Role of the fourth transmembrane domain in proton-coupled folate transporter function as assessed by the substituted cysteine accessibility method

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Abstract

The proton-coupled folate transporter (PCFT, SLC46A1) mediates folate transport across the apical brush-border membrane of the proximal small intestine and the basolateral membrane of choroid plexus ependymal cells. Two lossof-function mutations in PCFT, which are the basis for hereditary folate malabsorption, have been identified within the fourth transmembrane domain (TMD4) in subjects with this disorder. We have employed the substituted Cys accessibility method (SCAM) to study the accessibilities of all residues in TMD4 and their roles in folate substrate binding to the carrier. When residues 146-167 were replaced by Cys, all except R148C were expressed at the cell surface. Modification of five of these substituted Cys residues (positions 147, 152, 157, 158, and 161) by methanethiosulfonate (MTS) reagents led to reduction of PCFT function. All five residues could be labeled with N-biotinylaminoethyl-MTS, and this could be blocked by the highaffinity PCFT substrate pemetrexed. Pemetrexed also protected PCFT mutant function from inhibitory modification of the substituted Cys at positions 157, 158, and 161 by a MTS. The findings indicate that these five residues in TMD4 are accessible to the aqueous translocation pathway, play a role in folate substrate binding, and are likely located within or near the folate binding pocket. A homology model of PCFT places three of these residues, Phe157, Gly158, and Leu161, within a breakpoint in the midportion of TMD4, a region that likely participates in alterations in the PCFT conformational state during carrier cycling.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Cell Physiology
Volume304
Issue number12
DOIs
StatePublished - 2013

Fingerprint

Proton-Coupled Folate Transporter
Folic Acid
Pemetrexed
Cysteine
Carrier State
Choroid Plexus
Membranes
Microvilli
Small Intestine
Mutation

Keywords

  • Folate transporters
  • Folates
  • Heme carrier protein 1
  • Hereditary folate malabsorption
  • Homology model
  • Intestinal folate absorption
  • Proton-coupled folate transporter

ASJC Scopus subject areas

  • Cell Biology
  • Physiology

Cite this

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title = "Role of the fourth transmembrane domain in proton-coupled folate transporter function as assessed by the substituted cysteine accessibility method",
abstract = "The proton-coupled folate transporter (PCFT, SLC46A1) mediates folate transport across the apical brush-border membrane of the proximal small intestine and the basolateral membrane of choroid plexus ependymal cells. Two lossof-function mutations in PCFT, which are the basis for hereditary folate malabsorption, have been identified within the fourth transmembrane domain (TMD4) in subjects with this disorder. We have employed the substituted Cys accessibility method (SCAM) to study the accessibilities of all residues in TMD4 and their roles in folate substrate binding to the carrier. When residues 146-167 were replaced by Cys, all except R148C were expressed at the cell surface. Modification of five of these substituted Cys residues (positions 147, 152, 157, 158, and 161) by methanethiosulfonate (MTS) reagents led to reduction of PCFT function. All five residues could be labeled with N-biotinylaminoethyl-MTS, and this could be blocked by the highaffinity PCFT substrate pemetrexed. Pemetrexed also protected PCFT mutant function from inhibitory modification of the substituted Cys at positions 157, 158, and 161 by a MTS. The findings indicate that these five residues in TMD4 are accessible to the aqueous translocation pathway, play a role in folate substrate binding, and are likely located within or near the folate binding pocket. A homology model of PCFT places three of these residues, Phe157, Gly158, and Leu161, within a breakpoint in the midportion of TMD4, a region that likely participates in alterations in the PCFT conformational state during carrier cycling.",
keywords = "Folate transporters, Folates, Heme carrier protein 1, Hereditary folate malabsorption, Homology model, Intestinal folate absorption, Proton-coupled folate transporter",
author = "Shin, {Daniel Sanghoon} and Rongbao Zhao and Andras Fiser and Goldman, {I. David}",
year = "2013",
doi = "10.1152/ajpcell.00353.2012",
language = "English (US)",
volume = "304",
journal = "American Journal of Physiology - Renal Physiology",
issn = "1931-857X",
publisher = "American Physiological Society",
number = "12",

