The Region between Transmembrane Domains 1 and 2 of the Reduced Folate Carrier Forms Part of the Substrate-binding Pocket

A functional cysteine-less form of the hamster reduced folate carrier protein was generated by alanine replacement of the 14 cysteine residues. The predicted 12-transmembrane topology was examined by replacing selected amino acids, predicted to be exposed to the extracellular or cytosolic environments, with cysteines. The location of these cysteines was defined by their accessibility to biotin maleimide in the presence or absence of specific blocking agents. Amino acids predicted to be exposed to the extracellular environment (S46C, S179C, L300C, Y355C, and K430C) could be labeled with biotin maleimide; this modification could be blocked by prior treatment with nonpermeable reagents. Amino acids predicted to be within the cytosol (S152C, Cys224, and L475C) could be labeled only after streptolysin O permeabilization. In addition, the cysteine-less reduced folate carrier was exploited to evaluate a potential substrate-binding domain as suggested by previous studies. Nineteen cysteine replacements were generated between residues 39 and 75, a region located between the first and second transmembrane segments. From the biotinylation of these sites and the ability of various reagents to block this labeling, it appears that L41C, E45C, S46C, T49C, I66C, and L70C are exposed to the extracellular environment, whereas Q54C, Q61C, and T63C are slightly less accessible. Cysteines 39, 42, 44, 47, 51, and 73 were inefficiently biotinylated, suggesting that these sites are located in the membrane or within a tightly folded domain of the protein. Furthermore, biotinylation of cysteines 41, 46, 49, 70, and 71 could be prevented by prior treatment with either methotrexate or folinic acid, indicating that these sites form part of a substrate-binding pocket. A functional cysteine-less form of the hamster reduced folate carrier protein was generated by alanine replacement of the 14 cysteine residues. The predicted 12-transmembrane topology was examined by replacing selected amino acids, predicted to be exposed to the extracellular or cytosolic environments, with cysteines. The location of these cysteines was defined by their accessibility to biotin maleimide in the presence or absence of specific blocking agents. Amino acids predicted to be exposed to the extracellular environment (S46C, S179C, L300C, Y355C, and K430C) could be labeled with biotin maleimide; this modification could be blocked by prior treatment with nonpermeable reagents. Amino acids predicted to be within the cytosol (S152C, Cys224, and L475C) could be labeled only after streptolysin O permeabilization. In addition, the cysteine-less reduced folate carrier was exploited to evaluate a potential substrate-binding domain as suggested by previous studies. Nineteen cysteine replacements were generated between residues 39 and 75, a region located between the first and second transmembrane segments. From the biotinylation of these sites and the ability of various reagents to block this labeling, it appears that L41C, E45C, S46C, T49C, I66C, and L70C are exposed to the extracellular environment, whereas Q54C, Q61C, and T63C are slightly less accessible. Cysteines 39, 42, 44, 47, 51, and 73 were inefficiently biotinylated, suggesting that these sites are located in the membrane or within a tightly folded domain of the protein. Furthermore, biotinylation of cysteines 41, 46, 49, 70, and 71 could be prevented by prior treatment with either methotrexate or folinic acid, indicating that these sites form part of a substrate-binding pocket. The reduced folate carrier (RFC), 1The abbreviations used are: RFC, reduced folate carrier; TM, transmembrane domain; BM, biotin maleimide; LYI, lucifer yellow iodoacetamide; MTSET, (2-(trimethylammonium) ethyl)methanethiosulfonate bromide; NEM, N-ethylmaleimide; Mtx, methotrexate; EGFP, enhanced green fluorescent protein; PBS, phosphate-buffered saline.1The abbreviations used are: RFC, reduced folate carrier; TM, transmembrane domain; BM, biotin maleimide; LYI, lucifer yellow iodoacetamide; MTSET, (2-(trimethylammonium) ethyl)methanethiosulfonate bromide; NEM, N-ethylmaleimide; Mtx, methotrexate; EGFP, enhanced green fluorescent protein; PBS, phosphate-buffered saline. the major transporter for folates in mammalian cells (1Sirotnak F.M. Tolner B. Annu. Rev. Nutr. 1999; 19: 91-122Crossref PubMed Scopus (260) Google Scholar), is a low capacity carrier with a high affinity for substrate and a preference for reduced folates (2Sirotnak F.M. Cancer Res. 