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Functional Role of Arginine 373 in Substrate Translocation by the Reduced Folate Carrier

2002; Elsevier BV; Volume: 277; Issue: 44 Linguagem: Inglês

10.1074/jbc.m206459200

1083-351X

Heather Sadlish, Frederick M. Williams, Wayne F. Flintoff,

Iron Metabolism and Disorders

The reduced folate carrier (RFC) plays a critical role in the cellular uptake of folates. However, little is known regarding the mechanism used to transport substrates or the tertiary structure of the protein. Through the analysis of a Chinese hamster ovary cell line deficient in folate uptake, we have identified a single residue in TM10 (Arg-373) of RFC that appears to play a critical role in the translocation of substrate. Replacement of this position with various amino acids (KHQNA) diminished the rate of translocation by 16–50-fold, although substrate binding, protein stability, and localization were unaffected. Furthermore, the translocation capabilities of an R373C mutant in a cysteine-less form of the reduced folate carrier were enhanced 2.5-fold by the positively charged methanethiosulfonate reagent, confirming the essential role of a positive charge at this position. When considering the membrane-impermeable nature of this reagent, the data further suggest that the Arg-373 residue is located within the substrate translocation pathway of the RFC protein. Moreover, cross-linking analysis of the Arg-373 residue demonstrates that it is within 6 Å of residue Glu-394 (TM11), providing the first definitive tertiary structural information for this protein. The reduced folate carrier (RFC) plays a critical role in the cellular uptake of folates. However, little is known regarding the mechanism used to transport substrates or the tertiary structure of the protein. Through the analysis of a Chinese hamster ovary cell line deficient in folate uptake, we have identified a single residue in TM10 (Arg-373) of RFC that appears to play a critical role in the translocation of substrate. Replacement of this position with various amino acids (KHQNA) diminished the rate of translocation by 16–50-fold, although substrate binding, protein stability, and localization were unaffected. Furthermore, the translocation capabilities of an R373C mutant in a cysteine-less form of the reduced folate carrier were enhanced 2.5-fold by the positively charged methanethiosulfonate reagent, confirming the essential role of a positive charge at this position. When considering the membrane-impermeable nature of this reagent, the data further suggest that the Arg-373 residue is located within the substrate translocation pathway of the RFC protein. Moreover, cross-linking analysis of the Arg-373 residue demonstrates that it is within 6 Å of residue Glu-394 (TM11), providing the first definitive tertiary structural information for this protein. reduced folate carrier green fluorescent protein enhanced green fluorescent protein methanethiosulfonate sodium (2-sulfonatoethyl)methanethiosulfonate [2-(trimethylammonium)ethyl]methanethiosulfonate methotrexate N,N′-o-phenylenedimaleimide Chinese hamster ovary horseradish peroxidase phosphate-buffered saline amino acid transmembrane segment dihydrofolate reductase gene Folates are essential compounds required by mammalian organisms for numerous biosynthetic pathways, including the synthesis of pyrimidines, purines and several essential amino acids (1Lucock M. Mol. Genet. Metab. 2000; 71: 121-138Crossref PubMed Scopus (646) Google Scholar). These nutrients are transported into the cell primarily by the reduced folate carrier (RFC)1 system. This transporter has been implicated in clinical resistance to the chemotherapeutic drug, methotrexate (Mtx) (2Gorlick R. Goker E. Trippett T. Steinherz P. Elisseyeff Y. Mazumdar M. Flintoff W.F. Bertino J.R. Blood. 1997; 89: 1013-1018Crossref PubMed Google Scholar, 3Guo W. Healey J.H. Meyers P.A. Ladanyi M. Huvos A.G. Bertino J.R. Gorlick R. Clin. Cancer Res. 1999; 5: 621-627PubMed Google Scholar, 4Ma D. Huang H. Moscow J.A. Biochem. Biophys. Res. Commun. 2000; 279: 891-897Crossref PubMed Scopus (13) Google Scholar), further underlining the importance of characterizing the RFC protein and its mechanism of transport.The RFC protein is consistent with a predicted 12-transmembrane (TM) topology and cytoplasmically located N and C termini, as determined by epitope mapping (5Ferguson P.L. Flintoff W.F. J. Biol. Chem. 1999; 274: 16269-16278Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar) and cysteine scanning. 2W. F. Flintoff, H. Sadlish, and F. M. R. Williams, manuscript in preparation. 2W. F. Flintoff, H. Sadlish, and F. M. R. Williams, manuscript in preparation. Recent work has also demonstrated that these cytoplasmic termini, as well as the loop between TM6 and TM7, do not appear to play a direct role in protein function although they are essential for protein stability and trafficking (6Sadlish H. Williams F.F. Flintoff W.F. Biochem. J. 2002; 364: 777-786Crossref PubMed Scopus (0) Google Scholar, 7Sharina I.G. Zhao R. Wang Y. Babani S. Goldman I.D. Biochem. Pharmacol. 