Cation‐selectivity of the <scp>l</scp>‐glutamate transporters of <i>Escherichia coli, Bacillus stearothermophilus</i> and <i>Bacillus caldotenax</i>: dependence on the environment in which the proteins are expressed

Tolner, Berend; Ubbink-Kok, Trees; Poolman, Bert; Konings, Wil N.

doi:10.1111/j.1365-2958.1995.mmi_18010123.x

Cited by 51 publications

(52 citation statements)

References 36 publications

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“…Protons modulate the activity of a number of sodium channels and sodium-dependent transporters by competing with sodium in binding to the protein (Woodhull, 1973;Tolner et al, 1995). We investigated this idea as a possible mechanism for proton inhibition of glycine transport by measuring glycine transport currents using a fixed glycine concentration and varying the sodium concentration (Fig.…”

Section: Resultsmentioning

confidence: 99%

Molecular Basis for Proton Regulation of Glycine Transport by Glycine Transporter Subtype 1b

2000

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In the central nervous system, glycine is a coagonist with glutamate at the N-methyl-D-aspartate subtype of ionotropic glutamate receptors. The GLYT1b subtype of glycine transporters is expressed in similar regions of the brain as the excitatory N-methyl-D-aspartate receptors and has been postulated to regulate glycine concentrations within excitatory synapses. We have expressed GLYT1b in Xenopus laevis oocytes and used electrophysiological techniques to investigate the pH regulation of glycine transporter function. We found that H ϩ inhibits glycine transport by a noncompetitive mechanism, with half-maximal inhibition occurring at concentrations found in both physiological and pathological conditions. Charge-to-flux experiments revealed that the decreased current measured corresponds to a decreased influx of [ 3 H]glycine and that the proton inhibition of GLYT1b does not alter the coupling ratio of transport. The membrane potential does not affect proton inhibition of transport, suggesting that the site of action on GLYT1b is not within the electric field of the membrane. Mutation of histidine 421 to an alanine residue, in the fourth extracellular loop of GLYT1b, renders the transporter insensitive to regulation by pH, but does not seem to alter the kinetics of glycine transport. These results suggests that histidine 421 is responsible for mediating the inhibitory actions of protons. Proton modulation of GLYT1b may be an important factor in determining the dynamics of excitatory neurotransmission.

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Section: Resultsmentioning

confidence: 99%

Molecular Basis for Proton Regulation of Glycine Transport by Glycine Transporter Subtype 1b

2000

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“…Na ϩ -and H ϩ -dependent symport of glutamate was observed when the enzyme was investigated in B. stearothermophilus membrane vesicles, while in E. coli membrane vesicles, only H ϩ -driven transport was found (31). Experiments are in progress to determine the influence of detergents and lipids on EII Mtl 's stability and activity.…”

Section: Purification Of Iicb-his Mtl Membrane Vesicles Of E Coli mentioning

confidence: 99%

Cloning, expression, and isolation of the mannitol transport protein from the thermophilic bacterium Bacillus stearothermophilus

et al. 1996

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A mannitol phosphotransferase system (PTS) was identified in Bacillus stearothermophilus by in vitro complementation with Escherichia coli EI, HPr, and IIA Mtl . Degenerate primers based on regions of high amino acid similarity in the E. coli and Staphylococcus carnosus EII Mtl were used to develop a digoxigenin-labeled probe by PCR. Using this probe, we isolated three overlapping DNA fragments totaling 7.2 kb which contain the genes mtlA, mtlR, mtlF, and mtlD, encoding the mannitol IICB, a regulator, IIA, and a mannitol-1-phosphate dehydrogenase, respectively. The mtlA gene consists of 1,413 bp coding for a 471-amino-acid protein with a calculated mass of 50.1 kDa. The amino acid sequence shows high similarity with the sequence of IICB Mtl of S. carnosus and the IICB part of the IICBA Mtl s of E. coli and B. subtilis. The enzyme could be functionally expressed in E. coli by placing it behind the strong tac promoter. The rate of thermal inactivation at 60؇C of B. stearothermophilus IICB Mtl expressed in E. coli was two times lower than that of E. coli IICB Mtl . IICB Mtl in B. stearothermophilus is maximally active at 85؇C and thus very thermostable. The enzyme was purified on Ni-nitrilotriacetic acid resin to greater than 95% purity after six histidines were fused to the C-terminal part of the transporter.Many bacteria transport D-mannitol and other carbohydrates via a phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS). Two general cytoplasmic proteins, EI and HPr, are responsible for the transfer of the phosphoryl group from PEP to different sugar-specific PTS proteins (15,18,22 In general, the genes of proteins involved in the uptake and phosphorylation of a specific sugar are located in a single operon. To date, the mtlA genes of E. coli (13), S. carnosus (7), and Bacillus subtilis (1) and the cryptic mtlA gene of E. coli (29), all encoding homologous mannitol transporters, have been cloned. Our attempts to crystallize the E. coli protein have been unsuccessful, possibly because of limited stability. Since membrane proteins in thermophilic bacteria were found to be more stable than their mesophilic counterparts, a search for a mannitol PTS was done in these organisms. A mannitol PTS had been found in the obligate anaerobic thermophile Clostridium thermocellum (21). This paper reports the identification of a mannitol PTS in the aerobic thermophile Bacillus stearothermophilus and presents the cloning of the mannitol operon, sequencing of the mtlA gene, functional expression of B. stearothermophilus IICB Mtl in E. coli, and purification and partial characterization of the mannitol transporter. MATERIALS AND METHODSStrains and plasmids used in this study are listed in Table 1. Restriction enzymes, Taq DNA polymerase, Klenow enzyme, T4 DNA ligase, digoxigenin (DIG) labeling mix, and DIG nucleic acid detection kit were purchased from Boehringer Mannheim. The DNA sequencing kit was obtained from Pharmacia. Sodium deoxycholate (DOC), n-decyl-␤-D-maltopyranoside (d-maltopyranoside), PEP, and mannitol...

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“…In electron density maps we see non-protein density features in both crevices that may be bound lipid or detergent molecules. These crevices allow lipid molecules to access helical hairpins 1 and 2 (HP1, HP2), which are key functional regions of the transporter, and may provide a structural basis for understanding how lipids modulate the activity of bacterial and eukaryotic transporters [24][25][26] .…”

Section: Trimer Architecturementioning

confidence: 99%

Structure of a glutamate transporter homologue from Pyrococcus horikoshii

Yernool

Boudker

Jin

et al. 2004

Nature

768

1,151

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Cation‐selectivity of the l‐glutamate transporters of Escherichia coli, Bacillus stearothermophilus and Bacillus caldotenax: dependence on the environment in which the proteins are expressed

Cited by 51 publications

References 36 publications

Molecular Basis for Proton Regulation of Glycine Transport by Glycine Transporter Subtype 1b

Molecular Basis for Proton Regulation of Glycine Transport by Glycine Transporter Subtype 1b

Cloning, expression, and isolation of the mannitol transport protein from the thermophilic bacterium Bacillus stearothermophilus

Structure of a glutamate transporter homologue from Pyrococcus horikoshii

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