Abstract
The multicomponent phosphoenolpyruvate (PEP)-dependent sugar-transporting phosphotransferase system (PTS) in Escherichia coli takes up sugar substrates from the medium and concomitantly phosphorylates them, releasing sugar phosphates into the cytoplasm. We have recently provided evidence that many of the integral membrane PTS permeases interact with the fructose PTS (FruA/FruB) [1]. However, the biochemical and physiological significance of this finding was not known. We have carried out molecular genetic/biochemical/physiological studies that show that interactions of the fructose PTS often enhance, but sometimes inhibit the activities of other PTS transporters many fold, depending on the target PTS system under study. Thus, the glucose (Glc), mannose (Man), mannitol (Mtl) and N-acetylglucosamine (NAG) permeases exhibit enhanced in vivo sugar transport and sometimes in vitro PEP-dependent sugar phosphorylation activities while the galactitol (Gat) and trehalose (Tre) systems show inhibited activities. This is observed when the fructose system is induced to high levels and prevented when the fruA/fruB genes are deleted. Overexpression of the fruA and/or fruB genes in the absence of fructose induction during growth also enhances the rates of uptake of other hexoses. The β-galactosidase activities of man, mtl, and gat-lacZ transcriptional fusions and the sugar-specific transphosphorylation activities of these enzyme transporters were not affected either by frustose induction or by fruAB overexpression, showing that the rates of synthesis of the target PTS permeases were not altered. We thus suggest that specific protein-protein interactions within the cytoplasmic membrane regulate transport in vivo (and sometimes the PEP-dependent phosphorylation activities in vitro) of PTS permeases in a physiologically meaningful way that may help to provide a hierarchy of preferred PTS sugars. These observations appear to be applicable in principle to other types of transport systems as well.
Highlights
The prokaryotic phosphotransferase system (PTS) consists of two general energy coupling proteins, Enzyme I, (EI, PtsI) and HPr (HPr, PtsH), as well as the sugar-specificEnzyme II (EII) complexes [1,2,3,4,5]
It consists of energy-coupling phosphoryl transfer proteins (Enzyme I, HPr, and the sugarspecific IIA and IIB proteins or protein domains) as well as the PTS sugar transporters, the IIC
Prior to the present study, the enzyme IIC proteins had not been shown to function as parts of regulatory networks involving direct protein-protein interactions within the membrane
Summary
The prokaryotic phosphotransferase system (PTS) consists of two general energy coupling proteins, Enzyme I, (EI, PtsI) and HPr (HPr, PtsH), as well as the sugar-specific. The results of the studies reported here suggest that in the cytoplasmic membrane of E. coli, the transport of various PTS sugars is influenced by the presence of other PTS permeases in general, and the degree of activation or inhibition depends on the specific systems under study as well as their concentrations in the membrane. High level expression of the fructose EII complex activates the mannitol, N-acetyl glucosamine, glucose and mannose systems, but it inhibits the galactitol and trehalose systems in wild type cells These protein-protein interactions do not appreciably affect the sugar-P:sugar TP reactions or synthesis of the target. The more general conclusion is that integral membrane transport proteins in the cytoplasmic membranes of E. coli are in close contact and influence each other’s activities in the intact cell, either in one direction (activation) or the other (inhibition) In some cases, these effects appear to be highly specific, while in others they may be more general. This is, to the best of our knowledge, the first example where global proteomic analyses based on a complete interactome data set, have led to the discovery of regulating interactions influencing the enzymatic and transport activities of some of the interacting integral membrane proteins
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