2002; 63: 1717-1724Crossref PubMed Scopus (17) Google Scholar). The characterization of various mutations in the RFC protein using human, mouse, and hamster systems has demonstrated that single amino acid changes can lead to drastic alterations in substrate affinity (8Roy K. Tolner B. Chiao J.H. Sirotnak F.M. J. Biol. Chem. 1998; 273: 2526-2531Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 9Zhao R. Assaraf Y.G. Goldman I.D. J. Biol. Chem. 1998; 273: 7873-7879Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 10Tse A. Brigle K. Taylor S.M. Moran R.G. J. Biol. Chem. 1998; 273: 25953-25960Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 11Jansen G. Mauritz R. Drori S. Sprecher H. Kathmann I. Bunni M. Priest D.G. Noordhuis P. Schornagel J.H. Pinedo H.M. Peters G.J. Assaraf Y.G. J. Biol. Chem. 1998; 273: 30189-30198Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 12Zhao R. Gao F. Goldman I.D. Biochem. Pharmacol. 1999; 58: 1615-1624Crossref PubMed Scopus (29) Google Scholar, 13Zhao R. Gao F. Babani S. Goldman I.D. Clin. Cancer Res. 2000; 6: 3304-3311PubMed Google Scholar, 14Zhao R. Gao F. Wang P.J. Goldman I.D. Mol. Pharmacol. 2000; 57: 317-323PubMed Google Scholar, 15Sharina I.G. Zhao R. Wang Y. Babani S. Goldman I.D. Mol. Pharmacol. 2001; 59: 1022-1028Crossref PubMed Scopus (33) Google Scholar), substrate translocation (16Brigle K.E. Spinella M.J. Sierra E.E. Goldman I.D. J. Biol. Chem. 1995; 270: 22974-22979Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 17Zhao R. Assaraf Y.G. Goldman I.D. J. Biol. Chem. 1998; 273: 19065-19071Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), protein stability, and trafficking (18Wong S.C. Zhang L. Witt T.L. Proefke S.A. Bhushan A. Matherly L.H. J. Biol. Chem. 1999; 274: 10388-10394Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 19Sadlish H. Murray R.C. Williams F.M. Flintoff W.F. Biochem. J. 2000; 346: 509-518Crossref PubMed Google Scholar). Preliminary analysis has suggested an interaction between amino acid residues in TM2 and TM4 (20Liu X.Y. Matherly L.H. Biochem. J. 2001; 358: 511-516Crossref PubMed Scopus (22) Google Scholar) providing some insight regarding the tertiary structure. Overall, however, there is limited information available on the folding of the RFC protein or the mechanism of transporting folates.In this report, we examined the RFC protein encoded by a folate transport-deficient Chinese hamster ovary (CHO) line. The protein has a single point mutation in the predicted TM10, resulting in the substitution of arginine for histidine (R373H). Functional analysis of modified proteins with amino acid replacements for Arg-373 indicates that this residue plays a critical role in substrate translocation and may form part of the translocation pathway. Furthermore, the Arg-373 (TM10) residue and another (Glu-394; TM11) are shown to be within close proximity of each other, presenting the first definitive tertiary structural information for the RFC protein.DISCUSSIONA number of functionally disruptive mutations have been identified and characterized within the RFC protein, and yet the mechanism of substrate transport has remained undefined. In this study, a charged residue (Arg-373) that appears to have a key role in the efficiency of substrate translocation has been identified. Furthermore, this residue was demonstrated to be in close proximity to one located 21 residues downstream, providing the first delineation of the RFC protein tertiary structure.The Mtx-resistant phenotype of the MtxRII 4-5 cell line is the combined result of the loss of one rfc allele, possibly during the initial mutagenesis procedure, and the presence of a mutation in the remaining allele. This point mutation resulted in the substitution of histidine for arginine at position 373. As this amino acid is conserved within the RFC protein throughout eukaryotic organisms including theCaenorhabditis elegans homologue, it is likely to play a pivotal role in defining protein structure or function. Biochemical and functional analysis of an array of amino acids substituted into this position indicated that there are strict requirements to allow any degree of substrate transport. First, the amino acid at position 373 appears to require the ability to form hydrogen bonds, as the frequency of functional transfectants diminishes as the polar tendencies of the residues decline (Arg ∼ Lys > His > Gln > Ala). Second, the amino acid needs to be of a certain size, as exemplified by the transfection frequency of the R373Q and R373N mutant proteins. Although the asparagine residue differs by only a single carbon in length, the transfection frequency is significantly reduced from that of R373Q. However, the alanine substitution was also slightly better tolerated than the asparagine, suggesting that the ability to form hydrogen bonds can be detrimental if the residue is too small due to destabilizing polar interactions within the local environment.The transfection frequencies are drastically reduced for most of the alterations at aa 373 with the exception of lysine, a residue similar in size and charge to arginine. However, most of the other