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Papers In Press, published online ahead of print August 8, 2003
Department of Microbiology and Immunology, University of Western Ontario, London, Ontario N6A 5C1
Corresponding Author: flintoff{at}uwo.ca
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, K430C) could be labeled with biotin maleimide; this modification could be blocked by prior treatment with non-permeable reagents. Amino acids predicted to be within the cytosol (S152C, C224, L475C) could only be labeled after streptolysin O permeabilization. In addition, the cysteine-less RFC was exploited to evaluate a potential substrate binding domain as suggested by previous studies. Nineteen cysteine replacements were generated between residues 39 to 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 labelling, it appears that positions L41C, E45C, S46C, T49C, I66C, and L70C are exposed to the extracellular environment while postions Q54C, Q61C, and T63C are slightly less accessible. Cysteines at 39, 42, 44, 47, 51, and 73 are inefficiently biotinylated suggesting these sites are located in the membrane or within a tightly folded domain of the protein. Furthermore, biotinylation of cysteines at 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.
J. Biol. Chem, 10.1074/jbc.M302102200
Submitted on February 27, 2003
Revised on July 25, 2003
Accepted on August 8, 2003
The region between transmembrane domains 1 and 2 of the reduced folate carrier forms parts of the substrate binding pocket
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