Analysis of the Oligomeric State of Surface-Localized Proton-Coupled Folate Transporter by Blue Native Polyacrylamide Gel Electrophoresis

Abstract

Folate vitamins are essential for DNA replication and cellular proliferation. However, mammalian organisms are devoid of de novo folate biosynthesis and thus rely on dietary sources to meet their metabolic needs. The proton coupled folate transporter (PCFT/SLC46A1) has been recently identified as the molecular entity of the carrier mediated intestinal folate uptake pathway for folic acids from food sources. PCFT is also involved in the absorption of chemotherapeutically used antifolates. Currently, there is limited information about the structure and function of PCFT. Hydropathy analysis suggests that there are 10-12 transmembrane segments. Further, using the Substituted Cysteine Accessibility Method (SCAM) evidence was provided for a 12 transmembrane segment topology. Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) is a technique for separation of protein complexes in a native state with high resolution. We expressed PCFT in Xenopus laevis oocytes. Oocyte plasma membranes were polymerized to the vitelline membrane using ludox colloidal silica solution and polyacrylic acid, isolated by centrifugation, and plasma membrane proteins subsequently solubilized with digitonin and separated by BN-PAGE. The separation characteristics of native PCFT were compared to a molecular ruler produced by partial dissociation of homopentameric 5-hydroxytryptamine type 3A (5HT3A) receptors. Under native conditions, 5HT3A subunits largely migrated as a pentamer and PCFT only as a monomer. Treatment with denaturing agents generated a ladder of five bands for 5HT3A subunits, which consisted of monomer, dimer, trimer, tetramer and pentamer. Addition of crosslinking agents resulted in migration of 5HT3A subunits as a pentamer, even in the presence of denaturing agents. In contrast, crosslinking agents did not induce oligomeric assemblies of PCFT. These results indicate that functional plasma-membrane bound PCFT is a monomeric protein.