replacements (R373H, R373Q and R373A) yield cell lines similar to the wild-type in folinic acid requirements and Mtx sensitivity; this seems to be the result of increased EGFP fusion protein expression (3–6-fold) over Arg-373-EGFP lines. The low folinic acid growth conditions select for cells with a high level of protein expression, which can compensate for the decreased rate of substrate transport. Thus, in some cases in which clones were not selected for functionality but merely for G418 resistance carried by the plasmid, the protein expression levels are lower (i.e. R373N-EGFP). The low level of R373N-EGFP expression as compared with the other 373 mutant cell lines is reflected in the folinic acid requirements and Mtx sensitivity.Based on algorithms, epitope mapping (5Ferguson P.L. Flintoff W.F. J. Biol. Chem. 1999; 274: 16269-16278Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), and cysteine scanning,2 the RFC protein is consistent with a 12-TM topology with aa 373 predicted to be in the 10th transmembrane domain. It appears that the presence of this charged amino acid in the hydrophobic membrane environment may be tolerated as a result of accessibility to the extracellular environment, perhaps as part of the translocation pathway. The kinetic parameters of Mtx uptake demonstrate that any alteration to the Arg-373 residue leads to a 16–50-fold reduction in the efficiency of substrate translocation (V max), indicating that this residue has a vital role in the structure or function of the protein. As evaluated by protein stability, cellular distribution, and the portion of total molecules at the plasma membrane, the amino acid at position 373 does not significantly affect the folding or localization of RFC. However, the addition of the positively charged MTSET reagent to cells expressing an R373C-EGFP gene product led to a 2.5-fold increase in the amount of Mtx drug accumulation as compared with nontreated cells, confirming the functional importance of the positive charge at 373.The 373 residue is predicted to be located within a membrane-spanning segment of the RFC protein and thus should not be accessible to the extracellular environment. Preliminary experiments indicate that this is the case, as a cysteine at this position cannot be labeled by the membrane-impermeable biotin maleimide compound (data not shown). However, although the MTSET reagent is also membrane-impermeable, its small size enables the molecule to permeate protein pores. Taken together with the functional role of this amino acid in substrate transport, it appears the Arg-373 residue forms part of the translocation pathway.It is difficult to determine conclusively whether the Arg-373 residue interacts with another amino acid based on functional analyses of an array of mutant proteins. It is only with the replacement of Asp-86 or Glu-394 with alanines in combination with R373A to limit potential secondary effects that there is a positive influence on protein function. When transfected into the RFC-deficient cell line, each combination demonstrates an increased transfection frequency as compared with R373A-EGFP alone (3–13-fold), although it is still less than for Arg-373-EGFP. Furthermore, the Mtx uptake kinetics of the cell lines expressing the D86A,R373A-EFGP mutant indicate that this protein has a 5-fold better rate of translocation compared with R373A-EGFP alone. This is suggestive of an interaction between these two residues, although secondary structural effects may also play a role. In contrast, it appears that combining the E394A alteration with R373A has little functional effect on the rate of substrate translocation.The potential interactions between Arg-373 and the Asp-86 or Glu-394 residues were further examined using a technique previously utilized for the elucidation of membrane protein helix packing (30Sun J. Kaback H.R. Biochemistry. 1997; 36: 11959-11965Crossref PubMed Scopus (54) Google Scholar, 31Kim Y.M., Ye, L. Maloney P.C. J. Biol. Chem. 2001; 276: 36681-36686Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 32Ward S.D. Hamdan F.F. Bloodworth L.M. Wess J. J. Biol. Chem. 2002; 277: 2247-2257Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Cysteines replaced the residues of interest in a cysteine-less RFC backbone, and a protease site inserted between them was used to evaluate the effectiveness of the cross-linking agents. Previous work has indicated that removal of the cysteines has little effect on functionality of the RFC and thus, should not interfere with helix packing.2 Based on these data, it is clear that E394 is within 6 Å of the Arg-373 residue in the tertiary structure of the RFC protein. However, there was no apparent cross-linking between the D86C and R373C residues after the addition of the o-PDM cross-linker or under oxidative conditions.The demonstration that Glu-394 (TM11) is within 6 Å of Arg-373 (TM10) is highly significant, as it provides the first evidence for a tertiary structure for this protein. Furthermore, it reflects studies on the bacterial lactose permease protein, where TM10 and TM11 are also located adjacent to each other (33Frillingos S. Sahin-Toth M., Wu, J. Kaback H.R. FASEB J. 1998; 12: 1281-1299Crossref PubMed Scopus (318) Google Scholar). The two proteins are structurally similar in many respects, in that they are both polytopic 12-TM domain proteins with a large cytoplasmic loop between TM6 and TM7. As the helix packing of very few polytopic proteins has been documented, it is not clear whether there are common tertiary structural characteristics. A recent report of a potential charge-pair interaction in the human RFC protein (20Liu X.Y. Matherly L.H. Biochem. J. 2001; 358: 511-516Crossref PubMed Scopus (22) Google Scholar) implicated the residues corresponding to Asp-86 and Arg-131 in the hamster protein. In an attempt to confirm these observations in the hamster system, the same alterations were made (D86V,R131L). However, neither this, nor a double alanine mutant (D86A,R131A) was able to complement the MtxRII 5-3 cell line under restricted folinic acid growth conditions (data not shown). Based on these two opposing results, it appears that there may be differences in tertiary folding of the RFC protein between the human and hamster species. This is somewhat surprising, as there is a high degree of amino acid similarity (∼60%) between the two species as well as similar folate transport properties.The 373 position has strict charge and size requirements such that none of the amino acids tested was able to provide comparable substrate translocation efficiency. The data presented here suggest that the Arg-373 residue is accessible to the extracellular space and may directly interact with the substrate, although it does not appear to be part of the substrate-binding site. Furthermore, the close proximity of this residue to TM11 and the slight stabilizing effect of the D86A alteration suggest that Arg-373 may also have a structural role in defining the translocation pathway. As the structural nature of the arginine residue allows multiple and simultaneous interactions, it is not improbable that the Arg-373 position in RFC may have many different roles. Folates are essential compounds required by mammalian organisms for numerous biosynthetic pathways, including the synthesis of pyrimidines, purines and several essential amino acids (1Lucock M. Mol. Genet. Metab. 2000; 71: 121-138Crossref PubMed Scopus (646) Google Scholar). These nutrients are transported into the cell primarily by the reduced folate carrier (RFC)1 system. This transporter has been implicated in clinical resistance to the chemotherapeutic drug, methotrexate (Mtx) (2Gorlick R. Goker E. Trippett T. Steinherz P. Elisseyeff Y. Mazumdar M. Flintoff W.F. Bertino J.R. Blood. 1997; 89: 1013-1018Crossref PubMed Google Scholar, 3Guo W. Healey J.H. Meyers P.A. Ladanyi M. Huvos A.G. Bertino J.R. Gorlick R. Clin. Cancer Res. 1999; 5: 621-627PubMed Google Scholar, 4Ma D. Huang H. Moscow J.A. Biochem. Biophys. Res. Commun. 2000; 279: 891-897Crossref PubMed Scopus (13) Google Scholar), further underlining the importance of characterizing the RFC protein and its mechanism of transport. The RFC protein is consistent with a predicted 12-transmembrane (TM) topology and cytoplasmically located N and C termini, as determined by epitope mapping (5Ferguson P.L. Flintoff W.F. J. Biol. Chem. 1999; 274: 16269-16278Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar) and cysteine scanning. 2W. F. Flintoff, H. Sadlish, and F. M. R. Williams, manuscript in preparation. 2W. F. Flintoff, H. Sadlish, and F. M. R. Williams, manuscript in preparation. Recent work has also demonstrated that these cytoplasmic termini, as well as the loop between TM6 and TM7, do not appear to play a direct role in protein function although they are essential for protein stability and trafficking (6Sadlish H. Williams F.F. Flintoff W.F. Biochem. J. 2002; 364: 777-786Crossref PubMed Scopus (0) Google Scholar, 7Sharina I.G. Zhao R. Wang Y. Babani S. Goldman I.D. Biochem. Pharmacol. 2002; 63: 1717-1724Crossref PubMed Scopus (17) Google Scholar). The characterization of various mutations in the RFC protein using human, mouse, and hamster systems has demonstrated that single amino acid changes can lead to drastic alterations in substrate affinity (8Roy K. Tolner B. Chiao J.H. Sirotnak F.M. J. Biol. Chem. 1998; 273: 2526-2531Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 9Zhao R. Assaraf Y.G. Goldman I.D. J. Biol. Chem. 1998; 273: 7873-7879Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 10Tse A. Brigle K. Taylor S.M. Moran R.G. J. Biol. Chem. 1998; 273: 25953-25960Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 11Jansen G. Mauritz R. Drori S. Sprecher H. Kathmann I. Bunni M. Priest D.G. Noordhuis P. Schornagel J.H. Pinedo H.M. Peters G.J. Assaraf Y.G. J. Biol. Chem. 1998; 273: 30189-30198Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 12Zhao R. Gao F. Goldman I.D. Biochem. Pharmacol. 1999; 58: 1615-1624Crossref PubMed Scopus (29) Google Scholar, 13Zhao R. Gao F. Babani S. Goldman I.D. Clin. Cancer Res. 2000; 6: 3304-3311PubMed Google Scholar, 14Zhao R. Gao F. Wang P.J. Goldman I.D. Mol. Pharmacol. 