Expression of the UhpT sugar-phosphate transporter in 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 transcription, via binding to the 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 [γ-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∼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 P∼UhpA levels are low and/or that, to keep sufficient P∼UhpA accumulated to induce transcription, the promoter DNA itself is involved in stabilization/sequestration of P∼UhpA.


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