Identification of the GalP galactose transport protein of Escherichia coli.

Jones‐Mortimer, M. C.; Horne, P; Henderson, Peter J. F.

doi:10.1016/s0021-9258(18)32635-8

Cited by 42 publications

(7 citation statements)

References 38 publications

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“…To examine the ability of A8-35 to stabilize MP structures in the gas phase further, we investigated two α-helical membrane transporters, Mhp1 and GalP (Figure c). Mhp1 is a 54.6 kDa Na + coupled hydantoin transporter and a member of the nucleobase-cation-symport 1 (NCS1) family of transporters which are involved in nucleobase salvage pathways and vitamin influx. , The 12 transmembrane helix bundle of Mhp1 undergoes significant conformational changes as it transports its substrate. , GalP is the 51.7 kDa galactose-H + symporter from E. coli and is a member of the major facilitator superfamily (MFS) of transport proteins. − There is limited structural information available for this protein and other related members of the MFS; however, they are generally predicted to comprise 12 transmembrane helices, and structures have been proposed using homology modeling (Figure c).…”

Section: Resultsmentioning

confidence: 99%

Amphipols Outperform Dodecylmaltoside Micelles in Stabilizing Membrane Protein Structure in the Gas Phase

et al. 2014

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Noncovalent mass spectrometry (MS) is emerging as an invaluable technique to probe the structure, interactions, and dynamics of membrane proteins (MPs). However, maintaining native-like MP conformations in the gas phase using detergent solubilized proteins is often challenging and may limit structural analysis. Amphipols, such as the well characterized A8-35, are alternative reagents able to maintain the solubility of MPs in detergent-free solution. In this work, the ability of A8-35 to retain the structural integrity of MPs for interrogation by electrospray ionization-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS) is compared systematically with the commonly used detergent dodecylmaltoside. MPs from the two major structural classes were selected for analysis, including two β-barrel outer MPs, PagP and OmpT (20.2 and 33.5 kDa, respectively), and two α-helical proteins, Mhp1 and GalP (54.6 and 51.7 kDa, respectively). Evaluation of the rotationally averaged collision cross sections of the observed ions revealed that the native structures of detergent solubilized MPs were not always retained in the gas phase, with both collapsed and unfolded species being detected. In contrast, ESI-IMS-MS analysis of the amphipol solubilized MPs studied resulted in charge state distributions consistent with less gas phase induced unfolding, and the presence of lowly charged ions which exhibit collision cross sections comparable with those calculated from high resolution structural data. The data demonstrate that A8-35 can be more effective than dodecylmaltoside at maintaining native MP structure and interactions in the gas phase, permitting noncovalent ESI-IMS-MS analysis of MPs from the two major structural classes, while gas phase dissociation from dodecylmaltoside micelles leads to significant gas phase unfolding, especially for the α-helical MPs studied.

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Section: Resultsmentioning

confidence: 99%

Amphipols Outperform Dodecylmaltoside Micelles in Stabilizing Membrane Protein Structure in the Gas Phase

et al. 2014

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“…The protection by substrates was exploited to label the susceptible transport proteins, AraE, XylE and GalP, with radioactive A-ethylmaleimide; they were identified as proteins that migrated with an apparent relative molecular mass (Mr) of 35000-40000 in SDSpolyacrylamide gels (Macpherson et al 1981(Macpherson et al , 1983Henderson et al 1983;Henderson & Macpherson 1986;Davis 1986). By similar procedures the lactose/H+ transport protein was the first to be identified, with an apparent M r of about 30000 (Jones & Kennedy 1969).…”

Section: N-ethylmaleimidementioning

confidence: 99%

Homologous sugar transport proteins in Escherichia coli and their relatives in both prokaryotes and eukaryotes

Henderson

Maiden

1990

Phil. Trans. R. Soc. Lond. B

152

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Separate proteins for proton-linked transport of D-xylose, L-arabinose, D-galactose, L-rhamnose and L-fucose into Escherichia coli are being studied. By cloning and sequencing the appropriate genes, the amino acid sequences of proteins for D-xylose/H+ symport (XylE), L-arabinose/H+ symport (AraE), and part of the protein for D-galactose/H+ symport (GalP) have been determined. These are homologous, with at least 28% identical amino acid residues conserved in the aligned sequences, although their primary sequences are not similar to those of other E. coli transport proteins for lactose, melibiose, or D-glucose. However, they are equally homologous to the passive D-glucose transport proteins from yeast, rat brain, rat adipocytes, human erythrocytes, human liver, and a human hepatoma cell line. The substrate specificity of GalP from E. coli is similar to that of the mammalian glucose transporters. Furthermore, the activities of GalP, AraE and the mammalian glucose transporters are all inhibited by cytochalasin B and N-ethylmaleimide. Conserved residues in the aligned sequences of the bacterial and mammalian transporters are identified, and the possible roles of some in sugar binding, cation binding, cytochalasin binding, and reaction with N-ethylmaleimide are discussed. Each protein is independently predicted to form 12 hydrophobic, membrane-spanning alpha-helices with a central hydrophilic segment, also comprised of alpha-helix. This unifying structural model of the sugar transporters shares features with other ion-linked transport proteins for citrate or tetracycline.

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“…The results presented in this work contributed to the identification of the presumptive KDG permease and provided another example of characterization by cloning procedures of E. coli active transport systems energized by the proton motive force (8,15,16,37). The plasmids that we have constructed can now be used for large-scale purification of the gene product so that the properties of the KDG transport system reconstituted into lipid vesicles can be studied.…”

Section: Discussionmentioning

confidence: 84%

Construction and expression of hybrid plasmids containing the structural gene of the Escherichia coli K-12 3-deoxy-2-oxo-D-gluconate transport system

Mandrand‐Berthelot¹,

Ritzenthaler²,

Mata-Gilsinger³

1984

J Bacteriol

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The kdgT gene of Escherichia coli, which encodes for the 3-deoxy-2-oxo-D-gluconate transport system, was isolated as a ColE1-kdgT hybrid plasmid from the Clarke and Carbon bank. A restriction and genetic map of the min 88 region of the chromosome was established; by subcloning the restriction fragments into the plasmid vector pBR322, the kdgT gene was localized on a 1.4-megadalton PstI DNA fragment, and the direction of transcription of the gene was determined by making use of an in vitro gene fusion between kdgT and lacZ genes. Amplification of the gene product of kdgT was up to 14-fold the level found in a haploid strain. When plasmids bearing kdgT were expressed in an in vivo maxicell system, a specific polypeptide of 28,000 daltons appeared that was found to be associated with the membrane fraction.

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Identification of the GalP galactose transport protein of Escherichia coli.

Cited by 42 publications

References 38 publications

Amphipols Outperform Dodecylmaltoside Micelles in Stabilizing Membrane Protein Structure in the Gas Phase

Amphipols Outperform Dodecylmaltoside Micelles in Stabilizing Membrane Protein Structure in the Gas Phase

Homologous sugar transport proteins in Escherichia coli and their relatives in both prokaryotes and eukaryotes

Construction and expression of hybrid plasmids containing the structural gene of the Escherichia coli K-12 3-deoxy-2-oxo-D-gluconate transport system

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