2000; 57: 317-323PubMed Google Scholar, 15Sharina I.G. Zhao R. Wang Y. Babani S. Goldman I.D. Mol. Pharmacol. 2001; 59: 1022-1028Crossref PubMed Scopus (33) Google Scholar), substrate translocation (16Brigle K.E. Spinella M.J. Sierra E.E. Goldman I.D. J. Biol. Chem. 1995; 270: 22974-22979Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 17Zhao R. Assaraf Y.G. Goldman I.D. J. Biol. Chem. 1998; 273: 19065-19071Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), protein stability, and trafficking (18Wong S.C. Zhang L. Witt T.L. Proefke S.A. Bhushan A. Matherly L.H. J. Biol. Chem. 1999; 274: 10388-10394Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 19Sadlish H. Murray R.C. Williams F.M. Flintoff W.F. Biochem. J. 2000; 346: 509-518Crossref PubMed Google Scholar). Preliminary analysis has suggested an interaction between amino acid residues in TM2 and TM4 (20Liu X.Y. Matherly L.H. Biochem. J. 2001; 358: 511-516Crossref PubMed Scopus (22) Google Scholar) providing some insight regarding the tertiary structure. Overall, however, there is limited information available on the folding of the RFC protein or the mechanism of transporting folates. In this report, we examined the RFC protein encoded by a folate transport-deficient Chinese hamster ovary (CHO) line. The protein has a single point mutation in the predicted TM10, resulting in the substitution of arginine for histidine (R373H). Functional analysis of modified proteins with amino acid replacements for Arg-373 indicates that this residue plays a critical role in substrate translocation and may form part of the translocation pathway. Furthermore, the Arg-373 (TM10) residue and another (Glu-394; TM11) are shown to be within close proximity of each other, presenting the first definitive tertiary structural information for the RFC protein. DISCUSSIONA number of functionally disruptive mutations have been identified and characterized within the RFC protein, and yet the mechanism of substrate transport has remained undefined. In this study, a charged residue (Arg-373) that appears to have a key role in the efficiency of substrate translocation has been identified. Furthermore, this residue was demonstrated to be in close proximity to one located 21 residues downstream, providing the first delineation of the RFC protein tertiary structure.The Mtx-resistant phenotype of the MtxRII 4-5 cell line is the combined result of the loss of one rfc allele, possibly during the initial mutagenesis procedure, and the presence of a mutation in the remaining allele. This point mutation resulted in the substitution of histidine for arginine at position 373. As this amino acid is conserved within the RFC protein throughout eukaryotic organisms including theCaenorhabditis elegans homologue, it is likely to play a pivotal role in defining protein structure or function. Biochemical and functional analysis of an array of amino acids substituted into this position indicated that there are strict requirements to allow any degree of substrate transport. First, the amino acid at position 373 appears to require the ability to form hydrogen bonds, as the frequency of functional transfectants diminishes as the polar tendencies of the residues decline (Arg ∼ Lys > His > Gln > Ala). Second, the amino acid needs to be of a certain size, as exemplified by the transfection frequency of the R373Q and R373N mutant proteins. Although the asparagine residue differs by only a single carbon in length, the transfection frequency is significantly reduced from that of R373Q. However, the alanine substitution was also slightly better tolerated than the asparagine, suggesting that the ability to form hydrogen bonds can be detrimental if the residue is too small due to destabilizing polar interactions within the local environment.The transfection frequencies are drastically reduced for most of the alterations at aa 373 with the exception of lysine, a residue similar in size and charge to arginine. However, most of the other replacements (R373H, R373Q and R373A) yield cell lines similar to the wild-type in folinic acid requirements and Mtx sensitivity; this seems to be the result of increased EGFP fusion protein expression (3–6-fold) over Arg-373-EGFP lines. The low folinic acid growth conditions select for cells with a high level of protein expression, which can compensate for the decreased rate of substrate transport. Thus, in some cases in which clones were not selected for functionality but merely for G418 resistance carried by the plasmid, the protein expression levels are lower (i.e. R373N-EGFP). The low level of R373N-EGFP expression as compared with the other 373 mutant cell lines is reflected in the folinic acid requirements and Mtx sensitivity.Based on algorithms, epitope mapping (5Ferguson P.L. Flintoff W.F. J. Biol. Chem. 1999; 274: 16269-16278Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), and cysteine scanning,2 the RFC protein is consistent with a 12-TM topology with aa 373 predicted to be in the 10th transmembrane domain. It appears that the presence of this charged amino acid in the hydrophobic membrane environment may be tolerated as a result of accessibility