Oligomeric Structure of the Human Reduced Folate Carrier

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. 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 (1Stokstad E.L.R. Picciano M.F. Stokstad E.L.R. Greogory J.F. Folic Acid Metabolism in Health and Disease. Wiley-Liss, New York1990: 1-21Google Scholar). Because mammals cannot synthesize folates de novo, cellular uptake of these derivatives is essential for cell growth and tissue regeneration (2Sirotnak F.M. Tolner B. Annu. Rev. Nutr... 1999; 19: 91-122Google Scholar, 3Matherly L.H. Goldman I.D. Vitam. Horm... 2003; 66: 403-456Google Scholar). 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) 2The abbreviations used are: RFC, reduced folate carrier; hRFC, human RFC; Mtx, methotrexate; TMD, transmembrane domain; ER, endoplasmic reticulum; wt, wild type; MTS-3-MTS, 1,3-propanediyl bis-methanethiosulfonate; PBS, phosphate-buffered saline; DTT, dithiothreitol; HA, hemagglutinin; sulfo-NHS-SS-biotin, sulfo-N-hydroxysuccinimide (NHS)-SS-biotin.2The abbreviations used are: RFC, reduced folate carrier; hRFC, human RFC; Mtx, methotrexate; TMD, transmembrane domain; ER, endoplasmic reticulum; wt, wild type; MTS-3-MTS, 1,3-propanediyl bis-methanethiosulfonate; PBS, phosphate-buffered saline; DTT, dithiothreitol; HA, hemagglutinin; sulfo-NHS-SS-biotin, sulfo-N-hydroxysuccinimide (NHS)-SS-biotin. is widely considered to be the major transport system for folate co-factors in mammalian cells and tissues (3Matherly L.H. Goldman I.D. Vitam. Horm... 2003; 66: 403-456Google Scholar, 4Matherly L.H. Hou Z. Deng Y. Cancer Metastasis Rev... 2007; 26: 111-128Google Scholar). RFC plays a generalized role in folate transport and provides specialized tissue functions such as transport across the basolateral membrane of renal proximal tubules (5Kneuer C. Honscha K.U. Honscha W. Cell Tissue Res... 2005; 320: 517-524Google Scholar), transplacental transport of folates (6Sweiry J.H. Yudlievich D.L. Biochim. Biophys. Acta.. 1985; 821: 497-501Google Scholar), and folate transport across the blood-brain barrier (7Spector C. Johanson C. Pharm. Res... 2006; 23: 2515-2524Google Scholar), although the contribution of RFC to intestinal absorption of folates remains controversial (8Zhao R. Goldman I.D. Cancer Metastasis Rev... 2007; 26: 129-139Google Scholar, 9Balamurugan K. Said H.M. Am. J. Physiol... 2006; 291: C189-C193Google Scholar). Loss of RFC expression or function portends potentially profound physiologic and developmental consequences associated with folate deficiency (10Matherly L.H. Curr. Pharmacogenetics.. 2004; 2: 287-298Google Scholar). RFC is also a major transporter of antifolate drugs used for cancer chemotherapy such as methotrexate (Mtx), pemetrexed, and raltitrexed (4Matherly L.H. Hou Z. Deng Y. Cancer Metastasis Rev... 2007; 26: 111-128Google Scholar). 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 (4Matherly L.H. Hou Z. Deng Y. Cancer Metastasis Rev... 2007; 26: 111-128Google Scholar, 11Zhao R. Goldman I.D. Oncogene.. 2003; 22: 7431-7457Google Scholar). Reflecting its particular physiologic and pharmacologic importance, interest in RFC structure and function has been high. Since 1994, when murine RFC was first cloned (12Dixon K.H. Lanpher B.C. Chiu J. Kelley K. Cowan K.H. J. Biol. Chem... 1994; 269: 17-20Google Scholar), 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) (4Matherly L.H. Hou Z. Deng Y. Cancer Metastasis Rev... 2007; 26: 111-128Google Scholar, 13Matherly L.H. Hou Z. Vitam. Horm... 2008; 79: 145-184Google Scholar). 