Knockout of the folate transporter <Emphasis Type="Italic">folt-1</Emphasis> causes germline and somatic defects in <Emphasis Type="Italic">C. elegans</Emphasis>

Background  

The C. elegans gene folt-1 is an ortholog of the human reduced folate carrier gene. The FOLT-1 protein has been shown to transport folate and to be involved in uptake of exogenous folate by worms. A knockout mutation of the gene, folt-1(ok1460), was shown to cause sterility, and here we investigate the source of the sterility and the effect of the folt-1 knockout on somatic function.     

Cloning and developmental expression analysis of ltd-1, the Caenorhabditis elegans homologue of the mouse kyphoscoliosis (ky) gene

We have characterized the developmental expression pattern of the Caenorhabditis elegans homologue of the mouse ky gene. The Ky protein has a putative key function in muscle development and has homologues in invertebrates, fungi and a cyanobacterium. The C. elegans Ky homologue gene has been named ltd-1 for LIM and transglutaminase domains gene. The LTD-1::GFP construct is expressed in developing hypodermal cells from the twofold stage embryo through adulthood. These data define the ltd-1 gene as a novel marker for C. elegans epithelial cell development.     

Transmembrane domains 4, 5, 7, 8, and 10 of the human reduced folate carrier are important structural or functional components of the transmembrane channel for folate substrates

The human reduced folate carrier (hRFC) facilitates membrane transport of folates and antifolates. hRFC is characterized by 12 transmembrane domains (TMDs). To identify residues or domains involved in folate binding, we used substituted cysteine (Cys) accessibility methods (SCAM) with sodium (2-sulfonatoethyl)methanethiosulfonate (MTSES). We previously showed that residues in TMD11 of hRFC were involved in substrate binding, whereas those in TMD12 were not (Hou, Z., Stapels, S. E., Haska, C. L., and Matherly, L. H. (2005) J. Biol. Chem. 280, 36206-36213). In this study, 232 Cys-substituted mutants spanning TMDs 1-10 and conserved stretches within the TMD6-7 (residues 204-217) and TMD10-11 connecting loop domains were transiently expressed in hRFC-null HeLa cells. All Cys-substituted mutants showed moderate to high levels of expression on Western blots, and only nine mutants including R133C, I134C, A135C, Y136C, S138C, G163C, Y281C, R373C, and S313C were inactive for methotrexate transport. MTSES did not inhibit transport by any of the mutants in TMDs 1, 3, 6, and 9 or for positions 204-217. Whereas most of the mutants in TMDs 2, 4, 5, 7, 8, and 10, and in the TMD10-11 connecting loop were insensitive to MTSES, this reagent inhibited methotrexate transport (25-75%) by 26 mutants in these TMDs. For 13 of these (Y126C, S137C, V160C, S168C, W274C, S278C, V284C, V288C, A311C, T314C, Y376C, Q377C, and V380C), inhibition was prevented by leucovorin, another hRFC substrate. Combined with our previous findings, these results implicate amino acids in TMDs 4, 5, 7, 8, 10, and 11, but not in TMDs 1, 2, 3, 6, 9, or 12, as important structural or functional components of the putative hydrophilic cavity for binding of anionic folate substrates.     

Localization of a substrate binding domain of the human reduced folate carrier to transmembrane domain 11 by radioaffinity labeling and cysteine-substituted accessibility methods