to the extracellular environment, perhaps as part of the translocation pathway. The kinetic parameters of Mtx uptake demonstrate that any alteration to the Arg-373 residue leads to a 16–50-fold reduction in the efficiency of substrate translocation (V max), indicating that this residue has a vital role in the structure or function of the protein. As evaluated by protein stability, cellular distribution, and the portion of total molecules at the plasma membrane, the amino acid at position 373 does not significantly affect the folding or localization of RFC. However, the addition of the positively charged MTSET reagent to cells expressing an R373C-EGFP gene product led to a 2.5-fold increase in the amount of Mtx drug accumulation as compared with nontreated cells, confirming the functional importance of the positive charge at 373.The 373 residue is predicted to be located within a membrane-spanning segment of the RFC protein and thus should not be accessible to the extracellular environment. Preliminary experiments indicate that this is the case, as a cysteine at this position cannot be labeled by the membrane-impermeable biotin maleimide compound (data not shown). However, although the MTSET reagent is also membrane-impermeable, its small size enables the molecule to permeate protein pores. Taken together with the functional role of this amino acid in substrate transport, it appears the Arg-373 residue forms part of the translocation pathway.It is difficult to determine conclusively whether the Arg-373 residue interacts with another amino acid based on functional analyses of an array of mutant proteins. It is only with the replacement of Asp-86 or Glu-394 with alanines in combination with R373A to limit potential secondary effects that there is a positive influence on protein function. When transfected into the RFC-deficient cell line, each combination demonstrates an increased transfection frequency as compared with R373A-EGFP alone (3–13-fold), although it is still less than for Arg-373-EGFP. Furthermore, the Mtx uptake kinetics of the cell lines expressing the D86A,R373A-EFGP mutant indicate that this protein has a 5-fold better rate of translocation compared with R373A-EGFP alone. This is suggestive of an interaction between these two residues, although secondary structural effects may also play a role. In contrast, it appears that combining the E394A alteration with R373A has little functional effect on the rate of substrate translocation.The potential interactions between Arg-373 and the Asp-86 or Glu-394 residues were further examined using a technique previously utilized for the elucidation of membrane protein helix packing (30Sun J. Kaback H.R. Biochemistry. 1997; 36: 11959-11965Crossref PubMed Scopus (54) Google Scholar, 31Kim Y.M., Ye, L. Maloney P.C. J. Biol. Chem. 2001; 276: 36681-36686Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 32Ward S.D. Hamdan F.F. Bloodworth L.M. Wess J. J. Biol. Chem. 2002; 277: 2247-2257Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Cysteines replaced the residues of interest in a cysteine-less RFC backbone, and a protease site inserted between them was used to evaluate the effectiveness of the cross-linking agents. Previous work has indicated that removal of the cysteines has little effect on functionality of the RFC and thus, should not interfere with helix packing.2 Based on these data, it is clear that E394 is within 6 Å of the Arg-373 residue in the tertiary structure of the RFC protein. However, there was no apparent cross-linking between the D86C and R373C residues after the addition of the o-PDM cross-linker or under oxidative conditions.The demonstration that Glu-394 (TM11) is within 6 Å of Arg-373 (TM10) is highly significant, as it provides the first evidence for a tertiary structure for this protein. Furthermore, it reflects studies on the bacterial lactose permease protein, where TM10 and TM11 are also located adjacent to each other (33Frillingos S. Sahin-Toth M., Wu, J. Kaback H.R. FASEB J. 1998; 12: 1281-1299Crossref PubMed Scopus (318) Google Scholar). The two proteins are structurally similar in many respects, in that they are both polytopic 12-TM domain proteins with a large cytoplasmic loop between TM6 and TM7. As the helix packing of very few polytopic proteins has been documented, it is not clear whether there are common tertiary structural characteristics. A recent report of a potential charge-pair interaction in the human RFC protein (20Liu X.Y. Matherly L.H. Biochem. J. 2001; 358: 511-516Crossref PubMed Scopus (22) Google Scholar) implicated the residues corresponding to Asp-86 and Arg-131 in the hamster protein. In an attempt to confirm these observations in the hamster system, the same alterations were made (D86V,R131L). However, neither this, nor a double alanine mutant (D86A,R131A) was able to complement the MtxRII 5-3 cell line under restricted folinic acid growth conditions (data not shown). Based on these two opposing results, it appears that there may be differences in tertiary folding of the RFC protein between the human and hamster species. This is somewhat surprising, as