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 (13Matherly L.H. Hou Z. Vitam. Horm... 2008; 79: 145-184Google Scholar, 14Hou Z. Ye J. Haska C.L. Matherly L.H. J. Biol. Chem... 2006; 281: 33588-33596Google Scholar) 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 (15Veenhoff L.M. Heuberger E.H.M.L. Poolman B. Trends Biochem. Sci... 2002; 27: 242-249Google Scholar, 16Sitte H.H. Freissuth M. Eur. J. Pharmacol... 2003; 479: 229-236Google Scholar, 17Vinothkumar K.R. Raunser S. Jung H. Kuhlbrandt W. J. Biol. Chem... 2006; 281: 4795-4801Google Scholar, 18Regeer R.R. Nicke A. Markovich D. Int. J. Biochem. Cell Biol... 2007; 39: 2240-2251Google Scholar). For major facilitator superfamily proteins, both monomeric (e.g. LacY, GlpT, UhpT, and GLUT3) (19Abramson J. Smirnova I. Kasho V. Verner G. Kaback H.R. Iwata S. Science.. 2003; 301: 610-615Google Scholar, 20Huang Y. Lemieux M.J. Song J. Auer M. Wang D.N. Science.. 2003; 301: 616-620Google Scholar, 21Ambudkar S.V. Anantharam V. Maloney P.C. J. Biol. Chem... 1990; 265: 12287-12292Google Scholar, 22Burant C.F. Bell G.I. Biochemistry.. 1992; 31: 10414-10420Google Scholar) and oligomeric (e.g. LacS, AE1, GLUT1, and TetA) (23Veenhoff L.M. Heuberger E.H.M.L. Poolman B. EMBO J.. 2001; 20: 3056-3062Google Scholar, 24Dahl N.K. Jiang L. Chernova M.N. Stuart-Tilley A.K. Shmukler B.E. Alper S.L. J. Biol. Chem... 2003; 278: 44949-44958Google Scholar, 25Taylor A.M. Zhu Q. Casey J.R. Biochem. J... 2001; 359: 661-668Google Scholar, 26Zottola R.J. Cloherty E.K. Coderre P.E. Hansen A. Hebert D.N. Carruthers A. Biochemistry.. 1995; 34: 9734-9747Google Scholar, 27Yin C.C. ma-Ramos M.L. Borges-Walmsley M.I. Taylor R.W. Walmsley A.R. Levy S.B. Bullough P.A. Mol. Microbiol... 2000; 38: 482-492Google Scholar, 28Hickman R.K. Levy S.B. J. Bacteriol... 1988; 170: 1715-1720Google Scholar) 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. Reagents—[3′,5′,7-3H]Mtx (20 Ci/mmol) was purchased from Moravek Biochemicals (Brea, CA). Unlabeled Mtx was provided by the Drug Development Branch, NCI, National Institutes of Health (Bethesda, MD). Both labeled and unlabeled Mtx were purified by high pressure liquid chromatography prior to use (33Fry D.W. Yalowich J.C. Goldman I.D. J. Biol. Chem... 1982; 257: 1890-1896Google Scholar). Synthetic oligonucleotides were obtained from Invitrogen. Tissue culture reagents and supplies were purchased from assorted vendors with the exception of fetal bovine serum, which was purchased from Hyclone Technologies (Logan, UT). The cross-linking reagent, MTS-3-MTS (1,3-propanediyl bis-methanethiosulfonate), was purchased from Toronto Research Chemicals (Toronto, Canada). Generation of hRFC wt and Mutant Constructs—Full-length wt hRFCMyc-His10 was prepared by PCR from the full-length hRFC (pC43) (34Wong S.C. Proefke S.A. Bhushan A. Matherly L.H. J. Biol. Chem... 1995; 270: 17468-17475Google Scholar), using the P6 primer (35Witt T.L. Stapels S.E. Matherly L.H. J. Biol. Chem... 2004; 279: 46755-46763Google Scholar) and the antisense FL His10/AS primer (5′-cgctcgagGGATCCctggttcacattctg-3′). PCR conditions were 4 min at 95 °C for one cycle, followed by 35 cycles at 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 1 min. The amplicon was digested with BamHI and SfiI (New England Biolabs, Ipswich, MA) and ligated into BamHI/SfiI-digested truncated hRFCMyc-His10 in pCDNA3 (35Witt T.L. Stapels S.E. Matherly L.H. J. Biol. Chem... 2004; 279: 46755-46763Google Scholar). Deglycosylated full-length hRFCMyc-His10 (dghRFCMyc-His10) with Gln substituted for Asn at position 58 was generated by site-directed mutagenesis with the QuikChange™ kit (Stratagene, La Jolla, CA), using the wt hRFCMyc-His10 construct as template. The same strategy was used with full-length wt hRFCHA template (36Payton S.G. Haska C.L. Flatley R.M. Ge Y. Matherly L.H. Biochim. Biophys. Acta.. 2007; 1769: 131-138Google Scholar) to generate the S138C-hRFCHA, G163P-hRFCHA, and R373A-hRFCHA