Secondary solute transport in bacteria

Poolman, Bert; Konings, Wil N.

doi:10.1016/0005-2728(93)90003-x

Cited by 202 publications

(164 citation statements)

References 320 publications

(303 reference statements)

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“…20 amino acids in interhelix loop 10-11 of the LacS members of the GPH family (sequence motif Lys-x-x-His-x-x-Glu; Poolman et al, 1992;1995b). Among the conserved residues are a histidine (322 in LacY, 376 in LacS) and a glu-tamic acid residue (325 in LacY, 379 in LacS), which have similar properties in both of the galactoside transport proteins (Kaback, 1992;Poolman and Konings, 1993). For instance, glutamic acid-325 in LacY is believed to participate directly in galactoside-coupled proton translocation because sugar-H + symport is completely defective in neutral substitution mutants, whereas uncoupled sugar transport (uniport) can still be observed (Carrasco et al, 1986;1989).…”

Section: Interhelix Loop 10-11mentioning

confidence: 99%

“…Most of the sensible secondary coupling mechanisms that one can imagine in terms of direction of transport, reaction stoichiometry, electrogenic/ electrophoretic nature, and type of coupling solute have been discovered in the past two decades (Poolman and Konings, 1993). As more and more primary sequences of these proteins have become available it has become apparent that a single family of homologous transporters can be composed of uniporters, symporters and antiporters, while among the symporters and antiporters the reaction stoichiometries, type of coupling solute etc.…”

Section: Introductionmentioning

confidence: 99%

“…The largest number of mutants is available for the lactose-transport protein (LacY) of Escherichia coli (Kaback, 1990;Poolman and Konings, 1993), and the second best studied bacterial transporter in this respect is the melibiose-carrier protein (MelB) of E. coli Poolman and Konings, 1993). While LacY obligatorily couples the transport of galactosides to protons (Kaback, 1990), the MelB protein can use either Na + , Li + or H + as coupling ions ( Tsuchiya and Wilson, 1978;Wilson and Wilson, 1987).…”

Section: Introductionmentioning

confidence: 99%

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Cation and sugar selectivity determinants in a novel family of transport proteins

Poolman

Knol

Does

et al. 1996

Molecular Microbiology

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SummaryA new family of homologous membrane proteins that transport galactosides-pentoses-hexuronides (GPH) is described. By analysing the aligned amino acid sequences of the GPH family, and by exploiting their different specificities for cations and sugars, we have designed mutations that yield novel insights into the nature of ligand binding sites in membrane proteins. Mutants have been isolated/constructed in the melibiose transport proteins of Escherichia coli, Klebsiella pneumoniae and Salmonella typhimurium, and the lactose transport protein of Streptococcus thermophilus which facilitate uncoupled transport or have an altered cation and/or substrate specificity. Most of the mutations map in the amino-terminal region, in or near amphipathic a -helices II and IV, or in interhelix-loop 10-11 of the transport proteins. On the basis of the kinetic properties of these mutants, and the primary and secondary structure analyses presented here, we speculate on the cation binding pocket of this family of transporters. The regulation of the transporters through interaction with, or phosphorylation by, components of the phosphoenolpyruvate:sugar phosphotransferase system is also discussed.

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Section: Interhelix Loop 10-11mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Cation and sugar selectivity determinants in a novel family of transport proteins

Poolman

Knol

Does

et al. 1996

Molecular Microbiology

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171

147

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“…The lactose permease (LacY) 1 of Escherichia coli transduces the free energy stored in an electrochemical H ϩ gradient into a concentration gradient of D-galactopyranosides and vice versa (galactoside/H ϩ symport) (1)(2)(3)(4). LacY is a 12-transmembranehelix bundle with the N and C termini on the cytoplasmic face of the membrane (5)(6)(7).…”