there is a high degree of amino acid similarity (∼60%) between the two species as well as similar folate transport properties.The 373 position has strict charge and size requirements such that none of the amino acids tested was able to provide comparable substrate translocation efficiency. The data presented here suggest that the Arg-373 residue is accessible to the extracellular space and may directly interact with the substrate, although it does not appear to be part of the substrate-binding site. Furthermore, the close proximity of this residue to TM11 and the slight stabilizing effect of the D86A alteration suggest that Arg-373 may also have a structural role in defining the translocation pathway. As the structural nature of the arginine residue allows multiple and simultaneous interactions, it is not improbable that the Arg-373 position in RFC may have many different roles. A number of functionally disruptive mutations have been identified and characterized within the RFC protein, and yet the mechanism of substrate transport has remained undefined. In this study, a charged residue (Arg-373) that appears to have a key role in the efficiency of substrate translocation has been identified. Furthermore, this residue was demonstrated to be in close proximity to one located 21 residues downstream, providing the first delineation of the RFC protein tertiary structure. The Mtx-resistant phenotype of the MtxRII 4-5 cell line is the combined result of the loss of one rfc allele, possibly during the initial mutagenesis procedure, and the presence of a mutation in the remaining allele. This point mutation resulted in the substitution of histidine for arginine at position 373. As this amino acid is conserved within the RFC protein throughout eukaryotic organisms including theCaenorhabditis elegans homologue, it is likely to play a pivotal role in defining protein structure or function. Biochemical and functional analysis of an array of amino acids substituted into this position indicated that there are strict requirements to allow any degree of substrate transport. First, the amino acid at position 373 appears to require the ability to form hydrogen bonds, as the frequency of functional transfectants diminishes as the polar tendencies of the residues decline (Arg ∼ Lys > His > Gln > Ala). Second, the amino acid needs to be of a certain size, as exemplified by the transfection frequency of the R373Q and R373N mutant proteins. Although the asparagine residue differs by only a single carbon in length, the transfection frequency is significantly reduced from that of R373Q. However, the alanine substitution was also slightly better tolerated than the asparagine, suggesting that the ability to form hydrogen bonds can be detrimental if the residue is too small due to destabilizing polar interactions within the local environment. The transfection frequencies are drastically reduced for most of the alterations at aa 373 with the exception of lysine, a residue similar in size and charge to arginine. However, most of the other replacements (R373H, R373Q and R373A) yield cell lines similar to the wild-type in folinic acid requirements and Mtx sensitivity; this seems to be the result of increased EGFP fusion protein expression (3–6-fold) over Arg-373-EGFP lines. The low folinic acid growth conditions select for cells with a high level of protein expression, which can compensate for the decreased rate of substrate transport. Thus, in some cases in which clones were not selected for functionality but merely for G418 resistance carried by the plasmid, the protein expression levels are lower (i.e. R373N-EGFP). The low level of R373N-EGFP expression as compared with the other 373 mutant cell lines is reflected in the folinic acid requirements and Mtx sensitivity. Based on algorithms, epitope mapping (5Ferguson P.L. Flintoff W.F. J. Biol. Chem. 1999; 274: 16269-16278Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), and cysteine scanning,2 the RFC protein is consistent with a 12-TM topology with aa 373 predicted to be in the 10th transmembrane domain. It appears that the presence of this charged amino acid in the hydrophobic membrane environment may be tolerated as a result of accessibility to the extracellular environment, perhaps as part of the translocation pathway. The kinetic parameters of Mtx uptake demonstrate that any alteration to the Arg-373 residue leads to a 16–50-fold reduction in the efficiency of substrate translocation (V max), indicating that this residue has a vital role in the structure or function of the protein. As evaluated by protein stability, cellular distribution, and the portion of total molecules at the plasma membrane, the amino acid at position 373 does not significantly affect the folding or localization of RFC. However, the addition of the positively charged MTSET reagent to cells expressing an R373C-EGFP gene product led to a 2.5-fold increase in the amount of Mtx drug accumulation as compared with nontreated cells, confirming the functional importance of the positive charge at 373. The 373 residue is predicted to be located within a