mutants. Mutation primers were designed on the Stratagene web site. Sequences for the mutation primers are available upon request. All of the mutations were confirmed by automated DNA sequencing at the Wayne State University Sequencing Core. Cell Culture—hRFC-null Mtx-resistant HeLa cells, R5 R. S. M. Goldman I.D. Cancer Res... 2004; Scholar), were a of I. Goldman New R5 cells were as previously Z. Stapels S.E. Haska C.L. Matherly L.H. J. Biol. Chem... 2005; Scholar). wt and mutant hRFC were into R5 cells with Z. Stapels S.E. Haska C.L. Matherly L.H. J. Biol. Chem... 2005; Scholar). For in which with were with results for cells with a single DNA for the was by all the cells were for of membranes and Western For transport were and an R5 cells with were with phosphate-buffered and with MTS-3-MTS at a of for 30 min at 4 of used for was to the cells as a The reactions were by the of with cells were and at membrane were prepared by L.H. Cancer Res... Scholar), and were with and by and Western uptake of was min at °C in in (20 to with as previously Z. Stapels S.E. Haska C.L. Matherly L.H. J. Biol. Chem... 2005; Scholar). The levels of intracellular were expressed as of from of and protein R.J. J. Biol. Chem... Scholar) of the cell were as previously Z. Stapels S.E. Haska C.L. Matherly L.H. J. Biol. Chem... 2005; Scholar), using and CA). The were with Scholar) and on a in the of for Western and Western membrane and to membranes were as previously Z. Stapels S.E. Haska C.L. Matherly L.H. J. Biol. Chem... 2005; Scholar). and of proteins was with and with an used the the hRFC were and for an was a of hRFC with Cell was used to and surface proteins. the cells were with in for 30 min at 4 °C and with The was to the The was with for 1 at and the were with The proteins were with Scholar) and by For surface labeled proteins were with Z. Stapels S.E. Haska C.L. Matherly L.H. J. Biol. Chem... 2005; Scholar), which was used for but in the of confocal microscopy, R5 cells were and in wt and and wt wt and wt were as the cells were with with and with followed by with as previously (35Witt T.L. Stapels S.E. Matherly L.H. J. Biol. Chem... 2004; 279: 46755-46763Google Scholar). The used were and and and of were including and the ER, the endoplasmic kit was and and were also The were with a using a using the same for all of the was at the of the Cancer of hRFC by cross-linking is a for establishing of proteins in their G. S. G. S. Mol. Biol. Scholar) and has been used to study quaternary structures of membrane proteins (15Veenhoff L.M. Heuberger E.H.M.L. Poolman B. Trends Biochem. Sci... 2002; 27: 242-249Google Scholar). hRFC has including in the and in the and and in the intracellular and explore the possibility that hRFC as a higher order we used an MTS-3-MTS, of cross-linking to hRFC-null R5 HeLa cells with a hRFC construct epitope-tagged with a to position and in which the consensus at position 58 was to Gln cross-linking at 4 membranes were and the membrane proteins were by and Western with for using for these the to the of hRFC species of and was based on our that mutation of to Gln only on hRFC surface expression and transport function S.C. L. Proefke S.A. Matherly L.H. Biochim. Biophys. Acta.. Scholar). in of R5 cells with MTS-3-MTS in not in the cells with in of for which only monomeric labeled was The at and molecular masses of and as a species of membranes with and prior to cross-linking for and as in their and However, was that it as of hRFC in which MTS-3-MTS and are to Although the of and cross-linking of with proteins, the of these for masses of and that homo-oligomers of these results establish the of the existence of homo-oligomeric of hRFC oligomers with a homobifunctional The construct was transiently expressed