Two-Dimensional Crystallization ofEscherichia coliLactose Permease

Zhuang, Jianping; Privé, G.G.; G, Werner; Ringler, Philippe; Kaback, H. Ronald; Engel, Andréas

doi:10.1006/jsbi.1998.4059

Cited by 87 publications

(60 citation statements)

References 37 publications

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“…1). Finally, 7 of the 9 mutants are located toward the cytoplasmic surface, whereas only 2 are located toward the periplasmic surface, suggesting that the profile of LacY in the membrane may not be regular (28)(29)(30).…”

Section: Discussionmentioning

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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Section: Discussionmentioning

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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“…The r values 0.40±0.42 and 0.68 (Table 2) for GLUT1 in proteoliposomes and biotinylated red blood cells, respectively, could alternatively be explained in terms of GLUT1 trimers, each showing either one or two CB-binding sites, provided that this can be reconciled with the kinetics of glucose transport and inhibition. A chimeric protein consisting of the 12-a-helical lactose permease from Escherichia coli with cytochrome b 562 in the middle cytoplasmic loop formed trimers on two-dimensional crystallization [49].…”

Section: Discussionmentioning

confidence: 99%

Conversion between two cytochalasin B-binding states of the human GLUT1 glucose transporter

Gottschalk¹,

Lundqvist²,

Zeng³

et al. 2000

Eur J Biochem

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Two cytochalasin B-binding states of the human red blood cell facilitative glucose transporter GLUT1 were studied, one exhibiting one cytochalasin B-binding site on every second GLUT1 monomer (state 1) and the other showing one site per monomer (state 2). Quantitative affinity chromatography of cytochalasin B was performed on (a) biotinylated red blood cells, (b) cytoskeleton-depleted red blood cell membrane vesicles, and (c) GLUT1 proteoliposomes. The cells were adsorbed on streptavidin-derivatized gel beads, and the vesicles and proteoliposomes entrapped in dextran-grafted agarose gel beads. Cytochalasin B binding to free vesicles and proteoliposomes was analyzed by Hummel and Dreyer size-exclusion chromatography and ultracentrifugation. Analysis of the biotinylated cells indicated an equilibrium between the two GLUT1 states. GLUT1 in free membrane vesicles attained state 2, but was converted into state 1 on entrapment of the vesicles. Purification of GLUT1 in the presence of non-ionic detergent followed by reconstitution produced GLUT1 in state 1. This state was maintained after entrapment of the proteoliposomes. Finally, GLUT1 showed slightly higher affinity for cytochalasin B in state 1 than in state 2. In summary, the cytochalasin B-binding state of GLUT1 seemed to be affected by (a) biotinylation of the cell surface, (b) removal of the cytoskeleton at high pH and low ionic strength, (c) interaction between the dextran-grafted agarose gel matrix and the membrane vesicles, and (d) reconstitution to form proteoliposomes.Keywords: cytochalasin B; glucose transporter states; GLUT1; immobilized biomembrane affinity chromatography; red blood cell streptavidin±biotin immobilization.The facilitative glucose transporter GLUT1 [1±9] mediates passage of d-glucose and dehydroascorbic acid across plasma membranes. The transport is passive, driven by the concentration gradient of the substrate and a transport mechanism that is still under debate [6,8]. In the more generally accepted alternating conformation model', each separate GLUT1 monomer presents a translocation pathway, oscillating between a state exposing the sugar export site and another exposing the import site as described in the reviews [1,4,6,8]. In the`fixedsite carrier model' [10,11], the protein is regarded as a dimer of dimers exposing two sugar import sites and two export sites. Each of the latter two sites is overlapped by a binding site for the transport inhibitor cytochalasin B (CB) [10±13], and thus there are two binding sites for CB per tetramer (`state 1'). Alkaline reduction of the disulfides in the transporter has been reported to convert GLUT1 into another state with one CB-binding site per monomer [12,13]. Recently, Gottschalk et al. [14] showed by immobilized-biomembrane affinity chromatography (IBAC) on lectin-immobilized red blood cells that GLUT1 is converted from state 1 into a state showing a CB-binding site on every monomer (`state 2') by interaction between human red blood cells and polylysine. IBAC allows determination of ligand±membr...

