title: The region between transmembrane domains 1 and 2 of the reduced folate carrier forms part of the substrate-binding pocket.
abstract: 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.
authors.affiliation: Department of Microbiology and Immunology, University of Western Ontario, London, Ontario N6A 5C1, Canada. flintoff@uwo.ca
authors.forename: Wayne F
authors.initials: WF
authors.lastname: Flintoff
authors.forename: Frederick M R
authors.initials: FM
authors.lastname: Williams
authors.forename: Heather
authors.initials: H
authors.lastname: Sadlish
mesh_terms: D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins; D001710:Biotin / Q000737:chemistry / Q000494:pharmacology; D019921:Biotinylation; D002352:Carrier Proteins / Q000737:chemistry*; D002462:Cell Membrane / Q000378:metabolism*; D006224:Cricetinae; D003545:Cysteine / Q000737:chemistry; D003600:Cytosol / Q000378:metabolism; D005492:Folic Acid / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D026901:Membrane Transport Proteins; D008727:Methotrexate / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D058978:Reduced Folate Carrier Protein; D013301:Streptolysins / Q000494:pharmacology
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D008164:Luminescent Proteins; D026901:Membrane Transport Proteins; D011993:Recombinant Fusion Proteins; D058978:Reduced Folate Carrier Protein; D013301:Streptolysins; C025964:streptolysin O; D049452:Green Fluorescent Proteins; D012313:RNA; D001710:Biotin; D005492:Folic Acid; D003545:Cysteine; D000409:Alanine; D008727:Methotrexate