Regulation of bacterial sugar-H+ symport by phosphoenolpyruvate-dependent enzyme I/HPr-mediated phosphorylation.
The lactose-H+ symport protein (LacS) of Streptococcus thermophilus has a C-terminal hydrophilic domain that is homologous to IIA protein(s) domains of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). C-terminal truncation mutants were constructed and expressed in Escherichia coli and their properties were analyzed. Remarkably, the entire IIA domain (160 amino acids) could be deleted without significant effect on lactose-H+ symport and galactoside equilibrium exchange. Analysis of the LacS mutants in S. thermophilus cells suggested that transport is affected by PTS-mediated phosphorylation of the IIA domain. For further studies, membrane vesicles of S. thermophilus were fused with cytochrome c oxidase-containing liposomes, and, when appropriate, phosphoenolpyruvate (PEP) plus purified enzyme I and heat-stable protein HPr were incorporated into the hybrid membranes. Generation of a protonmotive force (delta p) in the hybrid membranes resulted in accumulation of lactose, whereas uptake of the PTS sugar sucrose was not observed. With PEP and the energy-coupling proteins enzyme I and HPr of the PTS on the inside, high rates of sucrose uptake were observed, whereas delta p-driven lactose uptake by wild-type LacS was inhibited. This inhibition was not observed with LacS(delta 160) and LacS(H552R), indicating that PEP-dependent enzyme I/HPr-mediated phosphorylation of the IIA domain (possibly the conserved His-552 residue) modulates lactose-H+ symport activity.
Department of Microbiology, University of Groningen, Haren, The Netherlands.
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B
Poolman
J
J
Knol
B
B
Mollet
B
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Nieuwenhuis
G
G
Sulter
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005816:Genetic Complementation Test; D007785:Lactose / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D010728:Phosphoenolpyruvate / Q000378:metabolism; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000378:metabolism*; D010766:Phosphorylation; D017384:Sequence Deletion / Q000502:physiology; D013291:Streptococcus / Q000201:enzymology*; D013395:Sucrose / Q000378:metabolism; D027981:Symporters
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008081:Liposomes; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; D013395:Sucrose; D010728:Phosphoenolpyruvate; C021499:lactose permease; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C030182:phosphocarrier protein HPr; D007785:Lactose