The monomeric state of the proton‐coupled folate transporter represents the functional unit in the plasma membrane

• Published in: The FEBS journal 2013-06, Vol.280 (12), p.2900-2915
• Main Authors: Duddempudi, Phaneendra K., Nakashe, Prachi, Blanton, Michael P., Jansen, Michaela
• Format: Article
• Language: English
• Subjects: Animals; Biological Transport; BN/PAGE; Calibration; Cell Membrane - metabolism; CHO Cells; Cricetinae; Electrophoresis, Polyacrylamide Gel - standards; Female; folic acid; Folic Acid - metabolism; Glycosylation; HeLa Cells; Humans; Hydrogen-Ion Concentration; Membrane Potentials; Membranes; Molecular biology; Molecular Weight; Oocytes - metabolism; Plasma; Protein Processing, Post-Translational; Protein Structure, Quaternary; Proteins; Proton-Coupled Folate Transporter - chemistry; Proton-Coupled Folate Transporter - metabolism; proton‐coupled folate transporter; quaternary structure; Reference Standards; transporter; two‐electrode voltage clamp; Vitamin B; Xenopus laevis; Xenopus laevis oocytes
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/febs.12293

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 [3H]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‐HT3A 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. Human proton‐coupled folate transporter (PCFT) was heterologously expressed in CHO cells and X. laevis oocytes. In both expression systems PCFT was functional and the predominant PCFT protomer on the plasma membrane was determined to be monomeric by both blue‐native PAGE and cross‐linking experiments.