Structure and Mechanism of the Glycerol-3-Phosphate Transporter from
            <i>Escherichia coli</i>

Huang, Yafei; Lemieux, M. Joanne; Song, Jinmei; Auer, Manfred; Wang, Da-Neng

doi:10.1126/science.1087619

Cited by 954 publications

(1,141 citation statements)

References 37 publications

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“…(ii) The citrate-binding site is located at the membrane/cytoplasm interface. The latter follows from the position of Arg-428 that is believed to be one of the residues to interact with the substrate (11,31) and is different from the situation in the LacY and GlpT structures where the substrates bind halfway up the membrane (12,13). Important consequences of this position of the substrate-binding site may be as follows: (i) the pore is wider in the outward facing conformation than in the case of LacY and GlpT, and (ii) isomerization from the outward to the inward facing conformation may pull apart the sites on the protein that coordinate the substrate molecule, thereby disrupting the binding site.…”

Section: Discussionmentioning

confidence: 94%

“…Under a constant flow of watersaturated air, and while stirring magnetically, 10 mM potassium ascorbate and 100 M PMS (final concentration) were added, and the proton motive force was allowed to develop for 2 min. Then, [1,[5][6][7][8][9][10][11][12][13][14] C]citrate (114 mCi/mmol Amersham Biosciences) was added to a final concentration of 4.4 M. The uptake was stopped by the addition of 2 ml of ice-cold 0.1 M LiCl, followed by immediate filtration over cellulose nitrate filters (0.45 m, pore size). The filters were washed once with 2 ml of the 0.1 M LiCl solution and assayed for radioactivity.…”

Section: Methodsmentioning

confidence: 99%

“…Recent crystal structures of the LacY and GlpT proteins, both secondary transporters from the major facilitator superfamily, support the alternating access model for the translocation mechanism of secondary transporters (12,13). In this model, the proteins contain single binding sites for substrate and co-ions that alternately are exposed to the two sides of the membrane during the catalytic cycle.…”

mentioning

confidence: 96%

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Alternating Access and a Pore-Loop Structure in the Na+-Citrate Transporter CitS of Klebsiella pneumoniae

Sobczak

Lolkema

2004

Journal of Biological Chemistry

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CitS of Klebsiella pneumoniae is a secondary transporter that transports citrate in symport with 2 Na ؉ ions. Reaction of Cys-398 and Cys-414, which are located in a cytoplasmic loop of the protein that is believed to be involved in catalysis, with thiol reagents resulted in significant inhibition of uptake activity. The reactivity of the two residues was determined in single Cys mutants in different catalytic states of the transporter and from both sides of the membrane. The single Cys mutants were shown to have the same transport stoichiometry as wild type CitS, but the C398S mutation was responsible for a 10-fold loss of affinity for Na ؉ . Both cysteine residues were accessible from the periplasmic as well as from the cytoplasmic side of the membrane by the membrane-impermeable thiol reagent [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MT-SET) suggesting that the residues are part of the translocation site. Binding of citrate to the outward facing binding site of the transporter resulted in partial protection against inactivation by N-ethylmaleimide, whereas binding to the inward facing binding site resulted in essentially complete protection. A 10-fold higher concentration of citrate was required at the cytoplasmic rather than at the periplasmic side of the membrane to promote protection. Only marginal effects of citrate binding were seen on reactivity with MTSET. Binding of Na ؉ at the periplasmic side of the transporter protected both Cys-398 and Cys-414 against reaction with the thiol reagents, whereas binding at the cytoplasmic side was less effective and discriminated between Cys-398 and Cys-414. A model is presented in which part of the cytoplasmic loop containing Cys-398 and Cys-414 folds back into the translocation pore as a pore-loop structure. The loop protrudes into the pore beyond the citrate-binding site that is situated at the membranecytoplasm interface.The secondary citrate transporter CitS of Klebsiella pneumoniae is a member of the bacterial 2-hydroxycarboxylate transporter (2HCT) 1 family. Members of the 2HCT family transport substrates that contain the 2-hydroxycarboxylate motif, as in citrate, malate, lactate, etc. (1). The family contains H ϩ and Na ϩ symporters that couple the translocation of the substrate to the co-ion as well as precursor/product exchangers that couple the uptake of the substrate to the excretion of a metabolic end product (2, 3). CitS is a Na ϩ symporter. It obligatorily couples the translocation of the divalent citrate anion (Hcit 2Ϫ ) to the translocation of two sodium ions and one proton (4, 5). The CitS protein is an integral membrane protein containing 446 amino acid residues (47.5 kDa). The protein is believed to consist of a bundle of 11 hydrophobic transmembrane segments (TMSs) with the N and C terminus located in the cytoplasm and periplasm, respectively ( Fig. 1) (6 -9). An additional hydrophobic segment, predicted to be transmembrane as well, was shown to reside in the periplasm between TMSs V and VI. The segment, termed Vb, and a stretch ...