1985; 45: 3992-4000PubMed Google Scholar). It is expressed as a membrane protein in a wide variety of tissues and cell types (1Sirotnak F.M. Tolner B. Annu. Rev. Nutr. 1999; 19: 91-122Crossref PubMed Scopus (260) Google Scholar, 3Wang Y. Zhao R. Russell R.G. Goldman I.D. Biochim. Biophys. Acta. 2001; 1513: 49-54Crossref PubMed Scopus (130) Google Scholar, 4Whetsine J.R. Flatley R.M. Matherly L.H. Biochem. J. 2002; 367: 629-640Crossref PubMed Google Scholar); and although highly conserved at the amino acid level, it varies in predicted size from 58 to 85 kDa depending upon the species (5Matherly L.H. Prog. Nucleic Acids Res. Mol. Biol. 2001; 67: 131-162Crossref PubMed Google Scholar, 6Zhao R. Gao F. Liu L. Goldman I.D. Biochim. Biophys. Acta. 2000; 1466: 7-10Crossref PubMed Scopus (16) Google Scholar, 7Sadlish H. Murray R.C. Williams F.M.R. Flintoff W.F. Biochem. J. 2000; 346: 509-518Crossref PubMed Google Scholar). Hydrophobicity analyses have indicated that the RFC protein has 12 transmembrane-spanning segments with the N and C termini located intracellularly and a large intracellular loop between transmembrane domain (TM) 6 and TM7 (8Ferguson P.L. Flintoff W.F. J. Biol. Chem. 1999; 274: 16269-16278Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Epitope insertions into the predicted major loops (8Ferguson P.L. Flintoff W.F. J. Biol. Chem. 1999; 274: 16269-16278Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 9Liu X.Y. Matherly L.H. Biochim. Biophys. Acta. 2002; 1564: 333-342Crossref PubMed Scopus (41) Google Scholar) and N-glycosylation scanning mutagenesis (9Liu X.Y. Matherly L.H. Biochim. Biophys. Acta. 2002; 1564: 333-342Crossref PubMed Scopus (41) Google Scholar) support this model, although the latter study suggests that the C-terminal portion of the protein may demonstrate an alternative topology. Various regions of the protein have been implicated in different aspects of function and biogenesis. For example, maintenance of both termini is important for ensuring the appropriate cellular localization of the RFC (10Sadlish H. Williams F.M.R. Flintoff W.F. Biochem. J. 2002; 364: 777-786Crossref PubMed Scopus (0) Google Scholar, 11Marchant J.S. Subramanian V.S. Parker I. Said H.M. J. Biol. Chem. 2002; 277: 33325-33333Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar), whereas the large intracellular loop between TM6 and TM7 is required for both protein stability and efficient substrate translocation (10Sadlish H. Williams F.M.R. Flintoff W.F. Biochem. J. 2002; 364: 777-786Crossref PubMed Scopus (0) Google Scholar, 12Sharina I.G. Zhao R. Wang Y. Babani S. Goldman I.D. Biochem. Pharmacol. 2002; 63: PubMed Scopus Google Scholar). Furthermore, amino acids in the predicted R. S. H. I. H.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, R. Goldman I.D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, L. I. Y. Biochem. J. 2002; 367: PubMed Scopus (70) Google Scholar) and Tolner B. F.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Matherly L.H. Biochim. Biophys. Acta. 2002; PubMed Scopus Google Scholar, R. Gao F. Goldman I.D. Biochem. Pharmacol. 1999; PubMed Scopus Google Scholar) have been implicated as important of substrate and scanning mutagenesis has been in various aspects of protein and function J.R. Y. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. PubMed Scopus (37) Google Scholar, J. PubMed Scopus Google Scholar, J. R. J.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Biochim. Biophys. Acta. 1999; PubMed Scopus Google Scholar). is the cysteine replacement of specific residues within a functional cysteine-less form of the protein. The modification of these replacement cysteines with a variety of reagents a to be as extracellular or cytosolic a is within the membrane or a tightly folded domain of the it may be J. PubMed Scopus Google Scholar, J. R. J.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Biochim. Biophys. Acta. 1999; PubMed Scopus Google Scholar). Furthermore, modification of these residues by the of or Biochim. Biophys. Acta. 1999; PubMed Scopus Google Scholar). In this of the 14 cysteine residues of the hamster RFC was with of these cysteines are conserved the hamster acids and and the cysteine-less form of the protein was residues were into the extracellular or cytosolic regions of the RFC, and the were with reagents. of sites the extracellular environment could be blocked by nonpermeable whereas intracellular sites required of the these are with the 12-transmembrane for the Furthermore, biotinylation of between and could be blocked by suggesting that this region part of the