title: The monomeric state of the proton-coupled folate transporter represents the functional unit in the plasma membrane.
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.
authors.affiliation: Department of Pharmacology and Neuroscience, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA.
authors.forename: Phaneendra K
authors.initials: PK
authors.lastname: Duddempudi
authors.forename: Prachi
authors.initials: P
authors.lastname: Nakashe
authors.forename: Michael P
authors.initials: MP
authors.lastname: Blanton
authors.forename: Michaela
authors.initials: M
authors.lastname: Jansen
mesh_terms: D000818:Animals; D001692:Biological Transport; D016466:CHO Cells; D002138:Calibration; D002462:Cell Membrane / Q000378:metabolism*; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel / Q000592:standards; D005260:Female; D005492:Folic Acid / Q000378:metabolism; D006031:Glycosylation; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008564:Membrane Potentials; D008970:Molecular Weight; D009865:Oocytes / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D020836:Protein Structure, Quaternary; D058979:Proton-Coupled Folate Transporter / Q000737:chemistry / Q000378:metabolism*; D012015:Reference Standards; D014982:Xenopus laevis
chemical_list: D058979:Proton-Coupled Folate Transporter; C505058:SLC46A1 protein, human; D005492:Folic Acid