The human reduced folate carrier (hRFC) mediates the membrane transport of reduced folates and classical anti-folates into mammalian cells. RFC is characterized by 12 transmembrane domains (TMDs), internally oriented N and C termini, and a large central linker connecting TMDs 1-6 and 7-12. By co-expression and N-hydroxysuccinimide methotrexate (Mtx) radioaffinity labeling of hRFC TMD 1-6 and TMD 7-12 half-molecules, combined with endoproteinase GluC digestion, a substrate binding domain was previously localized to within TMDs 8-12 (Witt, T. L., Stapels, S. E., and Matherly, L. H. (2004) J. Biol. Chem. 279, 46755-46763). In this report, this region was further refined to TMDs 11-12 by digestion with 2-nitro-5-thiocyanatobenzoic acid. A transportcompetent cysteine-less hRFC was used as a template to prepare single cysteine-replacement mutant constructs in which each residue from Glu-394 to Asp-420 of TMD 11 and Tyr-435 to His-457 of TMD 12 was replaced individually by a cysteine. The mutant constructs were transfected into hRFC-null HeLa cells. Most of the 50 single cysteine-substituted constructs were expressed at high levels on Western blots. With the exception of G401C hRFC, all mutants were active for Mtx transport. Treatment with sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES) had no effect on hRFC activity for all of the cysteine mutants within TMD 12 and for the majority of the cysteine mutants within TMD 11. However, MTSES inhibited Mtx uptake by the T404C, A407C, T408C, T412C, F416C, I417C, V418C, and S419C mutants by 25-65%. Losses of activity by MTSES treatment for T404C, A407C, T412C, and I417C hRFCs were appreciably reversed in the presence of excess leucovorin, a hRFC substrate. Our results strongly suggest that residues within TMD 11 are likely critical structural and/or functional components of the putative hRFC transmembrane channel for anionic folate and anti-folate substrates.     

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

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).     

Mechanism and regulation of folate uptake by human pancreatic epithelial MIA PaCa-2 cells

After the liver, the pancreas contains the second highest level of folate among human tissues, and folate deficiency adversely affects its physiological function. Despite that, nothing is currently known about the cellular mechanisms involved in folate uptake by cells of this important exocrine organ or about folate uptake regulation. We have begun to address these issues, and in this report we present the results of our findings on the mechanism of folate uptake by the human-derived pancreatic MIA PaCa-2 cells. Our results show folic acid uptake to be 1) temperature and energy dependent; 2) pH dependent, with a markedly higher uptake at acidic pH compared with neutral or alkaline pH; 3) Na⁺ independent; 4) saturable as a function of substrate concentration (apparent K_m = 0.762 ± 0.10 µM); 5) inhibited (with similar affinity) by reduced, substituted, and oxidized folate derivatives; and 6) sensitive to the inhibitory effect of anion transport inhibitors. RT-PCR and Western blot analysis showed expression of the human reduced folate carrier (hRFC) at the RNA and protein levels, respectively. The functional contribution of hRFC in carrier-mediated folate uptake was confirmed by gene silencing using gene-specific small interfering RNA. Evidence also was found suggesting that the folate uptake process by MIA PaCa-2 cells is regulated by cAMP- and protein tyrosine kinase (PTK)-mediated pathways. These studies demonstrate for the first time the involvement of a specialized, acidic pH-dependent, carrier-mediated mechanism for folate uptake by human pancreatic MIA PaCa-2 cells. The results also show the involvement of hRFC in the uptake process and suggest the possible involvement of intracellular cAMP- and PTK-mediated pathways in the regulation of folate uptake. human reduced folate carrier; small interfering RNA; transport regulation     

Intracellular trafficking and membrane targeting mechanisms of the human reduced folate carrier in Mammalian epithelial cells

The major pathway for cellular uptake of the water-soluble vitamin folic acid in mammalian cells is via a plasma membrane protein known as the reduced folate carrier (RFC). The molecular determinants that dictate plasma membrane expression of RFC as well as the cellular mechanisms that deliver RFC to the cell surface remain poorly defined. Therefore, we designed a series of fusion proteins of the human RFC (hRFC) with green fluorescent protein to image the targeting and trafficking dynamics of hRFC in living epithelial cells. We show that, in contrast to many other nutrient transporters, the molecular determinants that dictate hRFC plasma membrane expression reside within the hydrophobic backbone of the polypeptide and not within the cytoplasmic NH(2)- or COOH-terminal domains of the protein. Further, the integrity of the hRFC backbone is critical for export of the polypeptide from the endoplasmic reticulum to the cell surface. This trafficking is critically dependent on intact microtubules because microtubule disruption inhibits motility of hRFC-containing vesicles as well as final expression of hRFC in the plasma membrane. For the first time, these data define the mechanisms that control the intracellular trafficking and cell surface localization of hRFC within mammalian epithelia.     