substrate-binding pocket. and were from maleimide N lucifer yellow and fluorescent protein were from biotin was from and (2-(trimethylammonium) ethyl)methanethiosulfonate was from streptolysin and were from was from and by used as W.F. Biochem. Biophys. PubMed Scopus (37) Google Scholar). of RFC of the RFC with enhanced green fluorescent protein to C-terminal was H. Murray R.C. Williams F.M.R. Flintoff W.F. Biochem. J. 2000; 346: 509-518Crossref PubMed Google Scholar). The carrier was in the were used to the of the hamster RFC region with a by a was into the and into the and sites of A for the by and sites was generated and and after the was A second with by and sites and was into the of the and for The from this was into the RFC in the and for the for the protein in both of the and was with the RFC by the of the RFC and the replacement of residues with were in an hamster RFC to The of RFC function L. Matherly L.H. Biochim. Biophys. Acta. PubMed Scopus Google Scholar). For the of the mutagenesis was the mutagenesis was the and to the were for for and for of was with prior to In mutagenesis was a with a were used with appropriate and in to at the were and the was from in of were and used as with the in the second The was into with and into the appropriate or In this the with the replacement a region of in the were an to their of the into the cells was of in cells as F.M.R. Murray R.C. Flintoff W.F. J. Biol. Chem. Full Text PDF PubMed Google Scholar). cell is to it the and has F.M.R. Murray R.C. Flintoff W.F. J. Biol. Chem. Full Text PDF PubMed Google Scholar). with the cells were selected for in low of folinic acid or at low were were and in by from were for and folinic acid as Flintoff W.F. Mol. PubMed Scopus Google Scholar). For the is the that reduced to whereas for folinic acid the is the that with the analyses for the of and for were as W.F. Biochem. Biophys. PubMed Scopus (37) Google Scholar, Flintoff W.F. Mol. PubMed Scopus Google Scholar, H. Williams F.M.R. Flintoff W.F. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus (16) Google Scholar). is expressed as of of protein and as was a in as by J. A Scholar) and to membrane by the were with and exposed to as F.M.R. Murray R.C. Flintoff W.F. J. Biol. Chem. Full Text PDF PubMed Google Scholar). were and with cellular were from various of cells by in and at for at the was by and of the was as H. Murray R.C. Williams F.M.R. Flintoff W.F. Biochem. J. 2000; 346: 509-518Crossref PubMed Google Scholar). of the was in a by were and were the in the of the were used for and cells in and either the or residues at the sites were with phosphate-buffered and and with of and were at for in the absence or presence of or the indicated of either or folinic acid by the indicated of for these the cells were with and with The cells were as and of was The were at for was and the was for The was for at and with a of and were from the by for at with The were by and The were blocked in in and and were with a of in a were with and and were and with by In cells were with either or prior to with streptolysin O as by Full Text Full Text PDF PubMed Scopus Google Scholar). The were as biotin were at cell for replacement and for For of cells were with and with biotin for at were with and with of protein from cell were by and as was as were and as of the hamster RFC 14 with of these located in predicted transmembrane segments these are conserved acids and for and of the 14 cysteines are for mutagenesis was used to with previous indicated that the of the function of the RFC L. Matherly L.H. Biochim. Biophys. Acta. PubMed Scopus Google Scholar), were generated in an The cysteine-less protein was expressed with either or to the C to various functional have that the C-terminal RFC as a for carrier and cellular localization H. Murray R.C. Williams F.M.R. Flintoff W.F. Biochem. J. 2000; 346: 509-518Crossref PubMed Google Scholar, P.L. Flintoff W.F. J. Biol. Chem. 1999; 274: 16269-16278Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). of the the RFC was the was into hamster cells the RFC and selected for in low of folinic in the RFC was to the folate of the cells and cell although at a reduced with the or RFC of cell or cell are as is the the RFC the the with a N-glycosylation at the the with the N-glycosylation and the the protein with the N-glycosylation is expressed as the of of is the cell to acid is the folinic acid cell for is expressed as is expressed as of of The cell are as is the the RFC the the with a N-glycosylation at the the with the N-glycosylation and the the protein with the N-glycosylation is expressed