membrane-spanning segment of the RFC protein and thus should not be accessible to the extracellular environment. Preliminary experiments indicate that this is the case, as a cysteine at this position cannot be labeled by the membrane-impermeable biotin maleimide compound (data not shown). However, although the MTSET reagent is also membrane-impermeable, its small size enables the molecule to permeate protein pores. Taken together with the functional role of this amino acid in substrate transport, it appears the Arg-373 residue forms part of the translocation pathway. It is difficult to determine conclusively whether the Arg-373 residue interacts with another amino acid based on functional analyses of an array of mutant proteins. It is only with the replacement of Asp-86 or Glu-394 with alanines in combination with R373A to limit potential secondary effects that there is a positive influence on protein function. When transfected into the RFC-deficient cell line, each combination demonstrates an increased transfection frequency as compared with R373A-EGFP alone (3–13-fold), although it is still less than for Arg-373-EGFP. Furthermore, the Mtx uptake kinetics of the cell lines expressing the D86A,R373A-EFGP mutant indicate that this protein has a 5-fold better rate of translocation compared with R373A-EGFP alone. This is suggestive of an interaction between these two residues, although secondary structural effects may also play a role. In contrast, it appears that combining the E394A alteration with R373A has little functional effect on the rate of substrate translocation. The potential interactions between Arg-373 and the Asp-86 or Glu-394 residues were further examined using a technique previously utilized for the elucidation of membrane protein helix packing (30Sun J. Kaback H.R. Biochemistry. 1997; 36: 11959-11965Crossref PubMed Scopus (54) Google Scholar, 31Kim Y.M., Ye, L. Maloney P.C. J. Biol. Chem. 2001; 276: 36681-36686Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 32Ward S.D. Hamdan F.F. Bloodworth L.M. Wess J. J. Biol. Chem. 2002; 277: 2247-2257Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Cysteines replaced the residues of interest in a cysteine-less RFC backbone, and a protease site inserted between them was used to evaluate the effectiveness of the cross-linking agents. Previous work has indicated that removal of the cysteines has little effect on functionality of the RFC and thus, should not interfere with helix packing.2 Based on these data, it is clear that E394 is within 6 Å of the Arg-373 residue in the tertiary structure of the RFC protein. However, there was no apparent cross-linking between the D86C and R373C residues after the addition of the o-PDM cross-linker or under oxidative conditions. The demonstration that Glu-394 (TM11) is within 6 Å of Arg-373 (TM10) is highly significant, as it provides the first evidence for a tertiary structure for this protein. Furthermore, it reflects studies on the bacterial lactose permease protein, where TM10 and TM11 are also located adjacent to each other (33Frillingos S. Sahin-Toth M., Wu, J. Kaback H.R. FASEB J. 1998; 12: 1281-1299Crossref PubMed Scopus (318) Google Scholar). The two proteins are structurally similar in many respects, in that they are both polytopic 12-TM domain proteins with a large cytoplasmic loop between TM6 and TM7. As the helix packing of very few polytopic proteins has been documented, it is not clear whether there are common tertiary structural characteristics. A recent report of a potential charge-pair interaction in the human RFC protein (20Liu X.Y. Matherly L.H. Biochem. J. 2001; 358: 511-516Crossref PubMed Scopus (22) Google Scholar) implicated the residues corresponding to Asp-86 and Arg-131 in the hamster protein. In an attempt to confirm these observations in the hamster system, the same alterations were made (D86V,R131L). However, neither this, nor a double alanine mutant (D86A,R131A) was able to complement the MtxRII 5-3 cell line under restricted folinic acid growth conditions (data not shown). Based on these two opposing results, it appears that there may be differences in tertiary folding of the RFC protein between the human and hamster species. This is somewhat surprising, as there is a high degree of amino acid similarity (∼60%) between the two species as well as similar folate transport properties. The 373 position has strict charge and size requirements such that none of the amino acids tested was able to provide comparable substrate translocation efficiency. The data presented here suggest that the Arg-373 residue is accessible to the extracellular space and may directly interact with the substrate, although it does not appear to be part of the substrate-binding site. Furthermore, the close proximity of this residue to TM11 and the slight stabilizing effect of the D86A alteration suggest that Arg-373 may also have a structural role in defining the translocation pathway. As the structural nature of the arginine residue allows multiple and simultaneous interactions, it is not improbable that the Arg-373 position in RFC may have many different roles.