Oligomeric Structure of the Human Reduced Folate Carrier: IDENTIFICATION OF HOMO-OLIGOMERS AND DOMINANT-NEGATIVE EFFECTS ON CARRIER EXPRESSION AND FUNCTION*

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 protein in tumor cells results in antifolate resistance caused by incomplete inhibition of cellular enzyme targets and low levels of antifolate substrate for polyglutamate synthesis (4, 11).Reflecting its particular physiologic and pharmacologic importance, interest in RFC structure and function has been high. Since 1994, when murine RFC was first cloned (12), application of state-of-the-art molecular biology and biochemistry methods for characterizing polytopic membrane proteins has led to a progressively detailed picture of the molecular structure of the carrier, including its membrane topology, N-glycosylation, functionally or structurally important domains and amino acids, and packing of α-helix transmembrane domains (TMDs) (4, 13). Although no crystal structure for RFC has yet been reported, a detailed homology model for human RFC (hRFC) based on the bacterial lactose/proton symporter LacY and glycerol 3-phosphate/inorganic phosphate antiporter GlpT was generated (13, 14) that permits testing of hypotheses related to hRFC structure and mechanism in a manner not previously possible.Considerations of hRFC structure and mechanism to date have all been based on the notion that a single 591-amino acid hRFC molecule is sufficient to mediate concentrative uptake of folate and antifolate substrates. However, a growing literature suggests that quaternary structure involving the formation of higher order oligomers (e.g. dimers, tetramers, etc.) is commonly an important feature of the structure and function of many membrane transporters (15-18). For major facilitator superfamily proteins, both monomeric (e.g. LacY, GlpT, UhpT, and GLUT3) (19-22) and oligomeric (e.g. LacS, AE1, GLUT1, and TetA) (23-28) structures have been reported, establishing the lack of a clear structural consensus for these related proteins.In this report, we explore the question of whether hRFC exists as a homo-oligomeric species composed of multiple hRFC monomers. Based on results with an assortment of biochemical methods with wt and a collection of mutant hRFC proteins, we not only demonstrate the existence of oligomeric hRFC but also establish the probable importance of these higher order structures to intracellular trafficking and carrier function.     

Identification of lysine-411 in the human reduced folate carrier as an important determinant of substrate selectivity and carrier function by systematic site-directed mutagenesis

Site-directed mutagenesis was used to characterize the functional role of lysine-411, a conserved amino acid located in putative transmembrane domain (TMD) 11 of the human reduced folate carrier (hRFC). Lysine-411 was mutagenized to arginine, glutamate, and leucine, and the mutant constructs (K411R-, K411E-, and K411L-hRFC, respectively) were transfected into hRFC-deficient K562 cells. The mutant hRFC constructs were all expressed at high levels and restored 22-36% of the methotrexate (MTX) transport level in wild-type (K43-6) hRFC transfectants. Although 5-formyl tetrahydrofolate (5-CHO-H(4)PteGlu) uptake levels for both the K411E- and K411L-hRFCs were also impaired (approximately 33% and 28%, respectively), a complete restoration of the wild-type level was observed for K411R-hRFC. While loss of MTX transport activity for the K411R-hRFC transfectant was associated with an incomplete restoration of MTX sensitivity compared to K43-6 cells, these cells were similarly sensitive to Tomudex. The K411R-hRFC transfectants showed an approximately threefold decreased growth requirement for 5-CHO-H(4)PteGlu compared to K43-6 cells. The 5-CHO-H(4)PteGlu transport stimulation observed for the wild-type carrier in chloride-free buffer was also observed for K411R-hRFC, however, this response was decreased for the K411E- and K411L-hRFCs. The preservation of low levels of transport for the K411E- and K411L-hRFCs suggest that the amino acid at position 411 does not directly participate in the binding of anionic hRFC substrates. However, a functionally important role for a basic amino acid at position 411 was, nonetheless, implied by the increased MTX transport for wild-type hRFC over the K411 mutant hRFCs, and the highly selective uptake of 5-CHO-H(4)PteGlu over MTX for K411R-hRFC.