in hRFC-null HeLa R5 cells. The cells were with the MTS-3-MTS cross-linker at 4 °C for 30 min. cross-linking, membranes were and membrane proteins were by conditions and Western with In the of 4 were that were not in the of cross-linker of proteins with prior to cross-linking for and but no on and no molecular of and the monomeric hRFC we expressed full-length wt hRFCHA and wt hRFCMyc-His10 proteins in HeLa R5 cells or membranes were and with and Myc and protein The were followed by and Western in R5 cells, and hRFC proteins co-immunoprecipitated with or Myc 4 and However, this was not for hRFCHA and hRFCMyc-His10 expressed and with and 1 and or when were prior to and results were obtained a of expressed hRFCHA and hRFCMyc-His10 the existence of cell surface oligomeric hRFC, a was in which R5 cells were with wt hRFCHA and wt hRFCMyc-His10 and with sulfo-NHS-SS-biotin, which hRFC molecules at the cell surface so were and proteins were to and with The were with Myc in with a membrane particulate from the same by L.H. Cancer Res... Scholar) and by and Western with and that results were obtained for particulate and and wt that hRFCMyc-His10 with hRFCHA at the membrane surface. results establish that hRFC exists as a higher order hRFC monomers at the membrane surface. wt and Mutant hRFC for establishing the consequences of of transporter proteins co-expression of and inactive (23Veenhoff L.M. Heuberger E.H.M.L. Poolman B. EMBO J.. 2001; 20: 3056-3062Google Scholar, H. M. S. J. Biol. Chem... Scholar, G. S. 2000; Scholar, L. M. M. D. S. 2007; Scholar). By this the of a mutant not the activity of a wt transporter that is as a L. M. M. D. S. 2007; Scholar). However, or monomers the the of a mutant transport an of our of for hRFC Z. Ye J. Haska C.L. Matherly L.H. J. Biol. Chem... 2006; 281: 33588-33596Google Scholar), we hRFC with significant of transport activity of associated with decreased hRFC levels and and of or of a functionally important amino acid in expression of the are in the hRFC in transport and are in R5 HeLa cells with wt hRFCMyc-His10 and wt or mutant hRFCs, for with R5 cells. the cells were for with levels of cell surface hRFC by The single and R373A-hRFCHA the surface expression Although all of the were expressed at the membrane surface from its on S138C to be or wt hRFC with or wt transport hRFC and cell surface Mtx transport was decreased for all from of wt for to and of wt for and with wt hRFCMyc-His10 no on surface expression levels for and from in cells with mutant However, of wt transport activity in the of the S138C and were accompanied by of surface wt hRFCMyc-His10 protein and of the level for wt hRFCMyc-His10 with wt was not by in on with a not were no significant in the levels of wt hRFCMyc-His10 or R373A-hRFCHA the and R5 cells. these in surface carrier levels and transport function for the hRFC by of hRFC proteins hRFC oligomers were at the membrane surface Although are the levels of expression and the activity for wt hRFC in the S138C and this was for for which was no in levels of surface hRFC for wt or mutant a low level of transport expression for wt and mutant in single or R5 cells were confirmed by confocal and with and wt hRFCMyc-His10 and wt hRFCHA were both expressed at high levels at the cell surface or in with our surface biotinylation was no of R5 cells for mutant hRFCs, surface from low to and to the high levels for wt hRFCHA and In combined with wt was a to significant loss of and was no of R373A-hRFCHA on surface expression of wt hRFCMyc-His10 Collectively, these results demonstrate the formation of oligomers wt and mutant that be at the cell surface. For and a were of wt hRFC at the cell to for the mutants. that this of mutant hRFC and in the such that oligomers