as the of of The is the cell to The is the folinic acid cell for is expressed as is expressed as of of in a selected were for their to Mtx, for the for folinic acid, and for the with In cells this protein a slightly to and for folinic acid with the protein is in a in the affinity for as as a reduced of of and RFC was used to the of the in the in the of in cells was to that in cells the form of the carrier The of in the cell was that in the The size of the expressed for was the or the expressed for this is a of replacing the region with of different of from of cells indicated of protein in the cell The cells a protein that as a at to that H. Murray R.C. Williams F.M.R. Flintoff W.F. Biochem. J. 2000; 346: 509-518Crossref PubMed Google Scholar, H. Williams F.M.R. Flintoff W.F. Biochem. J. 2002; 364: 777-786Crossref PubMed Scopus (0) Google Scholar, H. Williams F.M.R. Flintoff W.F. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus (16) Google Scholar). The in of and H. Murray R.C. Williams F.M.R. Flintoff W.F. Biochem. J. 2000; 346: 509-518Crossref PubMed Google Scholar). or a protein of kDa in the of the N-glycosylation size is less the size of 85 kDa and is to of the protein cellular be prior to of protein the of protein that was expressed at the cell of cells or were with nonpermeable biotin protein is kDa in size it is protein were in the to protein the were for both cell the absence of cysteines and the of N-glycosylation to with the membrane localization of the RFC protein. of into the RFC of a cysteine-less the to the topology of the RFC scanning a less either or N-glycosylation selected sites within the RFC residues were with cysteines the were in the and into the cells to The C-terminal was with to the specific of the residues with reagents. was the residues for Annu. Rev. Biochem. 67: PubMed Scopus Google Scholar). In the with the cysteine were to the cell indicating that RFC was of the cysteine a protein that a ability to the cell the folinic acid the folinic acid were to the of was indicating that this protein may substrate less of and and in are the in folinic acid as the and and and and and The in are the in folinic acid as the in a of in accessibility of replacement residues predicted to be the extracellular environment was for functional cell these various were with in the presence or absence of the reagents and was biotinylation of the protein. For of the cysteine protein was after treatment with or BM, although and K430C) required the of biotinylation for of the replacements was by with LYI, a nonpermeable to the of protein biotinylation was for replacements at 46, and biotinylation was by only for the replacement and was by the with the prevented biotinylation at either these the extracellular location of the regions residues 46, and as by (8Ferguson P.L. Flintoff W.F. J. Biol. Chem. 1999; 274: 16269-16278Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 9Liu X.Y. Matherly L.H. Biochim. Biophys. Acta. 2002; 1564: 333-342Crossref PubMed Scopus (41) Google Scholar) and that is exposed to the extracellular environment, an to previous N-glycosylation scanning mutagenesis (9Liu X.Y. Matherly L.H. Biochim. Biophys. Acta. 2002; 1564: 333-342Crossref PubMed Scopus (41) Google Scholar). may be in a it is with biotin maleimide and is that to LYI, is to the of in with replacements in the predicted cytosolic regions and L475C) and the were examined in a as the replacements in the extracellular of the residues were indicating the used in this the cell of the cells with streptolysin O prior to treatment of the residues at these could be prevented by with the NEM, with the the cytosolic location of regions residues and as (8Ferguson P.L. Flintoff W.F. J. Biol. Chem. 1999; 274: 16269-16278Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 9Liu X.Y. Matherly L.H. Biochim. Biophys. Acta. 2002; 1564: 333-342Crossref PubMed Scopus (41) Google Scholar) and in a location as predicted by the 12-transmembrane of of exposed to the extracellular environment could be in substrate these regions are in substrate the biotinylation of residues at these sites be prevented by prior treatment with substrate could a in the protein that the of residues. cells the with extracellular residues were with either or folinic acid prior to in biotinylation of was by prior treatment with and was reduced by with folinic In the sites were of in the between and the of this substrate 46, a of were generated within the region from residues to indicated that of these were in the were with the N-glycosylation in a of these replacements were to the of the and were to the selected of folinic acid and were E45C, and were to only selected folinic acid indicating that these may the substrate less of replacements in the region between and in are the in folinic acid as the The in are the in folinic acid as the in a indicated that the protein from to the cell an the The expressed protein as a high at kDa that the form H. Murray R.C. Williams F.M.R. Flintoff W.F. Biochem. J. 2000; 346: 509-518Crossref PubMed Google Scholar, H. Williams F.M.R. Flintoff W.F. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus (16) Google Scholar), the form H. Murray R.C. Williams F.M.R. Flintoff W.F. Biochem. J. 2000; 346: 509-518Crossref PubMed Google Scholar, H. Williams F.M.R. Flintoff W.F. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus (16) Google Scholar), and the form H. Murray R.C. Williams F.M.R. Flintoff W.F. Biochem. J. 2000; 346: 509-518Crossref PubMed Google Scholar, H. Williams F.M.R. Flintoff W.F. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus (16) Google Scholar). The of these species the cell in the and appears to be of the form the whereas the form appears in the may and of the replacements at these although these were the accessibility of the replacement residues in these functional the were with in the presence or absence of the reagents and indicated in of the residues were E45C, S46C, T49C, Q54C, Q61C, I66C, and Furthermore, this biotinylation could be prevented by with either or for replacements indicating that these sites are exposed to the extracellular It is of to in S46C, and the form of the protein was that this could be suggests that the form is expressed at the of the In of the residues were labeled at a low and or at and at high of sites may be in the membrane or have that of of of in the between and that of the sites in the region between and were exposed to the extracellular environment the to a in substrate In an as for cells these replacements were with various of or folinic acid prior to to the ability of the substrate to the sites could be into types depending the ability of the substrate to the sites in biotinylation could be at substrate S46C, T49C, and in blocking at substrate and sites in biotinylation was prevented by substrate Q61C, and of the residues in was blocked in a that biotinylation at the of For the and folinic acid a of in the biotinylation was at and for For S46C, L41C, and the were for both with at and For the were at folinic acid, of the biotinylation was whereas at was required to the that L41C, S46C, T49C, and are in with whereas Q54C, Q61C, and are of folinic acid biotin of replacements in the region between and a at the indicated sites were with the indicated of folinic acid prior to biotinylation at the indicated in the to were and were as in the to a protein; protein; protein; the expressed to folinic acid for The of the are indicated in of substrate of biotinylation of replacements in the region between and The from 6 and were to of the substrate required for of folinic The In this a cysteine-less form of the hamster RFC was generated by alanine replacement of the 14 cysteine residues. The RFC protein was to to the cell membrane and to reduced folates with slightly with the indicating the for this amino acid in RFC a functional cysteine-less form of the RFC has been generated Matherly L.H. Biochem. J. PubMed Google Scholar). The of a functional RFC the topology of the protein to be by cysteine scanning a less and less or The predicted extracellular location of specific regions was by the ability of the nonpermeable reagents to block biotinylation of residues at 46, and of these residues were as required either high of biotin maleimide for and K430C) or a for blocking and these sites are exposed to the extracellular environment, it appears that these regions may a as a or that is with is that with biotin maleimide and a for The that between and is exposed to the extracellular environment is of as this region an (8Ferguson P.L. Flintoff W.F. J. Biol. Chem. 1999; 274: 16269-16278Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). this to a protein and the of the location of this in the the replacement in a functional protein that the was A previous study of the RFC protein (9Liu X.Y. Matherly L.H. Biochim. Biophys. Acta. 2002; 1564: 333-342Crossref PubMed Scopus (41) Google Scholar) suggested that the C-terminal of the protein may demonstrate alternative from the predicted 12-transmembrane model, the region between and is exposed to the intracellular is the absence of N-glycosylation at a into this N-glycosylation is to sites 12 residues from the membrane J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). it is that this predicted loop may be to the membrane to be In the used a less to demonstrate that the between and in the hamster RFC protein is exposed to the extracellular The only support for this alternative topology is the of a region between and in a (9Liu X.Y. Matherly L.H. Biochim. Biophys. Acta. 2002; 1564: 333-342Crossref PubMed Scopus (41) Google Scholar). it is that are in topology between the and hamster RFC this the high of amino acid and of these the used in this biotin maleimide the is by the of biotinylation of regions exposed to the cytosol and in the form of the is to in J. R. J.