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“…For single-particle analysis PM28 complexes were extracted, aligned, and classified. Briefly, a reference was established by selecting a well preserved particle and symmetrizing it 20-fold rotationally (34,35). Cross-correlation functions of this reference with images of digitized micrographs containing adsorbed particles of PM28 revealed correlation peaks at the particle positions, irrespective of their angular orientation.…”

Section: Methodsmentioning

confidence: 99%

Structural Characterization of Two Aquaporins Isolated from Native Spinach Leaf Plasma Membranes

Fotiadis

Jenö

Mini

et al. 2001

Journal of Biological Chemistry

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Two members of the aquaporin family, PM28A and a new one, PM28C, were isolated and shown to be the major constituents of spinach leaf plasma membranes. These two isoforms were identified and characterized by matrix-assisted laser desorption ionization-mass spectrometry. Edman degradation yielded the amino acid sequence of two domains belonging to the new isoform. PM28B, a previously described isoform, was not found in our preparations. Scanning transmission electron microscopy mass analysis revealed both PM28 isoforms to be tetrameric. Two types of particles, a larger and a smaller one, were found by transmission electron microscopy of negatively stained solubilized proteins and by atomic force microscopy of PM28 two-dimensional crystals. The ratio of larger to smaller particles observed by transmission electron microscopy and single particle analysis correlated with the ratio of PM28A to PM28C determined by matrix-assisted laser desorption ionization-mass spectrometry. The absence of PM28B and the ratio of PM28A to PM28C indicate that these plasma membrane intrinsic proteins are differentially expressed in spinach leaves. These findings suggest that differential expression of the various aquaporin isoforms may regulate the water flux across the plasma membrane, in addition to the known mechanism of regulation by phosphorylation.Water is the universal solvent and most important molecule for life. Immense water volumes pass across the membranes of all living cells, especially across the plasma membranes of plants (1). Simple diffusion of water through the lipid bilayer has an activation energy of Ͼ10 kcal/mol and cannot explain the rapid water flow through human red cell membranes found to exhibit an activation energy of Ͻ5 kcal/mol. This led to the hypothesis that water pores must exist (2). The discovery (3) and cloning (4) of the first known water channel, aquaporin-1 (AQP1), 1 from human erythrocytes and the demonstration of water transport in Xenopus oocytes expressing its complementary RNA (5) confirmed this hypothesis.Since then, a large number of channel-forming integral proteins homologous to AQP1 have been found in all forms of life (6). This membrane protein family was initially named the MIP family after its first sequenced member, the major intrinsic protein (MIP) of bovine lens fiber cells (7). Multiple sequence alignment and phylogenetic analysis of 164 members of the MIP family, now frequently referred to as aquaporin super family, revealed 16 subfamilies that form two distinct clusters, the aquaporin (AQP) cluster and the glycerol facilitator-like cluster (8). The AQPs are highly specific for water, whereas the glycerol facilitators allow the passage of small, nonionic molecules such as glycerol and urea (9). In addition, ovine MIP tetramers have been found to form a groove and tongue contact via their extracellular surfaces, lending support to a dual function of the protein, as a water channel and as a cell to cell adhesion molecule in the eye lens (10).Most members of the aquaporin super family...

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Two-Dimensional Crystallization ofEscherichia coliLactose Permease

Cited by 87 publications

References 37 publications

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

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

Conversion between two cytochalasin B-binding states of the human GLUT1 glucose transporter

Structural Characterization of Two Aquaporins Isolated from Native Spinach Leaf Plasma Membranes

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