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

Abstract

The reduced folate carrier (RFC) is the major transport system for folates in mammals. We previously demonstrated the existence of human RFC (hRFC) homo-oligomers and established the importance of these higher order structures to intracellular trafficking and carrier function. In this report, we examined the operational significance of hRFC oligomerization and the minimal functional unit for transport. In negative dominance experiments, multimeric transporters composed of different ratios of active (either wild type (WT) or cysteine-less (CLFL)) and inactive (either inherently inactive (Y281L and R373A) due to mutation, or resulting from inactivation of the Y126C mutant by (2-sulfonatoethyl) methanethiosulfonate (MTSES)) hRFC monomers were expressed in hRFC-null HeLa (R5) cells, and residual WT or CLFL activity was measured. In either case, residual transport activity with increasing levels of inactive mutant correlated linearly with the fraction of WT or CLFL hRFC in plasma membranes. When active covalent hRFC dimers, generated by fusing CLFL and Y126C monomers, were expressed in R5 cells and treated with MTSES, transport activity of the CLFL-CLFL dimer was unaffected, whereas Y126C-Y126C was potently (64%) inhibited; heterodimeric CLFL-Y126C and Y126C-CLFL were only partly (27 and 23%, respectively) inhibited by MTSES. In contrast to Y126C-Y126C, trans-stimulation of methotrexate uptake by intracellular folates for Y126C-CLFL and CLFL-Y126C was nominally affected by MTSES. Collectively, these results strongly support the notion that each hRFC monomer comprises a single translocation pathway for anionic folate substrates and functions independently of other monomers (i.e. despite an oligomeric structure, hRFC functions as a monomer).

Transmembrane Topology and Oligomeric Structure of the High-affinity Choline Transporter

“…Instead, the result from E451Q co-expression supports the concept that each CHT1 monomer within the homo-oligomer can function independently of the other monomer(s). The lack of a dominant-negative effect by co-expression of a WT and an inactive mutant protein has been reported for other homo-oligomeric transporters, such as Na ϩ /H ϩ exchanger 1 (34), renal type IIa Na ϩ /P i cotransporter (35), and reduced folate carrier (36). It remains to be elucidated how I89A, and not the E451Q monomer, impairs substrate translocation or reorientation of the WT monomer via oligomerization.…”

Identification and Functional Impact of Homo-oligomers of the Human Proton-coupled Folate Transporter

“…However, if there is functional interaction between hPCFT monomers, total transport activity should increase or decrease quadratically with the increasing fraction of active hPCFT. These methods are completely analogous to those previously described for numerous other oligomeric transporters, including hRFC (34,(51)(52)(53)(54).…”

“…If hPCFT monomers function independently (i.e. monomeric hPCFT is the "minimal functional unit"), transport activity should closely reflect the amounts of WT hPCFT and increase linearly with increasing ratios of WT to total surface hPCFT forms (34,(51)(52)(53)(54). However, if there is functional interaction between hPCFT monomers, total transport activity should increase or decrease quadratically with the increasing fraction of active hPCFT.…”

“…(34,(51)(52)(53)(54). However, if interactions (positive or negative) occur between WT and mutant monomers, the results should conform to a distinctly non-linear (quadratic) pattern, depending on whether the effect of WT/mutant co-association is dominant-positive or -negative and on the oligomerization state (dimer, trimer, etc.)…”

Biology of the Major Facilitative Folate Transporters SLC19A1 and SLC46A1

“…The results showed that there are substantial differences between structurally diverse RFC (anti)folate substrates in their binding to RFCs with mutated Ser313, and in inducing conformational changes involving the proximal end of TMD8, determined by protein cross-linking. Thus, there is direct support for an essential role of TMD8 and Ser313 in binding and/or membrane translocation of (anti)folate substrates (Hou, Wu, et al, 2010). …”