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Am J Physiol Cell Physiol. 2016 Jul 1;311(1):C150-7. doi: 10.1152/ajpcell.00084.2016. Epub 2016 Jun 1.

Role of the tryptophan residues in proton-coupled folate transporter (PCFT-SLC46A1) function.

Author information

1
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York;
2
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York; Department of Medicine, Albert Einstein College of Medicine, Bronx, New York;
3
Department of Systems and Computational Biology; Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York.
4
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York; Department of Medicine, Albert Einstein College of Medicine, Bronx, New York; i.david.goldman@einstein.yu.edu.

Abstract

The proton-coupled folate transporter (PCFT) mediates folate absorption across the brush-border membrane of the proximal small intestine and is required for folate transport across the choroid plexus into the cerebrospinal fluid. In this study, the functional role and accessibility of the seven PCFT Trp residues were assessed by the substituted-cysteine accessibility method. Six Trp residues at a lipid-aqueous interface tolerated Cys substitution in terms of protein stability and function. W85C, W202C, and W213C were accessible to N-biotinyl aminoethylmethanethiosulfonate; W48C and W299C were accessible only after treatment with dithiotreitol (DTT), consistent with modification of these residues by an endogenous thiol-reacting molecule and their extracellular location. Neither W107C nor W333C was accessible (even after DTT) consistent with their cytoplasmic orientation. Biotinylation was blocked by pemetrexed only for the W48C (after DTT), W85C, W202C residues. Function was impaired only for the W299C PCFT mutant located in the 4th external loop between the 7th and 8th transmembrane helices. Despite its aqueous location, function could only be fully preserved with Phe and, to a lesser extent, Ala substitutions. There was a 6.5-fold decrease in the pemetrexed influx Vmax and a 3.5- and 6-fold decrease in the influx Kt and Ki, respectively, for the W299S PCFT. The data indicate that the hydrophobicity of the W299 residue is important for function suggesting that during the transport cycle this residue interacts with the lipid membrane thereby impacting on the oscillation of the carrier and, indirectly, on the folate binding pocket.

KEYWORDS:

HFM; PCFT; folate; hereditary folate malabsorption; methotrexate; pemetrexed; proton-coupled folate transporter; solute transporter; substituted cysteine accessibility assay

PMID:
27251438
PMCID:
PMC4967132
DOI:
10.1152/ajpcell.00084.2016
[Indexed for MEDLINE]
Free PMC Article

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