title: Inhibition of the phosphoenolpyruvate:lactose phosphotransferase system and activation of a cytoplasmic sugar-phosphate phosphatase in Lactococcus lactis by ATP-dependent metabolite-activated phosphorylation of serine 46 in the phosphocarrier protein HPr.
abstract: Lactococcus lactis takes up lactose and the nonmetabolizable lactose analogue, thiomethyl-beta-galactoside (TMG), via the phosphoenolpyruvate:sugar phosphotransferase system (PTS) which couples sugar transport to sugar phosphorylation. Earlier studies had shown that TMG-phosphate, previously accumulated in L. lactis cells, is rapidly dephosphorylated in the cytoplasm and effluxes from the cells upon addition of glucose and that glucose inhibits further uptake of TMG. We have developed a vesicular system to analyze this regulatory mechanism and have used electroporation to shock proteins and membrane-impermeable metabolites into the vesicles. Uptake of TMG was dependent on an energy source, effectively provided by intravesicular phosphoenolpyruvate at low concentrations or extravesicular phosphoenolpyruvate at high concentrations. TMG uptake into osmotically shocked vesicles was only weakly inhibited, and expulsion of preaccumulated TMG was only slightly stimulated upon addition of glucose. Intravesicular (but not extravesicular) wild-type HPr of Bacillus subtilis completely restored the regulatory behavior observed in vivo when glucose was present in the external medium. Glucose could be replaced by intravesicular (but not extravesicular) fructose 1,6-diphosphate, gluconate 6-phosphate, or 2-phosphoglycerate, but not by other phosphorylated metabolites, in agreement with the allosteric activating effects of these compounds on HPr(Ser) kinase measured in vitro. Intravesicular mutant HPr(S46A) protein could not promote regulation of lactose permease activity when electroporated into the vesicles regardless of the presence or absence of glucose or the various phosphorylated metabolites, but the HPr(S46D) mutant protein promoted regulation, even in the absence of glucose or a metabolite, and HPr(H15A) was more effective than the wild-type protein in promoting regulation. Intravesicular wild-type and H15A HPrs, but not the S46A or S46D mutant proteins, were found to be phosphorylated by ATP under the conditions which promoted TMG efflux. In toluenized vesicles, the conditions which promoted TMG efflux also promoted TMG-P hydrolysis. These results establish for the first time that HPr serine phosphorylation by the ATP-dependent metabolite-activated HPr kinase regulates the expulsion of intracellular sugar-phosphate as well as the uptake of sugar via the PTS in L. lactis.
authors.affiliation: Department of Biology, University of California at San Diego, La Jolla 92093-0116.
authors.forename: J J
authors.initials: JJ
authors.lastname: Ye
authors.forename: J
authors.initials: J
authors.lastname: Reizer
authors.forename: X
authors.initials: X
authors.lastname: Cui
authors.forename: M H
authors.initials: MH
authors.lastname: Saier
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology; D001426:Bacterial Proteins / Q000378:metabolism; D001692:Biological Transport; D050260:Carbohydrate Metabolism; D003593:Cytoplasm / Q000201:enzymology; D004789:Enzyme Activation; D005947:Glucose / Q000378:metabolism; D007700:Kinetics; D013294:Lactococcus lactis / Q000201:enzymology*; D008756:Methylgalactosides / Q000138:chemical synthesis / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D010766:Phosphorylation; D017354:Point Mutation; D012694:Serine; D013379:Substrate Specificity; D013862:Thiogalactosides / Q000138:chemical synthesis / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D008756:Methylgalactosides; D013862:Thiogalactosides; C009468:thiomethylgalactoside; D012694:Serine; D000255:Adenosine Triphosphate; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C030182:phosphocarrier protein HPr; C029022:phosphoenolpyruvate-lactose dependent phosphotransferase system; D010744:Phosphoric Monoester Hydrolases; C022700:sugar-phosphatase; D005947:Glucose