Transmembrane segment 5 of the dipeptide transporter hPepT1 forms a part of the substrate translocation pathway

Kulkarni, Ashutosh A.; Haworth, Ian S.; Lee, Vincent H.L.

doi:10.1016/s0006-291x(03)00926-4

Cited by 31 publications

(42 citation statements)

References 27 publications

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“…When human PEPT1 was mutated at each of these sites, transport function was lost or significantly diminished. In addition to identification of those functionally important residues in PEPT1, it has been proposed that the N-terminal one-half including TM7, TM8, and TM9 is the region of PEPT1 responsible for proton and substrate recognition (Doring et al, 1996;Fei et al, 1998;Terada et al, 2000), and a recent study with cysteine mutagenesis on PEPT1 revealed that TM5 may be part of the substrate translocation pathway (Kulkarni et al, 2003). Additionally, it has been speculated that the C-terminal region, although not directly involved in substrate recognition and binding, may be important for either protein trafficking or functional regulation (Doring et al, 1996).…”

Section: Discussionmentioning

confidence: 99%

Genetic Polymorphisms in Human Proton-Dependent Dipeptide Transporter PEPT1: Implications for the Functional Role of Pro⁵⁸⁶

Zhang¹,

Fu²,

Pak³

et al. 2004

J Pharmacol Exp Ther

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The human proton-dependent dipeptide transporter (PEPT1, gene SLC15A1) is important for intestinal absorption of di-and tripeptides and a variety of peptidomimetic compounds. Using a DNA polymorphism discovery panel of 44 ethnically diverse individuals, nine nonsynonymous and four synonymous coding-region single-nucleotide polymorphisms (SNPs) were identified in PEPT1. HeLa cells were transiently transfected with plasmids constructed by site-directed mutagenesis for each of the nine nonsynonymous variants. Quantitative polymerase chain reaction showed that the mRNA transcription level of all of the mutants was comparable with the mRNA transcription level of the reference sequence in transfected HeLa cells. Functional analysis in transiently transfected HeLa cells revealed that all nonsynonymous variants retained similar pH-dependent activity and K t values for [glycyl-1,2-14 C]glycylsarcosine (GlySar) uptake as the reference PEPT1. In addition, a group of seven peptide-like drugs showed inhibitory effect on Gly-Sar uptake by these variants comparable with the reference, suggesting conserved drug recognition. Of the nine nonsynonymous SNPs, a single SNP (P586L) demonstrated significantly reduced transport capacity as evidenced by a much lower V max value. This was consistent with lower immunoactive protein level (Western analysis) and lower plasma membrane expression (immunocytochemical analysis). Therefore, Pro 586 may have profound effect on PEPT1 translation, degradation, and/or membrane insertion.

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Section: Discussionmentioning

confidence: 99%

Genetic Polymorphisms in Human Proton-Dependent Dipeptide Transporter PEPT1: Implications for the Functional Role of Pro⁵⁸⁶

Zhang¹,

Fu²,

Pak³

et al. 2004

J Pharmacol Exp Ther

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“…In addition, chimeric studies on human PTR members PEPT1 and PEPT2 have revealed that helices 2-5 as well as 7 contain residues that mediate substrate recognition (9). This is supported by cysteine scanning mutagenesis of residues in helices 5 and 7 of PEPT1, which suggests that residues in these helices line the substrate translocation pathway (8,10).…”

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confidence: 90%

“…The first such motif is found in the first predicted transmembrane helix, whereas the second is found in loop 2-3 and corresponds to the motif GXXX(D/E)(R/K)[X]G[X](R/K)(R/K), where [X] is either one or two amino acids, which is also found in many MFS transporters (5,6). The third motif, the PTR signature motif FXXFYXX-INXGS, is located in the fifth predicted transmembrane helix of PTR members, and mutation of these residues leads to reduced transport activity or changes in substrate selectivity (7,8). In addition, chimeric studies on human PTR members PEPT1 and PEPT2 have revealed that helices 2-5 as well as 7 contain residues that mediate substrate recognition (9).…”

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confidence: 99%

Random Mutagenesis of the Prokaryotic Peptide Transporter YdgR Identifies Potential Periplasmic Gating Residues

Malle

Zhou

Neuhold

et al. 2011

Journal of Biological Chemistry

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The peptide transporter (PTR) family represents a group of proton-coupled secondary transporters responsible for bulk uptake of amino acids in the form of di-and tripeptides, an essential process employed across species ranging from bacteria to humans. To identify amino acids critical for peptide transport in a prokaryotic PTR member, we have screened a library of mutants of the Escherichia coli peptide transporter YdgR using a high-throughput substrate uptake assay. We have identified 35 single point mutations that result in a full or partial loss of transport activity. Additional analysis, including homology modeling based on the crystal structure of the Shewanella oneidensis peptide transporter PepT so , identifies Glu 56 and Arg 305 as potential periplasmic gating residues. In addition to providing new insights into transport by members of the PTR family, these mutants provide valuable tools for further study of the mechanism of peptide transport.

