Topology of allosteric regulation of lactose permease

Abstract

Sugar transport by some permeases in Escherichia coli is allosterically regulated by the phosphorylation state of the intracellular regulatory protein, enzyme IIA glc of the phosphoenolpyruvate:sugar phosphotransferase system. A sensitive radiochemical assay for the interaction of enzyme IIA glc with membrane-associated lactose permease was used to characterize the binding reaction. The N-and C-termini, as well as five hydrophilic loops in the permease, are exposed on the cytoplasmic surface of the membrane and it has been proposed that the central cytoplasmic loop of lactose permease is the major determinant for interaction with enzyme IIA glc . Lactose permease mutants with polyhistidine insertions in cytoplasmic loops IV͞V and VI͞VII and periplasmic loop VII͞VIII retain transport activity and therefore substrate binding, but do not bind enzyme IIA glc , indicating that these regions of lactose permease may be involved in recognition of enzyme IIA glc . Taken together, these results suggest that interaction of lactose permease with substrate promotes a conformational change that brings several cytoplasmic loops into an arrangement optimal for interaction with the regulatory protein, enzyme IIA glc . A topological map of the proposed interaction is presented.Bacteria use a wide range of carbon sources that are transported across the cytoplasmic membrane by a variety of specific transport systems. The phosphoenolpyruvate:sugar phosphotransferase system (PTS), a multifunctional system consisting of several protein components, is widespread in bacteria. The primary function of the PTS is the concomitant phosphorylation and translocation of a variety of sugar substrates from the medium (1, 2). In the case of glucose transport in Gram-negative bacteria, three soluble proteins (enzyme I, HPr, and enzyme IIA glc ) and one membrane-bound protein (enzyme IIB, C glc ) are required for transport. In Escherichia coli, the PTS also regulates uptake of various non-PTS sugars such as lactose, maltose, melibiose, galactose, and raffinose (inhibition of non-PTS inducer transport by PTS sugars is termed ''inducer exclusion''), as well as the phosphorylation of glycerol and the synthesis of cyclic AMP (catabolite repression) (2-6). These regulatory roles of the PTS have been proposed to be mediated by the glucosespecific enzyme IIA glc , and the currently accepted model suggests that the phosphorylation state of IIA glc determines whether or not it binds to a target protein (2).E. coli lactose permease (lac permease) is an important model for the study of membrane transport proteins. Lac permease is one of the most extensively investigated secondary active transport proteins that transduce free energy stored in an electrochemical ion gradient into a solute concentration gradient (7-9). It is a hydrophobic, polytopic membrane protein that catalyzes the coupled stoichiometric translocation of ␤-galactosides and H ϩ . All available evidence indicates strongly that lac permease is comprised of a bundle of 12 transmembrane ␣-h...

How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria

“…The binding of the sugar induces a structural change, which exposes the substrate binding site to the cytoplasm ("inward"). To explain the observation that EIIA Glc binds to LacY with higher affinity when its substrate is present (597,624,800,825), the "outward"/ "inward" transition is assumed to make the EIIA Glc interaction site more easily accessible (800,823). Evidence supporting this concept comes from studies of a Cys154Gly mutant LacY.…”

“…For example, EIIA Glc binds strongly to the lactose transporter LacY only in the presence of a ␤-galactoside (597,624,800,825). This mechanism ensures that EIIA Glc , the concentration of which is fairly constant in E. coli and S. enterica serovar Typhimurium cells, will not be wasted on useless binding, i.e., under conditions in which the substrate is not present in the environment.…”

“…The major determinants of substrate binding are located at the interfaces of helices IV, V, and VIII (300,444,754). In contrast, the cytoplasmic loop between helices IV and V together with the flanking helical domains and some residues in the large cytoplasmic loop connecting helices VI and VII are important for the interaction of LacY with EIIA Glc (338,800,823). Based on the analysis of various lacY mutants, a model for LacY function in galactoside transport has been proposed (3,391,942).…”

Escherichia coli enzyme IIA Ntr regulates the K + transporter TrkA

“…leucine toxicity ͉ nitrogen-metabolic phosphotransferase system (PTS) ͉ potassium transporter TrkA ͉ protein-protein interaction ͉ signal transduction T he well defined phosphotransferase system (PTS) is composed of two general cytoplasmic proteins, enzyme I (EI) and histidine phosphocarrier protein (HPr), and some sugarspecific components collectively known as enzymes II (1). The carbohydrate PTS, especially for glucose uptake, occupies a central position in bacterial physiology as a result of the identification of multiple regulatory functions superimposed on the transport functions, such as regulation of chemotaxis by EI (2); regulation of glycogen breakdown by HPr (3, 4); regulation of the global repressor Mlc by the membrane-bound glucose transporter EIICB Glc (5-7); and regulation of carbohydrate transport and metabolism (1,8,9), the metabolic flux between fermentation and respiration (10), and adenylyl cyclase activity (11) by EIIA Glc . These regulatory functions of the carbohydrate PTS depend on the phosphorylation state of the involved components, which have been shown to increase in the absence and decrease in the presence of a PTS sugar substrate.…”

Expression of ptsG Encoding the Major Glucose Transporter Is Regulated by ArcA in Escherichia coli

“…In addition to sugar transport, multiple roles are exerted by the PTS and these include chemoreception (3), catabolite repression (4), carbohydrate transport and metabolism (1,5,6), carbon storage (7,8), and the coordination of carbon and nitrogen metabolism (9). More recently, we found that EIIA Glc of the PTS also regulates the flux between respiration and fermentation pathways by sensing the available sugar species via a phosphorylation state-dependent interaction with the fermentation/respiration switch protein FrsA (10).…”