Glucose-6-phosphate-dependent phosphoryl flow through the Uhp two-component regulatory system.
Expression of the UhpT sugar-phosphate transporter in Escherichia coli is regulated at the transcriptional level via the UhpABC signalling cascade. Sensing of extracellular glucose 6-phosphate (G6P), by membrane-bound UhpC, modulates a second membrane-bound protein, UhpB, resulting in autophosphorylation of a conserved histidine residue in the cytoplasmic (transmitter) domain of the latter. Subsequently, this phosphoryl group is transferred to a conserved aspartate residue in the response-regulator UhpA, which then initiates uhpT transcription, via binding to the uhpT promoter region. This study demonstrates the hypothesized transmembrane signal transfer in an ISO membrane set-up, i.e. in a suspension of UhpBC-enriched membrane vesicles, UhpB autophosphorylation is stimulated, in the presence of [gamma-(32)P]ATP, upon intra-vesicular sensing of G6P by UhpC. Subsequently, upon addition of UhpA, very rapid and transient UhpA phosphorylation takes place. When P approximately UhpA is added to G6P-induced UhpBC-enriched membrane vesicles, rapid UhpA dephosphorylation occurs. So, in the G6P-activated state, UhpB phosphatase activity dominates over kinase activity, even in the presence of saturating amounts of G6P. This may imply that maximal in vivo P approximately UhpA levels are low and/or that, to keep sufficient P approximately UhpA accumulated to induce uhpT transcription, the uhpT promoter DNA itself is involved in stabilization/sequestration of P approximately UhpA.
Verhamme
D T
DT
Swammerdam Institute for Life Sciences, University of Amsterdam, Nieuwe Achtergracht 166, 1018 WV Amsterdam, The Netherlands.
Arents
J C
JC
Postma
P W
PW
Crielaard
W
W
Hellingwerf
K J
KJ
D001426:Bacterial Proteins / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000502:physiology*; D029968:Escherichia coli Proteins; D019298:Glucose-6-Phosphate / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D026901:Membrane Transport Proteins; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D010770:Phosphotransferases; D011494:Protein Kinases / Q000378:metabolism; D015398:Signal Transduction
D001426:Bacterial Proteins; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C489848:UhpA protein, E coli; C490131:uhpC protein, E coli; C080370:UhpA protein, Bacteria; C086586:uhpB protein, Bacteria; C086587:uhpC protein, bacteria; D019298:Glucose-6-Phosphate; D010770:Phosphotransferases; D011494:Protein Kinases; D010749:Phosphoprotein Phosphatases