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Full Text Full Text PDF PubMed Scopus Google Scholar), is in to J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The for this are may be cell of the cell membrane the biotinylation of sites predicted to be in cytosolic exposed biotinylation of residues in transmembrane regions has been to be to the of amino acids in this environment to biotin maleimide J. R. J.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), these the that sites and are in regions exposed to the cytosol and in a The in this with the (8Ferguson P.L. Flintoff W.F. J. Biol. Chem. 1999; 274: 16269-16278Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 9Liu X.Y. Matherly L.H. Biochim. Biophys. Acta. 2002; 1564: 333-342Crossref PubMed Scopus (41) Google Scholar) support the 12-transmembrane for the reduced folate indicated that a region in the predicted at residues may be in substrate and R. S. H. I. H.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, R. Goldman I.D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, L. I. Y. Biochem. J. 2002; 367: PubMed Scopus (70) Google Scholar). In this used scanning mutagenesis to that sites within a region from residues 39 to form a substrate or domain residues at sites S46C, T49C, and were with biotin and this modification could be prevented by prior treatment with the nonpermeable that these sites are and exposed to the extracellular environment, in to the of various that this region in R. S. H. I. H.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, R. Goldman I.D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, L. I. Y. Biochem. J. 2002; 367: PubMed Scopus (70) Google Scholar, R. I.G. Goldman I.D. Mol. Pharmacol. 1999; PubMed Scopus Google Scholar). Cysteines at sites Q61C, and required a of biotin maleimide for efficient Furthermore, of these sites Q61C, and required a nonpermeable to block suggesting that these regions may the cell and may a as a or that The and L70C sites required a high of biotin maleimide for efficient labeling, although could be blocked by although these sites are exposed to the extracellular environment, may have that In cysteines at sites and were either labeled or labeled at may be to a of the domain that or these sites are within the membrane J. R. J.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). could be biotinylation could be suggesting that it is may be of the as this replacement a a that to a in the amino acid it is that the of the region is of of replacements in the region between and in a The that biotinylation of residues at sites S46C, T49C, and be prevented by prior treatment with that this region a in substrate these of the carrier be in to the substrate to the with biotin was at of although blocking of L70C and required of It is to that of folinic acid were required to block suggesting that is a accessibility at this was for the folinic acid was a of biotinylation appears to be located the amino acid in this region residues and with Furthermore, the size of the that these are in to In a region between residues and have a in substrate although are exposed to the extracellular this region are a of sites that are exposed and and that the potential of this It is of to that is exposed to the extracellular environment, biotinylation is blocked by the of substrate a in the protein that the biotinylation to be The support to the that this region has a in substrate R. S. H. I. H.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, R. Goldman I.D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, L. I. Y. Biochem. J. 2002; 367: PubMed Scopus (70) Google Scholar, Matherly L.H. Biochem. J. PubMed Google Scholar). The of a functional RFC protein in the and of this and the size of the loops between the transmembrane segments. Furthermore, this be in with to the of various residues within this J. R. for

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