Mutational analysis of the Escherichia coli K-12 TolA N-terminal region and characterization of its TolQ-interacting domain by genetic suppression.
The Tol-Pal proteins of Escherichia coli are involved in maintaining outer membrane integrity. They form two complexes in the cell envelope. Transmembrane domains of TolQ, TolR, and TolA interact in the cytoplasmic membrane, while TolB and Pal form a complex near the outer membrane. The N-terminal transmembrane domain of TolA anchors the protein to the cytoplasmic membrane and interacts with TolQ and TolR. Extensive mutagenesis of the N-terminal part of TolA was carried out to characterize the residues involved in such processes. Mutations affecting the function of TolA resulted in a lack or an alteration in TolA-TolQ or TolR-TolA interactions but did not affect the formation of TolQ-TolR complexes. Our results confirmed the importance of residues serine 18 and histidine 22, which are part of an SHLS motif highly conserved in the TolA and the related TonB proteins from different organisms. Genetic suppression experiments were performed to restore the functional activity of some tolA mutants. The suppressor mutations all affected the first transmembrane helix of TolQ. These results confirmed the essential role of the transmembrane domain of TolA in triggering interactions with TolQ and TolR.
Germon
P
P
Laboratoire de Microbiologie et Génétique Moléculaire, CNRS-Université Lyon I, F-69622 Villeurbanne Cedex, France.
Clavel
T
T
Vianney
A
A
Portalier
R
R
Lazzaroni
J C
JC
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D013489:Suppression, Genetic
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; C062093:tolA protein, E coli; C060792:tolQ protein, E coli