Chapter 5 Structure and Function of the Reduced Folate Carrier

Matherly, Larry H.; Hou, Zhanjun

doi:10.1016/s0083-6729(08)00405-6

Cited by 71 publications

(29 citation statements)

References 141 publications

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“…The molecular identity of the systems involved in intestinal folate absorption has been delineated, with both the RFC (reduced folate carrier, the product of the SLC19A1 gene) [82–84] and the PCFT (the product of the SLC46A1 gene) [75,85] involved (see detailed reviews in [8,9,80,81]). hRFC (human RFC) (a 591 amino acid protein) is predicted to have 12 TMDs together with a number of putative protein kinase phosphorylation sites, and one N-glycosylation motif (indeed, the latter site appears to be glycosylated).…”

Section: Folatementioning

confidence: 99%

“…hRFC (human RFC) (a 591 amino acid protein) is predicted to have 12 TMDs together with a number of putative protein kinase phosphorylation sites, and one N-glycosylation motif (indeed, the latter site appears to be glycosylated). hRFC shares a high degree of homology with that of other mammals [85]. The RFC protein is expressed at the apical membrane domain of intestinal epithelial cells and functions at neutral pH [85–87].…”

Section: Folatementioning

confidence: 99%

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Intestinal absorption of water-soluble vitamins in health and disease

Said

2011

Biochemical Journal

335

239

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Our knowledge of the mechanisms and regulation of intestinal absorption of water-soluble vitamins under normal physiological conditions, and of the factors/conditions that affect and interfere with theses processes has been significantly expanded in recent years as a result of the availability of a host of valuable molecular/cellular tools. Although structurally and functionally unrelated, the water-soluble vitamins share the feature of being essential for normal cellular functions, growth and development, and that their deficiency leads to a variety of clinical abnormalities that range from anaemia to growth retardation and neurological disorders. Humans cannot synthesize water-soluble vitamins (with the exception of some endogenous synthesis of niacin) and must obtain these micronutrients from exogenous sources. Thus body homoeostasis of these micronutrients depends on their normal absorption in the intestine. Interference with absorption, which occurs in a variety of conditions (e.g. congenital defects in the digestive or absorptive system, intestinal disease/resection, drug interaction and chronic alcohol use), leads to the development of deficiency (and sub-optimal status) and results in clinical abnormalities. It is well established now that intestinal absorption of the water-soluble vitamins ascorbate, biotin, folate, niacin, pantothenic acid, pyridoxine, riboflavin and thiamin is via specific carrier-mediated processes. These processes are regulated by a variety of factors and conditions, and the regulation involves transcriptional and/or post-transcriptional mechanisms. Also well recognized now is the fact that the large intestine possesses specific and efficient uptake systems to absorb a number of water-soluble vitamins that are synthesized by the normal microflora. This source may contribute to total body vitamin nutrition, and especially towards the cellular nutrition and health of the local colonocytes. The present review aims to outline our current understanding of the mechanisms involved in intestinal absorption of water-soluble vitamins, their regulation, the cell biology of the carriers involved and the factors that negatively affect these absorptive events.

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Section: Folatementioning

confidence: 99%

Section: Folatementioning

confidence: 99%

Intestinal absorption of water-soluble vitamins in health and disease

Said

2011

Biochemical Journal

335

239

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“…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 ␣-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.…”

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confidence: 99%

Oligomeric Structure of the Human Reduced Folate Carrier

Hou

Matherly

2009

Journal of Biological Chemistry

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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...

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“…Since its cloning in the mid 1990s, RFC structure and function have been studied extensively (4,8). Using state-of-the-art molecular biology and biochemistry methods for characterizing polytopic membrane proteins, a detailed picture of the molecular structure of human RFC (hRFC) has emerged, including its membrane topology, N-glycosylation, functionally or structurally important domains and amino acids, and packing of ␣-helix transmembrane domains (4,8).…”

mentioning

confidence: 99%

“…Using state-of-the-art molecular biology and biochemistry methods for characterizing polytopic membrane proteins, a detailed picture of the molecular structure of human RFC (hRFC) has emerged, including its membrane topology, N-glycosylation, functionally or structurally important domains and amino acids, and packing of ␣-helix transmembrane domains (4,8). To test hypotheses related to structure and mechanism, a three-dimensional homology model for the 591-amino acid hRFC polypeptide was generated, based on solved structures for the bacterial lactose/ proton symporter LacY and glycerol 3-phosphate/inorganic phosphate antiporter GlpT as well as biochemical data for hRFC (8,9).…”

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confidence: 99%

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

Hou

Cherian

et al. 2010

Journal of Biological Chemistry

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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).

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Chapter 5 Structure and Function of the Reduced Folate Carrier

Cited by 71 publications

References 141 publications

Intestinal absorption of water-soluble vitamins in health and disease

Intestinal absorption of water-soluble vitamins in health and disease

Oligomeric Structure of the Human Reduced Folate Carrier

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

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