ATP-DEPENDENT PHOSPHORYLATION OF SERINE-46 IN THE PHOSPHOCARRIER PROTEIN HPR REGULATES LACTOSE H+ SYMPORT IN LACTOBACILLUS-BREVIS/

Lactobacillus brevis takes up lactose and the nonmetabolizable lactose analogue thiomethyl beta-galactoside (TMG) by a permease-catalyzed la ctose/H+ symport mechanism. Earlier studies have shown that TMG, previ ously accumulated in L. brevis cells, rapidly 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 m echanism and have used electroporation to shock proteins and membrane- impermeant metabolites into the vesicles. Uptake of TMG was dependent on an energy source, effectively provided by intravesicular ATP or ext ravesicular arginine. TMG uptake into these vesicles was not inhibited , and preaccumulated TMG did not efflux from them upon addition of glu cose. Intravesicular but not extravesicular wild-type phosphocarrier p rotein HPr of Bacillus subtilis restored regulation. Glucose could be replaced by intravesicular (but not extravesicular) fructose 1,6-bisph osphate, gluconate 6-phosphate, or 2 -phosphoglycerate, but not by oth er phosphorylated metabolites, in agreement with the allosteric activa ting effects of these compounds on HPr(Ser) kinase measured in vitro. Intravesicular serine-46 --> alanine mutant HPr could not promote regu lation of lactose permease activity when electroporated into the vesic les with or without glucose or the various phosphorylated metabolites, but the serine-46 --> aspartate mutant HPr promoted regulation, even in the absence of glucose or a metabolite. HPr(Ser-P) appears to conve rt the lactose/H+ symporter into a sugar uniporter. These results esta blish that HPr serine phosphorylation by the ATP-dependent metabolite- activated HPr kinase regulates lactose permease activity in L. brevis. A direct allosteric mechanism is proposed.