Membrane topology and mutational analysis of the TolQ protein of Escherichia coli required for the uptake of macromolecules and cell envelope integrity.
TolQ is a 230-amino-acid protein required to maintain the integrity of the bacterial envelope and to facilitate the import of both filamentous bacteriophage and group A colicins. Cellular fractionation experiments showed TolQ to be localized to the cytoplasmic membrane. Bacteria expressing a series of TolQ-beta-galactosidase and TolQ-alkaline phosphatase fusion proteins were analyzed for the appropriate enzyme activity, membrane location, and sensitivity to exogenously added protease. The results are consistent with TolQ being an integral cytoplasmic membrane protein with three membrane-spanning regions. The amino-terminal 19 residues as well as a small loop in the 155 to 170 residue region appear exposed in the periplasm, while the carboxy terminus and a large loop after the first transmembrane region are cytoplasmic. Amino-terminal sequence analysis of TolQ purified from the membrane revealed the presence of the initiating formyl methionine group, suggesting a rapid translocation of the amino-terminal region across the cytoplasmic membrane. Analysis of various tolQ mutant strains suggests that the third transmembrane region as well as parts of the large cytoplasmic loop are necessary for activity.
Vianney
A
A
Laboratoire de Microbiologie et Génétique Moléculaire, Centre National de la Recherche Scientifique, Université Claude Bernard Lyon I, Villeurbanne, France.
Lewin
T M
TM
Beyer
W F
WF
Lazzaroni
J C
JC
Portalier
R
R
Webster
R E
RE
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000648:ultrastructure*; D001692:Biological Transport; D002918:Chymotrypsin / Q000494:pharmacology; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000378:metabolism / Q000648:ultrastructure*; D029968:Escherichia coli Proteins; D005816:Genetic Complementation Test; D008565:Membrane Proteins / Q000648:ultrastructure*; D008969:Molecular Sequence Data
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; C060792:tolQ protein, E coli; D002918:Chymotrypsin