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TY - JOUR

T1 - Role of the fourth transmembrane domain in proton-coupled folate transporter function as assessed by the substituted cysteine accessibility method

AU - Shin, Daniel Sanghoon

AU - Zhao, Rongbao

AU - Fiser, Andras

AU - Goldman, I. David

PY - 2013

Y1 - 2013

N2 - The proton-coupled folate transporter (PCFT, SLC46A1) mediates folate transport across the apical brush-border membrane of the proximal small intestine and the basolateral membrane of choroid plexus ependymal cells. Two lossof-function mutations in PCFT, which are the basis for hereditary folate malabsorption, have been identified within the fourth transmembrane domain (TMD4) in subjects with this disorder. We have employed the substituted Cys accessibility method (SCAM) to study the accessibilities of all residues in TMD4 and their roles in folate substrate binding to the carrier. When residues 146-167 were replaced by Cys, all except R148C were expressed at the cell surface. Modification of five of these substituted Cys residues (positions 147, 152, 157, 158, and 161) by methanethiosulfonate (MTS) reagents led to reduction of PCFT function. All five residues could be labeled with N-biotinylaminoethyl-MTS, and this could be blocked by the highaffinity PCFT substrate pemetrexed. Pemetrexed also protected PCFT mutant function from inhibitory modification of the substituted Cys at positions 157, 158, and 161 by a MTS. The findings indicate that these five residues in TMD4 are accessible to the aqueous translocation pathway, play a role in folate substrate binding, and are likely located within or near the folate binding pocket. A homology model of PCFT places three of these residues, Phe157, Gly158, and Leu161, within a breakpoint in the midportion of TMD4, a region that likely participates in alterations in the PCFT conformational state during carrier cycling.

AB - The proton-coupled folate transporter (PCFT, SLC46A1) mediates folate transport across the apical brush-border membrane of the proximal small intestine and the basolateral membrane of choroid plexus ependymal cells. Two lossof-function mutations in PCFT, which are the basis for hereditary folate malabsorption, have been identified within the fourth transmembrane domain (TMD4) in subjects with this disorder. We have employed the substituted Cys accessibility method (SCAM) to study the accessibilities of all residues in TMD4 and their roles in folate substrate binding to the carrier. When residues 146-167 were replaced by Cys, all except R148C were expressed at the cell surface. Modification of five of these substituted Cys residues (positions 147, 152, 157, 158, and 161) by methanethiosulfonate (MTS) reagents led to reduction of PCFT function. All five residues could be labeled with N-biotinylaminoethyl-MTS, and this could be blocked by the highaffinity PCFT substrate pemetrexed. Pemetrexed also protected PCFT mutant function from inhibitory modification of the substituted Cys at positions 157, 158, and 161 by a MTS. The findings indicate that these five residues in TMD4 are accessible to the aqueous translocation pathway, play a role in folate substrate binding, and are likely located within or near the folate binding pocket. A homology model of PCFT places three of these residues, Phe157, Gly158, and Leu161, within a breakpoint in the midportion of TMD4, a region that likely participates in alterations in the PCFT conformational state during carrier cycling.

KW - Folate transporters

KW - Folates

KW - Heme carrier protein 1

KW - Hereditary folate malabsorption

KW - Homology model

KW - Intestinal folate absorption

KW - Proton-coupled folate transporter

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U2 - 10.1152/ajpcell.00353.2012

DO - 10.1152/ajpcell.00353.2012

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VL - 304

JO - American Journal of Physiology - Renal Physiology

JF - American Journal of Physiology - Renal Physiology

SN - 1931-857X

IS - 12

ER -