Cooperative binding of lactose and the phosphorylated phosphocarrier protein HPr(Ser-P) to the lactose/H+ symport permease of Lactobacillus brevis.
Lactobacillus brevis accumulates lactose and nonmetabolizable lactose analogues via sugar/H+ symport, but addition of glucose to the extracellular medium results in rapid efflux of the free sugar from the cells due to the uncoupling of sugar transport from proton transport. By using vesicles of L. brevis cells, we recently showed that these regulatory/effects could be attributed to the metabolite-activated ATP-dependent protein kinase-catalyzed phosphorylation of serine-46 in the phosphocarrier protein HPr [HPr(Ser-P)] of the phosphotransferase system and that a mutant form of HPr with the serine-46-->aspartate replacement ([S46D]HPr) is apparently locked in the seryl phosphorylated conformation. We here demonstrate that [S46D]HPr binds directly to inside-out membrane vesicles of L. brevis that contain the lactose permease. Sugar substrates of the permease markedly and specifically stimulate binding of [S46D]HPr to the membranes while certain transport inhibitors such as N-ethylmaleimide block binding. The pH dependency for binding follows that for transport. Wild-type HPr and the [S46A]HPr mutant protein did not appreciably compete with [S46D]HPr for binding to the permease. These results provide evidence for the direct interaction of HPr(Ser-P) with an allosteric site on the lactose/proton symporter of L. brevis for the purpose of regulating sugar accumulation in response to the metabolic needs of the cell.
Department of Biology, University of California at San Diego, La Jolla 92093-0116.
J J
JJ
Ye
M H
MH
Saier
D000494:Allosteric Regulation; D001426:Bacterial Proteins; D001667:Binding, Competitive; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005033:Ethylmaleimide / Q000494:pharmacology; D007457:Iodine Radioisotopes; D007544:Isopropyl Thiogalactoside / Q000378:metabolism; D007553:Isotope Labeling; D007778:Lactobacillus / Q000201:enzymology / Q000378:metabolism*; D007785:Lactose / Q000378:metabolism*; D008553:Melibiose / Q000378:metabolism; D008566:Membranes / Q000378:metabolism; D009004:Monosaccharide Transport Proteins; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000378:metabolism*; D010766:Phosphorylation; D010768:Phosphoserine; D011485:Protein Binding; D027981:Symporters; D013862:Thiogalactosides / Q000378:metabolism
D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D007457:Iodine Radioisotopes; C050221:LacY protein, E coli; D009004:Monosaccharide Transport Proteins; D027981:Symporters; D013862:Thiogalactosides; D010768:Phosphoserine; D007544:Isopropyl Thiogalactoside; D008553:Melibiose; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C030182:phosphocarrier protein HPr; D007785:Lactose; D005033:Ethylmaleimide