the same were for including L. Nicke A. S. H. A. H.H. H. V. J. Biol. Chem... 2008; Scholar, B. C. Biochem. J... 2008; Scholar), Q. Z. J. Biol. Chem... 2006; 281: Scholar), and the transporter S. 2008; Scholar). to a dominant-negative that wt carrier function of on intracellular trafficking and surface wt hRFC in the of Mutant explore the for the loss of surface wt hRFC in the of the we surface expression and intracellular of wt hRFC in the and of by confocal our to and and hRFC proteins transiently expressed in R5 cells, and with or and or the with the hRFC we used an membrane protein and with R5 cells were used as Both and wt hRFC proteins or in and were expressed at high levels at the cell surface with intracellular was for R5 cells for S138C-hRFCHA, or in with wt a low level of mutant protein was to the cell surface and as in our significant to be at in in with the wt hRFCMyc-His10 with was also although was a low level of cell surface as results are with the of wt and proteins to be not it not the system and is trafficking to the In the of a mutant hRFC such as both wt and mutant proteins are in a loss of wt carrier protein at the cell surface and decreased transport function. the present study the existence of higher order homo-oligomers for hRFC, the major membrane transporter of folates and in mammalian cells and tissues (3Matherly L.H. Goldman I.D. Vitam. Horm... 2003; 66: 403-456Google Scholar, 4Matherly L.H. Hou Z. Deng Y. Cancer Metastasis Rev... 2007; 26: 111-128Google Scholar). and Myc epitope-tagged hRFC proteins were from both membrane particulate and surface-enriched membrane from HeLa R5 that oligomeric hRFC is expressed at the membrane surface it of folate co-factors and By co-expression of wt and inactive mutant S138C hRFCs, a dominant-negative phenotype was demonstrated involving greatly decreased surface expression of wt carrier of impaired intracellular trafficking and in the an mutant hRFC with a protein such as Q. Z. J. Biol. Chem... 2006; 281: Scholar) that of wt carrier and be or For another mutant (R373A), expression of oligomeric hRFC was accompanied by a significant and disproportionate loss of wt activity unrelated to levels of surface carrier. Although the mechanism for this is by site-directed was in carrier function R. Wang Y. S. Goldman I.D. Mol. Pharmacol... 2001; Scholar, H. F.M. J. Biol. Chem... 2002; Scholar) and facilitate substrate the of folate Y. Hou Z. Wang L. C. J. A. Matherly L.H. Mol. Pharmacol... 2008; Scholar). The present results important into hRFC also and significant For although hRFC species with molecular masses of hRFC homo-oligomers were by chemical cross-linking in our the of the major hRFC structural (e.g. dimers, tetramers, etc.) is it not yet whether oligomeric hRFC is composed of a single hRFC monomeric a transmembrane such that are multiple or whether a single transmembrane for is by multiple monomeric hRFC Although our dominant-negative establish both a structural and hRFC it remains to be whether monomeric hRFC in mediate or transport by or whether a monomers (15Veenhoff L.M. Heuberger E.H.M.L. Poolman B. Trends Biochem. Sci... 2002; 27: 242-249Google Scholar) is are also possible. For loss of wt hRFC function also be to a that results in and of wt from a hRFC are to these The existence of homo-oligomeric hRFC has from a role in antifolate resistance and the possibility of mutant and wt that in impaired function or intracellular to the possibility of oligomeric carrier function or trafficking with molecules that as to intracellular trafficking and surface be the of I. Goldman for of hRFC-null R5 HeLa cells. The of K. in the of the confocal and the of and Deng in the with the and are

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