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Section: Discussionmentioning

confidence: 94%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 96%

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Alternating Access and a Pore-Loop Structure in the Na+-Citrate Transporter CitS of Klebsiella pneumoniae

Sobczak

Lolkema

2004

Journal of Biological Chemistry

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“…The difference relates rather to the length of loops connecting transmembrane domains than the length of the domains themselves (Liang et al 1995, Miyamoto 1996, Fei et al 2000. Although no crystallographic structure of PepT1 protein is known, a probable model of PepT1 protein was created (Abramson et al 2003, Huang et al 2003) using appropriate software, and crystallography of similar transporter proteins of E. coli LacY (crystallized bound to the substrate), and GlpT (crystallized without a substrate). PepT1 shows an α-helix structure, and consists of 12 functional transmembrane domains, nonlinearly distributed within the cell membrane.…”

Section: Discussionmentioning

confidence: 99%

Cloning Two PepT1 cDNA Fragments of Common Carp, <I>Cyprinus Carpio</I> (Actinopterygii: Cypriniformes: Cyprinidae)

Ostaszewska¹,

Szatkowska²,

Verri³

et al. 2009

Acta Icth et Piscat

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Background. Common carp, Cyprinus carpio, is a model organism within Teleostei. Oligopeptides are a new and promising alternative source of amino acids in animal as well as in human nutrition. In common carp, the membrane protein that transports oligopeptides across the enterocyte membrane is encoded by the gene PepT1 (SLC15A1). The aim of this paper was to sequence the PepT1 (SLC15A1) in common carp. Materials and Methods. Intestine samples were isolated from six-week old common carp. Total RNA was isolated using a Trizol method. Reverse transcription was used to synthesize cDNA. Two different pairs of primers were designed, according to the zebrafish (Danio rerio) PepT1 sequence, and used for PCR. The amplified DNA was isolated by electrophoresis, cloned (pCRII-TOPO vectors), sequenced, and subjected to in silico analysis. Results. Two nucleotide fragments of the PepT1 gene were obtained and analyzed using bioinformatic tools. Both fragments showed a high degree of homology with the known PepT1 genes of other teleosts, mammals, and birds. High homology of the PepT1 gene, and similar primary protein structure among the aforementioned taxa probably reflects the conservative function of the PepT1 protein product. Both fragments of the PepT1 gene were deposited in GenBank (FJ556590; FJ529670). Conclusions. The sequenced fragments of the common carp PepT1 gene will allow evaluation of PepT1 expression in the intestines of fish fed diets containing various forms of protein, which is an issue of importance regarding fish nutrition and its import for aquaculture.

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“…E. coli의 경우 구조 분석을 통해 막 횡단 단백질 GlpT가 하나의 기질 결합 부위를 가지고 있으며, 기 질접근을 교체하는 방식으로 수송한다는 사실이 알려졌다 (Huang et al, 2003). 많은 세균들이 GlpT를 갖고 있는데, E. coli의 경우 glpT 유전자는 glp 오페론에 포함되어 있으며, glycerol-3-P이 제공되는 조건에서 glycerol-3-P이 전사 억제자 GlpR의 DNA 결합력을 감소시켜 오페론의 발현이 증가하는 것 으로 알려졌다 (Lemieux et al, 2004).…”

Section: Glpt 시스템unclassified

Bacterial Phosphate Homeostasis: Role of Phosphate Transporters

Park¹,

Bang²

2012

The Korean Journal of Microbiology

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Structure and Mechanism of the Glycerol-3-Phosphate Transporter from Escherichia coli

Cited by 954 publications

References 37 publications

Alternating Access and a Pore-Loop Structure in the Na+-Citrate Transporter CitS of Klebsiella pneumoniae

Alternating Access and a Pore-Loop Structure in the Na+-Citrate Transporter CitS of Klebsiella pneumoniae

Cloning Two PepT1 cDNA Fragments of Common Carp, <I>Cyprinus Carpio</I> (Actinopterygii: Cypriniformes: Cyprinidae)

Bacterial Phosphate Homeostasis: Role of Phosphate Transporters

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