Connection of transport and sensing by UhpC, the sensor for external glucose-6-phosphate in Escherichia coli.
UhpC is a membrane-bound sensor protein in Escherichia coli required for recognizing external glucose-6-phosphate (Glc6P) and induction of the transport protein UhpT. Recently, it was shown that UhpC is also able to transport Glc6P. In this study we investigated whether these transport and sensing activities are obligatorily coupled in UhpC. We expressed a His-UhpC protein in a UhpC-deficient E. coli strain and verified that this construct does not alter the basic biochemical properties of the Glc6P sensor system. The effects of arginine replacements, mutations of the central loop, and introduction of a salt bridge in UhpC on transport and sensing were compared. The exchanges R46C, R266C and R149C moderately affected transport by UhpC but strongly decreased the sensing ability. This suggested that the affinity for Glc6P as a transported substrate is uncoupled in UhpC from its affinity for Glc6P as an inducer. Four of the 11 arginine mutants showed a constitutive phenotype but had near wild-type transport activity suggesting that Glc6P can be transported by a molecule locked in the inducing conformation. Introduction of an intrahelical salt bridge increased the transport activity of UhpC but abolished sensing. Three conserved residues from the central loop were mutated and although none of these showed transport, one exhibited increased affinity for sensing. Taken together, these data show that transport by UhpC is not required for sensing, that conserved arginine residues are important for sensing and not for transport, and that residues located in the central hydrophilic loop are critical for transport and for sensing.
Schwöppe
Christian
C
Pflanzenphysiologie, Universität Kaiserslautern, Kaiserslautern, Germany.
Winkler
Herbert H
HH
Neuhaus
H Ekkehard
HE
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001692:Biological Transport / Q000502:physiology; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D017124:Conserved Sequence; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism*; D005816:Genetic Complementation Test; D019298:Glucose-6-Phosphate / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C490131:uhpC protein, E coli; C086587:uhpC protein, bacteria; D019298:Glucose-6-Phosphate