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“…The PepT1 gene encodes a protein of 707 amino acids that spans 12 transmembranes, for which there is no direct structural information except for the confirmation of the membrane topology (4). Indirect approaches to elucidating the protein structure have led to the proposal of a template for transported substrates (5), and computer modeling of transmembrane regions by Lee and coworkers (6,7) has resulted in a number of suggested models. One residue of interest is Arg-282 because it is highly conserved as a positively charged residue between species and also represents a charged residue in a transmembrane region (TM)7.…”

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confidence: 99%

Site-directed Mutation of Arginine 282 to Glutamate Uncouples the Movement of Peptides and Protons by the Rabbit Proton-peptide Cotransporter PepT1

Meredith

2004

Journal of Biological Chemistry

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A conserved positive residue in the seventh transmembrane domain of the mammalian proton-coupled diand tripeptide transporter PepT1 has been shown by site-directed mutagenesis to be a key residue for protein function. Substitution of arginine 282 with a glutamate residue (R282E-PepT1) gave a protein at the plasma membrane of Xenopus laevis oocytes that was able to transport the non-hydrolyzable dipeptide [3 H]D-Phe-LGln, although unlike the wild type, the rate of transport by R282E-PepT1 was independent of the extracellular pH level, and the substrate could not be accumulated above equilibrium. The binding affinity of the mutant transport protein was unchanged from the wild type. Thus, R282E-Pept1 appears to have been changed from a proton-driven to a facilitated transporter for peptides. In addition, peptide transport by R282E-PepT1 still induced depolarization as measured by microelectrode recordings of membrane potential. A more detailed study by two-electrode voltage clamping revealed that R282E-PepT1 behaved as a peptide-gated non-selective cation channel with the ion selectivity series lithium > sodium > N-methyl-D-glucamine at pH 7.4. There was also a proton conductance (comparing pH 7.4 and 8.4), and at pH 5.5 the predominant conductance was for potassium ions. Therefore, it can be concluded that changing arginine 282 to a glutamate not only uncouples the cotransport of protons and peptides of the wild-type PepT1 but also creates a peptide-gated cation channel in the protein.

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Transmembrane segment 5 of the dipeptide transporter hPepT1 forms a part of the substrate translocation pathway

Cited by 31 publications

References 27 publications

Genetic Polymorphisms in Human Proton-Dependent Dipeptide Transporter PEPT1: Implications for the Functional Role of Pro⁵⁸⁶

Genetic Polymorphisms in Human Proton-Dependent Dipeptide Transporter PEPT1: Implications for the Functional Role of Pro⁵⁸⁶

Random Mutagenesis of the Prokaryotic Peptide Transporter YdgR Identifies Potential Periplasmic Gating Residues

Site-directed Mutation of Arginine 282 to Glutamate Uncouples the Movement of Peptides and Protons by the Rabbit Proton-peptide Cotransporter PepT1

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Transmembrane segment 5 of the dipeptide transporter hPepT1 forms a part of the substrate translocation pathway

Cited by 31 publications

References 27 publications

Genetic Polymorphisms in Human Proton-Dependent Dipeptide Transporter PEPT1: Implications for the Functional Role of Pro586

Genetic Polymorphisms in Human Proton-Dependent Dipeptide Transporter PEPT1: Implications for the Functional Role of Pro586

Random Mutagenesis of the Prokaryotic Peptide Transporter YdgR Identifies Potential Periplasmic Gating Residues

Site-directed Mutation of Arginine 282 to Glutamate Uncouples the Movement of Peptides and Protons by the Rabbit Proton-peptide Cotransporter PepT1

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Genetic Polymorphisms in Human Proton-Dependent Dipeptide Transporter PEPT1: Implications for the Functional Role of Pro⁵⁸⁶

Genetic Polymorphisms in Human Proton-Dependent Dipeptide Transporter PEPT1: Implications for the Functional Role of Pro⁵⁸⁶