutions, creating a generic transmembrane domain retaining only the essential SH residues and their relative spatial relationships. TonB derivatives containing up to 14 alanyl substitutions still properly partitioned to the cytoplasmic membrane, retained the ability to interact with ExbB, to be energized, and to participate in crosstalk. Replacement of residues 12-20 caused a decrease in TonB protein function, but that decrease was not the result of instability of those derivatives. In contrast, changes to residues 21-27 did affect the stability of TonB derivatives. These data indicated that the amino terminal residues in the transmembrane domain of the energy-transducers play an important, as yet unidentified role for proper energization of the energy-transducer by either energy-harvesting complex. Further, system specificity between the transducers and their respective energy-harvesting complexes did not appear to involve the residues of the TonB/TolA transmembrane domain.
School Location:USA - Ohio
Source Type:Master's Thesis
Date of Publication:01/01/2006