mentioning

confidence: 99%

Probing the Mechanism of a Membrane Transport Protein with Affinity Inactivators

Guan

Sahin–Tóth

Kálai

et al. 2003

Journal of Biological Chemistry

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Affinity inactivators are useful for probing catalytic mechanisms. Here we describe the synthesis and properties of methanethiosulfonyl (MTS) galactose or glucose derivatives with respect to a well studied membrane transport protein, the lactose permease of Escherichia coli. The MTS-galactose derivatives behave as affinity inactivators of a functional mutant with Ala 122 3 Cys in a background otherwise devoid of Cys residues. A proton electrochemical gradient (⌬ H ؉) markedly increases the rate of reaction between Cys 122 and MTS-galactose derivatives; nonspecific labeling with the corresponding MTS-glucose derivatives is unaffected. When the Ala 122 3 Cys mutation is combined with a mutation (Cys 154 3 Gly) that blocks transport but increases binding affinity, discrimination between the MTS-galactose and -glucose derivatives is abolished, and ⌬ H ؉ has no effect. The results provide strong confirmation that the non-galactosyl moiety of permease substrates abuts Ala 122 in helix IV. In addition, the findings demonstrate that the MTS-galactose derivatives do not react with the Cys residue at position 122 upon binding per se but at a subsequent step in the overall transport mechanism. Thus, these inactivators behave as unique suicide substrates.The lactose permease (LacY) 1 of Escherichia coli transduces the free energy stored in an electrochemical H ϩ gradient into a concentration gradient of D-galactopyranosides and vice versa (galactoside/H ϩ symport) (1-4). LacY is a 12-transmembranehelix bundle with the N and C termini on the cytoplasmic face of the membrane (5-7). Several lines of evidence indicate that it is both functionally (8) and structurally a monomer (9 -11).Analysis of an extensive library of mutants, particularly Cys replacement mutants (12), with a battery of site-directed biophysical and biochemical techniques has led to the formulation of a teriary structure model (49) that includes tilts as well as a working model for the transport mechanism (13).LacY is in close proximity to the nongalactosyl portion of ligand (24). Affinity labeling is based upon specific recognition of an inactivating ligand at the binding site. Because Cys 148 (helix V), which is in close proximity to Ala 122 , makes direct contact with the galactopyranosyl moiety of the substrate, numerous attempts to develop an affinity reagent specific for this side chain have failed. On the other hand, Ala 122 abuts the nongalactosyl moiety of disaccharide substrates, and alkyation of a Cys residue at this position blocks binding of disaccharides with no effect on galactose transport or binding. Therefore, methanethiosulfonyl (MTS) galactoside with an ethylene linkage between the galactopyranoside ring and MTS and MTS-5-galactose with a carbamide linkage were synthesized as potential affinity reagents for the single-Cys A122C mutant in LacY with the corresponding glucosides as controls. EXPERIMENTAL PROCEDURES ReagentsMTS-galactoside and MTS-glucoside-MTS-galactoside and MTSglucoside were synthesized as shown in the synthetic scheme (Sch...

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“…bioenergetics ͉ transport ͉ membrane protein ͉ structure T he lactose permease of Escherichia coli (LacY) is a paradigm for membrane transport proteins that couple free energy stored in electrochemical ion gradients into solute concentration gradients (1)(2)(3)(4). Thus, LacY catalyzes the coupled stoichiometric translocation of galactosides and H ϩ (lactose͞H ϩ symport), by using the free energy released from the downhill translocation of H ϩ to drive galactoside accumulation and vice versa.…”

mentioning

confidence: 99%

Surface-exposed positions in the transmembrane helices of the lactose permease of Escherichia coli determined by intermolecular thiol cross-linking

Guan

Murphy

Kaback

2002

Proc. Natl. Acad. Sci. U.S.A.

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bioenergetics ͉ transport ͉ membrane protein ͉ structure T he lactose permease of Escherichia coli (LacY) is a paradigm for membrane transport proteins that couple free energy stored in electrochemical ion gradients into solute concentration gradients (1-4). Thus, LacY catalyzes the coupled stoichiometric translocation of galactosides and H ϩ (lactose͞H ϩ symport), by using the free energy released from the downhill translocation of H ϩ to drive galactoside accumulation and vice versa. The protein has been solubilized from the membrane, purified in a completely functional state (reviewed in ref. 5) and shown to function as a monomer (6). LacY has 12 transmembrane helices with the N and C termini on the cytoplasmic face of the membrane ( Fig. 1; refs. 7-9).In a functional LacY mutant devoid of native Cys residues (Cys-less LacY), each residue has been replaced with Cys or other residues (reviewed in ref. 10). Systematic study of singleCys and other site-directed mutants has led to the identification of functionally essential residues (10) as well as a working model for the mechanism of lactose͞H ϩ symport (11,12). Analysis of the mutant library with a battery of site-directed biophysical and biochemical techniques has also led to the formulation of a helix-packing model of LacY ( Fig. 6; reviewed in ref. 13) In addition to other methods, intramolecular thiol crosslinking has been used for estimating helix packing, tilts, and ligand-induced conformational changes in LacY (12). While studying cross-linking of helix VI with homobifunctional thiol cross-linking agents in nonoverlapping, contiguous peptides corresponding to the N-and C-terminal halves of LacY (N 6 ͞C 6 split LacY), we observed (14) that certain paired-Cys mutants form C 6 ͞C 6 homodimers. The observation raised the possibility that such an approach might be useful for identifying residues in transmembrane helices that are surface exposed.In this study, homodimer formation induced by a homobifunctional thiol cross-linking agent 5 Å in length is observed with 9 single-Cys mutants in transmembrane helices of 250 mutants tested. Seven mutants are located at the cytoplasmic ends and 2 at periplasmic ends of transmembrane helices, and the positions are distributed around the periphery of the 12-helix bundle. The results are consistent with the current helix-packing model and suggest that Cys residues on helical surfaces exposed to the low dielectric of the membrane are unreactive. Moreover, two positions on opposite sides of the molecule cross-link at similar rates with sigmoid time courses, and cross-linking is markedly decreased at low temperature. The findings provide further evidence for the conclusion that LacY is a monomer, because homodimer formation appears to be a stochastic process involving random collisions within the plane of the membrane. Experimental ProceduresLacY Mutants. Construction of all single-Cys lacY mutants in plasmid pT7-5 has been described (15-24). Given mutants contain a 100-residue biotin acceptor domain (BAD) in the middle cy...

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Secondary solute transport in bacteria

Cited by 202 publications

References 320 publications

Cation and sugar selectivity determinants in a novel family of transport proteins

Cation and sugar selectivity determinants in a novel family of transport proteins

Probing the Mechanism of a Membrane Transport Protein with Affinity Inactivators

Surface-exposed positions in the transmembrane helices of the lactose permease of Escherichia coli determined by intermolecular thiol cross-linking

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