Protein:Protein Interactions in the Cytoplasmic Membrane Influencing Sugar Transport and Phosphorylation Activities of theE. coliPhosphotransferase System

Abstract

26The multicomponent phosphoenolpyruvate-dependent sugar-transporting 27 phosphotransferase system (PTS) in Escherichia coli takes up sugar substrates and 28 concomitantly phosphorylates them. We have recently provided evidence that many of the 29 integral membrane PTS permeases interact with the fructose PTS (FruA/FruB) [1]. However, the 30 biochemical and physiological significance of this finding was not known. We have carried out 31 molecular genetic/biochemical/physiological studies that show that interactions of the fructose 32 PTS often enhance, but sometimes inhibit the activities of other PTS transporters many fold, 33 depending on the target PTS system under study. Thus, the glucose, mannose, mannitol and N-34 acetylglucosamine permeases exhibit enhanced in vivo sugar transport and sometimes in vitro 35 PEP-dependent sugar phosphorylation activities while the galactitol and trehalose systems show 36 inhibited activities. This is observed when the fructose system is induced to high levels and 37 prevented when the fruA/fruB genes are deleted. Overexpression of the fruA and/or fruB genes in 38 the absence of fructose induction during growth also enhances the rates of uptake of other 39 hexoses. The β-galactosidase activities of man, mtl, and gat-lacZ transcriptional fusions and the 40 sugar-specific transphosphorylation activities of these enzyme transporters were not affected 41 either by frustose induction or fruAB overexpression, showing that the rates of synthesis and 42 protein levels in the membrane of the target PTS permeases were not altered. We thus suggest 43 that specific protein-protein interactions within the cytoplasmic membrane regulate transport in 44 vivo (and sometimes the PEP-dependent phosphorylation activities in vitro of PTS permeases) in 45 a physiologically meaningful way that may help to provide a hierarchy of preferred PTS sugars.46 These observations appear to be applicable in principle to other types of transport systems as 47 well. 63 complexes. Sugars transported via the PTS in various organisms include aldo-and keto-hexoses, 64 amino sugars and their N-acetylated derivatives, hexitols, pentoses, pentitols and a variety of 65 disaccharides, oligosaccharides and glycosides [8]. In Escherichia coli, there are many Enzyme 66 II (EII) complexes, some of which are still not functionally characterized [11]. The EIIs we will 67 be concerned with in this report, in addition to the three described above, are specific for sugars 68 such as galactitol (Gat), glucose (Glc) and the non-metabolizable glucose analogue, methyl α-69 glucoside (αMG), trehalose (Tre), and mannose (Man) [this system also transports Glc, 70 glucosamine (Glm) and 2-deoxyglucose (2DG)]. These systems are tabulated in Table 1 with 71 their protein abbreviations, protein domain orders and TC numbers (TC #s) in the Transporter 72 Classification Database (TCDB) [12-15]. 77 reaction that depends only on IIB and IIC [16-19]. All of these reactions have been used to 78 investigate the consequences of integral membrane pro...