Functional Characterization of Cysteine Residues in GlpT, the Glycerol 3-Phosphate Transporter of
            <i>Escherichia coli</i>

Fann, Mon Chou; Busch, Anne; Maloney, Peter C.

doi:10.1128/jb.185.13.3863-3870.2003

Cited by 20 publications

(18 citation statements)

References 39 publications

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“…Cysteine substitution mutagenesis was used to allocate residue(s) of TM5 to the permeation pathway, using operational criteria established earlier -in particular, that the residue must be accessible to hydrophilic probes from either membrane surface (25,26). Use of proteoliposomes is especially helpful in such work, since after reconstitution by detergent dilution, MFS family members such as OxlT, GlpT and UhpT are found in both inside-out and right-side orientations, each of which has equivalent activity (16,24,34). Accordingly, substantial inhibition (≥ 80%) by an externally added probe reflects accessibility from both entrance and exit points of the permeation pathway.…”

Section: Discussionmentioning

confidence: 99%

Cysteine Scanning Mutagenesis of TM5 Reveals Conformational Changes in OxlT, the Oxalate Transporter of Oxalobacter formigenes

Wang

McKinney

et al. 2008

Biochemistry

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We constructed a single cysteine panel encompassing TM5 of the oxalate transporter, OxlT. The 25 positions encompassed by TM5 were largely tolerant of mutagenesis, and functional product was recovered for 21 of the derived variants. For these derivatives, thiol-directed MTS-linked agents (MTSEA, MTSCE, and MTSES) were used as probes of transporter function, yielding eleven mutants that responded to probe treatment, as indicated by effects on oxalate transport. Further study identified three biochemical phenotypes among these responders. Group 1 included seven mutants, exemplified by G151C, displaying substrate protection against probe inhibition. Group 2 was comprised of a single mutant, P156C, which had unexpected behavior. In this case, we observed increased activity if weak acid/base or neutral probes were used, while exposure to probes introducing a fixed charge led to decreased function. In both instances, the presence of substrate prevented the observed response. Group 3 contained three mutants (eg, S143C) in which probe sensitivity was increased by the presence of substrate. The finding of substrate-protectable probe modification in Groups 1 and 2 suggests that TM5 lies on the permeation pathway, as do its structural counterparts, TM2, TM8 and TM11. In addition, we speculate that substrate binding facilitates TM5 conformational changes that allow new regions become accessible to MTS-linked probes (Group 3). These biochemical data are consistent with the recently developed OxlT homology model. KeywordsMembrane transport; Reconstitution; Permeation pathway; Antiport; Major Facilitator SuperfamilyThe membrane transporter OxlT is found in Oxalobacter formigenes, a Gram-negative anaerobe that resides in the mammalian intestine (1). In O. formigenes, OxlT mediates the heterologous exchange of external oxalate 2− and internal formate 1− , the product of oxalate decarboxylation (1,2). The phenomenological coupling between the vectorial and scalar reactions leads to generation of a proton-motive force that drives ATP synthesis and other membrane activities (2,3). Subsequent work has indicated that this organizational scheme is found in a large number of other microbes, both Gram-negative and Gram-positive (4).Bioinformatic analysis shows that OxlT belongs to the Major Facilitator Superfamily (MSF) (5), the largest collection of secondary carrier-type transporters. It is also apparent that while individual members of MFS may exhibit a broad range of substrate specificity, including sugars, antibiotics and neurotransmitters, as a group they share an architectural theme in which a central loop of variable size connects a pair of domains, each of which usually contains six transmembrane helices , and in some cases is replicated at the cytoplasmic end of TM8, as in OxlT and GlpT (5-7).New insight into structure/function relationships within the MFS has been offered by crystal structures obtained for four separate examples. Initially, a 6.5Å resolution structure of OxlT, obtained by electron crystallography, es...

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Section: Discussionmentioning

confidence: 99%

Cysteine Scanning Mutagenesis of TM5 Reveals Conformational Changes in OxlT, the Oxalate Transporter of Oxalobacter formigenes

Wang

McKinney

et al. 2008

Biochemistry

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“…Successful results were achieved using DDM-mediated reconstitution of partially purified (about 85-90% pure according to SDS-PAGE analysis) His-tagged GlpT into liposomes consisting of 3:1 E. coli total lipid/ phosphatidylcholine. The presence of a His tag does not affect the activity of the transporter (16). Previous experiments have demonstrated that GlpT reconstitutes into liposomes in a random orientation (15).…”

Section: Reconstitution Of Glptmentioning

confidence: 99%

“…For whole-cell transport assays, E. coli strain SH1200 cells were grown overnight in M63 minimum medium (22) with 0.2% glucose as the carbon source, along with antibiotics and amino acids (each 50 μg/mL) (16). After overnight growth, cells were diluted 100-fold into the same medium and cultured at 35 °C to an OD 660 of 0.4-0.8.…”

Section: Transport Assays Using E Coli Whole Cellsmentioning

confidence: 99%

“…For whole-cell transport assays, E. coli strain SH1200 (glpT phoR ugpA704::Tn10) (obtained from W. Boos, University of Konstanz, Germany) served as the host to the plasmids used in this part of the study (16). Strain SH1200 fails to transport G3P because of defects in GlpT and UgpT, the ATP-dependent G3P transporter.…”

