Soluble sugar permeases of the phosphotransferase system in Escherichia coli: evidence for two physically distinct forms of the proteins in vivo

The bacterial phosphoenolpyruvate-dependent phosphotransferase system (PTS) consists of a set of cytoplasmic energy-coupling proteins and various integral membrane permeases/sugar phosphotransferases, each specific for a different sugar. We have conducted biochemical analyses of three PTS permeases (enzymes II), the glucose permease (IIGlc), the mannitol permease (IIMtl) and the mannose permease (IIMan). These enzymes each catalyse two vectorial/chemical reactions, sugar phosphorylation using phosphoenolpyruvate (PEP) as the phosphoryl donor, dependent on enzyme I, HPr and IIA as well as IIBC (the PEP reaction), and transphosphorylation using a sugar phosphate (glucose-6-P for IIGlc and IIMan; mannitol-1-P for IIMtl) as the phosphoryl donor, dependent only on IIBC (the TP reaction). When crude extracts of French-pressed or osmotically shocked Escherichia coli cells are centrifuged in an ultracentrifuge at high speed, 5-20% of the enzyme II activity remains in the high-speed supernatant, and passage through a gel filtration column gives two activity peaks, one in the void volume exhibiting high PEP-dependent and TP activities, and a second included peak with high PEP-dependent activity and high (IIMan), moderate (IIGlc) or negligible (IIMtl) TP activities. Both log and stationary phase cells exhibit comparable relative amounts of pelletable and soluble enzyme II activities, but long-term exposure of cells to chloramphenicol results in selective loss of the soluble fraction with retention of much of the pelleted activity concomitant with extensive protein degradation. Short-term exposure of cells to chloramphenicol results in increased activities in both fractions, possibly because of increased lipid association, with more activation in the soluble fraction than in the pelleted fraction. Western blot analyses show that the soluble IIGlc exhibits a subunit size of about 45 kDa, and all three soluble enzymes II elute from the gel filtration column with apparent molecular weights of 40-50 kDa. We propose that enzymes II of the PTS exist in two physically distinct forms in the E. coli cell, one tightly integrated into the membrane and one either soluble or loosely associated with the membrane. We also propose that the membrane-integrated enzymes II are largely dimeric, whereas the soluble enzymes II, retarded during passage through a gel filtration column, are largely monomeric.