Oligomeric Structure of the Human Reduced Folate Carrier

Abstract

The ubiquitously expressed reduced folate carrier (RFC) is the major transport system for folate cofactors in mammalian cells and tissues. Previous considerations of RFC structure and mechanism were based on the notion that RFC monomers were sufficient to mediate transport of folate and antifolate substrates. The present study examines the possibility that human RFC (hRFC) exists as higher order homo-oligomers. By chemical cross-linking, transiently expressed hRFC in hRFC-null HeLa (R5) cells with the homobifunctional cross-linker 1,3-propanediyl bis-methanethiosulfonate and Western blotting, hRFC species with molecular masses of hRFC homo-oligomers were identified. Hemagglutinin-and Myc epitope-tagged hRFC proteins expressed in R5 cells were co-immunoprecipitated from both membrane particulate and surface-enriched membrane fractions, indicating that oligomeric hRFC is expressed at the cell surface. By co-expression of wild type and inactive mutant S138C hRFCs, combined with surface biotinylation and confocal microscopy, a dominant-negative phenotype was demonstrated involving greatly decreased cell surface expression of both mutant and wild type carrier caused by impaired intracellular trafficking. For another hRFC mutant (R373A), expression of oligomeric wild type-mutant hRFC was accompanied by a significant and disproportionate loss of wild type activity unrelated to the level of surface carrier. Collectively, our results demonstrate the existence of hRFC homo-oligomers. They also establish the likely importance of these higher order hRFC structures to intracellular trafficking and carrier function.Folates are members of the B class of vitamins that are required for the synthesis of nucleotide precursors, serine, and methionine in one-carbon transfer reactions (1). Because mammals cannot synthesize folates de novo, cellular uptake of these derivatives is essential for cell growth and tissue regeneration (2, 3). Folates are hydrophilic anionic molecules that do not cross biological membranes by diffusion alone, so it is not surprising that sophisticated membrane transport systems have evolved to facilitate their accumulation by mammalian cells.The ubiquitously expressed reduced folate carrier (RFC) 2 is widely considered to be the major transport system for folate co-factors in mammalian cells and tissues (3, 4). RFC plays a generalized role in folate transport and provides specialized tissue functions such as transport across the basolateral membrane of renal proximal tubules (5), transplacental transport of folates (6), and folate transport across the blood-brain barrier (7), although the contribution of RFC to intestinal absorption of folates remains controversial (8, 9). Loss of RFC expression or function portends potentially profound physiologic and developmental consequences associated with folate deficiency (10). RFC is also a major transporter of antifolate drugs used for cancer chemotherapy such as methotrexate (Mtx), pemetrexed, and raltitrexed (4). Loss of RFC expression or synthesis of mutant RFC...

Identification of the Minimal Functional Unit of the Homo-oligomeric Human Reduced Folate Carrier

“…For the homodimeric NhaA Na ϩ /H ϩ antiporter from Escherichia coli, monomers are fully functional, yet the dimer appears to be beneficial under extreme stress conditions at alkaline pH in the presence of Na ϩ or Li ϩ (14). Our recent study presented compelling evidence that hRFC exists as a homo-oligomer (15). (i) hRFC was cross-linked with homobifunctional cross-linkers to form higher order complexes with molecular masses approximating those of dimers, trimers, and tetramers.…”

“…The Cell Surface Labeling Accessory Pack (Thermo Scientific) with sulfo-N-hydroxysuccinimide-SS-biotin (sulfo-NHS-SS-biotin) was used to biotinylate and isolate surface proteins (15). The biotinylated surface proteins were isolated with immobilized NeutrAvidin TM and eluted with 10 mM Tris-Cl (pH 7.5), containing 0.5% SDS, 50 mM dithiothreitol, and protease inhibitor mixture.…”

“…Additional controls included monomeric Myc-and HA-tagged constructs expressed individually and immunoprecipitated with HA and Myc antibodies, respectively. These were described in our previous report (15). The samples were eluted with 3ϫ SDS-PAGE sample buffer (28) and fractionated on a 7.5% polyacrylamide gel in the presence of SDS for Western blotting.…”

“…To generate HA-tagged CLFL hRFC, a DNA fragment encoding the HA epitope following the stop codon was inserted after Gln-586 of CLFL hRFC, as previously described for HA-tagged full-length WT hRFC (23). QuikChange TM mutagenesis was also used with HA-tagged full-length WT hRFC template (23) to generate the HA-tagged Y281L hRFC and R373A hRFC (15). This approach was likewise used to generate Y126C in the Cysless background from CLFL hRFC (Myc-His 10 ) as template.…”

“…Generation of CLFL hRFC and Mutant Constructs-CLFL hRFC was generated from Myc-His 10 -tagged full-length hRFC in pCDNA3.1 (15) by subcloning and site-directed mutagenesis with the QuikChange TM kit (Agilent Technologies, La Jolla, CA). The first seven cysteines (Cys-30, -33, -220, -246, -365, -396, and -458) were mutated to serines by substituting a fragment (nucleotides 1-1611) from our previous Cys-less hRFC construct, which was truncated at amino acid 538 (22), using XhoI/SfiI digestions (New England Biolabs, Ipswich, MA).…”