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).
Highlights
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]
We examined the operational significance of human RFC (hRFC) oligomerization and the minimal functional unit for transport
The present study significantly expands upon our recent report that hRFC, the major folate membrane transporter in human cells and tissues, exists as a homo-oligomer [15]
Summary
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]. Many secondary transporters exist as higher order oligomers (e.g. dimers (NhaA and LacS) or trimers (AcrB, Glt, BetP, and CaiT)), unambiguous evidence for monomer structures (LacY and GlpT) was reported [10, 11]. Minimal Functional Unit of hRFC (ER) and intracellular trafficking [12], in only a few cases have clear functional or regulatory roles for transporter oligomerization been established. It was shown for the Streptococcus thermophilus lactose transporter LacS that individual monomers cooperate with each other during the transport cycle, implying that oligomerization is essential for transport function [13]. For the homodimeric NhaA Naϩ/Hϩ 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 Naϩ or Liϩ [14]
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