Flintoff Wayne F, Williams Frederick M R, Sadlish Heather
Department of Microbiology and Immunology, University of Western Ontario, London, Ontario N6A 5C1, Canada.
J Biol Chem. 2003 Oct 17;278(42):40867-76. doi: 10.1074/jbc.M302102200. Epub 2003 Aug 8.
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.
通过丙氨酸替代14个半胱氨酸残基，生成了仓鼠还原型叶酸载体蛋白的无功能半胱氨酸形式。通过用半胱氨酸替代预测暴露于细胞外或胞质环境的选定氨基酸，研究了预测的12跨膜拓扑结构。这些半胱氨酸的位置通过它们在存在或不存在特定封闭剂的情况下对生物素马来酰亚胺的可及性来定义。预测暴露于细胞外环境的氨基酸（S46C、S179C、L300C、Y355C和K430C）可以用生物素马来酰亚胺标记；这种修饰可以通过用非渗透性试剂预先处理来阻断。预测位于胞质溶胶内的氨基酸（S152C、Cys224和L475C）只有在链球菌溶血素O通透后才能被标记。此外，如先前研究所暗示的，利用无半胱氨酸的还原型叶酸载体来评估潜在的底物结合结构域。在位于第一和第二跨膜区段之间的39至75位残基之间产生了19个半胱氨酸替代。从这些位点的生物素化以及各种试剂阻断这种标记的能力来看，似乎L41C、E45C、S46C、T49C、I66C和L70C暴露于细胞外环境，而Q54C、Q61C和T63C的可及性略低。半胱氨酸39、42、44、47、51和73的生物素化效率低下，表明这些位点位于膜内或蛋白质的紧密折叠结构域内。此外，用甲氨蝶呤或亚叶酸预先处理可以阻止半胱氨酸41、46、49、70和71的生物素化，表明这些位点构成底物结合口袋的一部分。
