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• W2048360829 endingPage "2915" @default.
• W2048360829 startingPage "2900" @default.
• W2048360829 abstract "Folic acid is an essential vitamin required for de novo biosynthesis of nucleotides and amino acids. The proton-coupled folate transporter (PCFT; SLC46A1) has been identified as the major contributor for intestinal folate uptake. It is also involved in folate transport across the blood-brain barrier and into solid tumors. PCFT belongs to the major facilitator superfamily. Major facilitator superfamily members can exist in either monomeric or homo-oligomeric form. Here, we utilized blue native polyacrylamide gel electrophoresis (BN/PAGE) and crosslinking with bi-functional chemicals to investigate the quaternary structure of human PCFT after heterologous expression in Xenopus laevis oocytes and CHO cells. PCFT was expressed in the plasma membrane in both expression systems. The functionality of the utilized PCFT construct was confirmed in oocytes by folic acid induced currents at acidic pH. For both the oocyte and CHO expression system [(3)H]folic acid uptake studies indicated that PCFT was functional. To analyze the oligomeric state of PCFT in the plasma membrane, plasma membranes were isolated by polymerization with colloidal silica and polyacrylic acid and subsequent centrifugation. The digitonin-solubilized non-denatured PCFT migrated during BN/PAGE as a monomer, as judged by comparison with a membrane protein (5-HT(3A) receptor) of known pentameric assembly that was used to create a molecular sizing ladder. The chemical crosslinkers glutaraldehyde and dimethyl adipimidate were not able to covalently link potential higher order PCFT structures to form oligomers that were stable following SDS treatment. Together, our results demonstrate that plasma-membrane PCFT functions as a monomeric protein." @default.
• W2048360829 created "2016-06-24" @default.
• W2048360829 creator A5009088187 @default.
• W2048360829 creator A5023236961 @default.
• W2048360829 creator A5052760661 @default.
• W2048360829 creator A5079957780 @default.
• W2048360829 date "2013-05-28" @default.
• W2048360829 modified "2023-10-03" @default.
• W2048360829 title "The monomeric state of the proton-coupled folate transporter represents the functional unit in the plasma membrane" @default.
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• W2048360829 doi "https://doi.org/10.1111/febs.12293" @default.
• W2048360829 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/23601781" @default.
• W2048360829 hasPublicationYear "2013" @default.
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