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 digitoninsolubilized 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.

Analysis of lipid-composition changes in plasma membrane microdomains

“…After mechanical cell lysis, the SCMs can be separated by centrifugation, due to their high density. However, purities and yields of the membranes can vary in different laboratories because of the different experimental conditions used, including variations in buffer systems (26)(27)(28)(29)(30)(31)(32)(33)(34). In addition, a relatively large number of cells were needed in previous plasma membrane lipidomics studies using this method ( 30,31 ).…”

“…The cationic colloidal silica beads method was used for preparing plasma membranes (26)(27)(28)(29)(30)(31)(32)(33)(34). Briefl y, this step was performed on ice using ice-cold buffers.…”

The Major Facilitative Folate Transporters Solute Carrier 19A1 and Solute Carrier 46A1: Biology and Role in Antifolate Chemotherapy of Cancer

“…In contrast to the aforementioned evidence that oligomeric hPCFT is structurally and functionally important, a study by Duddempudi et al (2013) suggested that hPCFT may not be oligomeric when expressed in Chinese hamster ovary cells or Xenopus oocytes and isolated from plasma membranes prepared by polymerization with colloidal silica and polyacrylic acid. As this latter report used entirely different methods and metrics than the earlier study by Hou et al (2012), it is not possible to reconcile their disparate conclusions.…”

Biology of the Major Facilitative Folate Transporters SLC19A1 and SLC46A1

“…Duddempudi, Nakashe, Blanton, and Jansen (2013) suggested that PCFT may not oligomerize in plasma membranes prepared by polymerization with colloidal silica and polyacrylic acid from Xenopus oocytes or in Chinese hamster ovary cells. However, this conclusion was based on a limited number of methods.…”