Section: Experimental Procedures Bacterial Strains and Plasmidsmentioning

confidence: 99%

“…The substrate translocation pore is located between the N-and C-terminal domains ( Figure 1A). This structural information, in combination with an opus of previous biochemical, thermodynamic, and kinetic studies on both GlpT and LacY (12)(13)(14)(15)(16)(17), and the closely related E. coli organic phosphate/inorganic phosphate antiporter, UhpT (18,19), have allowed us to construct a model for substrate translocation by GlpT (8,20) (parts B and C of Figure 1). This single-binding site, alternating-access mechanism, which involves a rocker-switch-type movement of the N-and C-terminal domains of the protein, is dependent upon binding-site reorientation between an inward (C i ) and outward (C o ) facing conformation.…”

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Kinetic Evidence Is Consistent with the Rocker-Switch Mechanism of Membrane Transport by GlpT

et al. 2007

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Secondary active transport of substrate across the cell membrane is crucial to many cellular and physiological processes. The crystal structure of one member of the secondary active transporter family, the sn-glycerol-3-phosphate (G3P) transporter (GlpT) of the inner membrane of Escherichia coli, suggests a mechanism for substrate translocation across the membrane that involves a rockerswitch-type movement of the protein. This rocker-switch mechanism makes two specific predictions with respect to kinetic behavior: the transport rate increases with the temperature, whereas the binding affinity of the transporter to a substrate is temperature-independent. In this work, we directly tested these two predictions by transport kinetics and substrate-binding experiments, integrating the data on this single system into a coherent set of observations. The transport kinetics of the physiologically relevant G3P-phosphate antiport reaction were characterized at different temperatures using both E. coli whole cells and GlpT reconstituted into proteoliposomes. Substrate-binding affinity of the transporter was measured using tryptophan fluorescence quenching in detergent solution. Indeed, the substrate transport velocity of GlpT increased dramatically with temperature. In contrast, neither the apparent Michaelis constant (K m ) nor the apparent substrate-binding dissociation constant (K d ) showed temperature dependence. Moreover, GlpT-catalyzed G3P translocation exhibited a completely linear Arrhenius function with an activation energy of 35.2 kJ mol −1 for the transporter reconstituted into proteoliposomes, suggesting that the substrate-loaded transporter is delicately poised between the inward-and outward-facing conformations. When these results are taken together, they are in agreement with a rocker-switch mechanism for GlpT.Secondary active membrane transporter proteins use an electrochemical gradient generated across the membrane by primary active transport as the driving force for substrate translocation (1,2). Over 100 families of secondary active membrane transporters have been identified to date, the largest of which is the major facilitator superfamily (MFS), 1 with over 5000 known members (3-5). The MFS, members of which are ubiquitous in all three kingdoms of life, includes many medically and pharmaceutically important transporters, such as the efflux pumps that confer resistance to antibiotics in bacteria and chemotherapeutic drugs in humans (6,7). † This work was supported in part by NIH Grant . The work of P.C.M. was supported by NIH Grant GM-24195. * To whom correspondence should be addressed. Telephone: (212) 263-8951. E-mail: wang@saturn.med.nyu.edu. ‡ New York University School of Medicine. § Johns Hopkins School of Medicine.1 Abbreviations: GlpT, glycerol-3-phosphate transporter; G3P, glycerol-3-phosphate; P i , inorganic phosphate; MFS, major facilitator superfamily; E a , activation energy; DDM, N-dodecyl-β-D-maltoside. Over the years, extensive efforts have been made to understand the molecular ...

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The Structure and Function of OxlT, the Oxalate Transporter of Oxalobacter formigenes

Iyalomhe

Khantwal

Kang³

2014

J Membrane Biol

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OxlT, the oxalate transporter of Oxalobacter formigenes, is a member of the Major Facilitator Superfamily of transporters (MFS), one of the largest groups of membrane proteins with substantial relevance to solute transport physiology, pharmacology, and possible drug development. MFS proteins transport a wide range of substrates such as organic and inorganic anions, sugars, drugs, and neurotransmitters. This review succinctly summarizes experimental work on a model MFS protein, OxlT, beginning with its identification as an electrogenic oxalate/formate exchanger, its three-dimensional structure, and discussion of biochemical and biophysical data that have shed further light on its structure and function. We also discuss the structure and function of OxlT in relation to notable MFS carriers such as LacY and GlpT.

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Functional Characterization of Cysteine Residues in GlpT, the Glycerol 3-Phosphate Transporter of Escherichia coli

Cited by 20 publications

References 39 publications

Cysteine Scanning Mutagenesis of TM5 Reveals Conformational Changes in OxlT, the Oxalate Transporter of Oxalobacter formigenes

Cysteine Scanning Mutagenesis of TM5 Reveals Conformational Changes in OxlT, the Oxalate Transporter of Oxalobacter formigenes

Kinetic Evidence Is Consistent with the Rocker-Switch Mechanism of Membrane Transport by GlpT

The Structure and Function of OxlT, the Oxalate Transporter of Oxalobacter formigenes

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