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Analysis of the membrane topology for transmembrane domains 7-12 of the human reduced folate carrier by scanning cysteine accessibility methods.

机译:通过扫描半胱氨酸可及性方法分析人还原叶酸载体跨膜域7-12的膜拓扑。

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摘要

The hRFC (human reduced folate carrier) is the major membrane transporter for both reduced folates and antifolates in human tissues and tumours. The primary amino acid sequence of hRFC predicts a membrane topology involving 12 TMDs (transmembrane domains) with cytosolic oriented N- and C-termini, and a large internal loop connecting TMDs 6 and 7. Previous studies using haemagglutinin epitope insertion and scanning glycosylation mutagenesis methods verified portions of the predicted topology model, including TMDs 1-8 and the N- and C-termini of hRFC. However, the topology structure for TMDs 9-12 remains controversial. To further determine the membrane topology of the hRFC protein, single cysteine residues were introduced into the predicted extracellular or cytoplasmic loops of a fully functional cysteine-less hRFC expressed in transport impaired MtxRIIOua(R)2-4 Chinese hamster ovary cells. The membrane orientations of the substituted cysteines were determined by treatments with the thiol reagents 3-(N-maleimidylpropionyl)-biocytin (biotin maleimide) and 4-acetamido-4'maleimidylstilbene-2,2'-disulphonic acid (stilbenedisulphonate maleimide; SM) or N-ethylmaleimide, combined with the cell-permeabilizing reagent SLO (streptolysin O). We found that cysteine residues placed in the predicted extracellular loops between TMDs 7 and 8 (position 301), 9 and 10 (360), and 11 and 12 (429) could be biotinylated with 200 microM biotin maleimide, and labelling could be blocked with SM. However, biotinylation of cysteines placed in the predicted intracellular loops between TMDs 8 and 9 (position 332) and TMDs 10 and 11 (position 388) was only detected after cell permeabilization with SLO and was abolished by pre-treatment with N -ethylmaleimide. These results strongly support a 12-TMD topology structure for the hRFC protein.
机译:hRFC(人类还原性叶酸载体)是人体组织和肿瘤中还原性叶酸和抗叶酸的主要膜转运蛋白。 hRFC的主要氨基酸序列可预测涉及12个TMD(跨膜结构域)和胞质定向N-和C-末端的大分子膜拓扑结构,以及连接TMD 6和7的大内部环。以前使用血凝素表位插入和扫描糖基化诱变方法进行的研究验证的预测拓扑模型部分,包括TMD 1-8和hRFC的N和C末端。但是,TMD 9-12的拓扑结构仍存在争议。为了进一步确定hRFC蛋白的膜拓扑,将单个半胱氨酸残基引入到运输受损的MtxRIIOua(R)2-4中国仓鼠卵巢细胞中表达的功能齐全的无半胱氨酸的hRFC的预测细胞外或细胞质环中。通过用硫醇试剂3-(N-马来酰亚胺基丙酰基)-生物cytin(生物素马来酰亚胺)和4-乙酰氨基-4'马来酰亚胺基苯乙烯-2,2'-二磺酸(stilbenedisulphonate maleimide; SM)处理来确定取代的半胱氨酸的膜取向或N-乙基马来酰亚胺,与细胞通透性试剂SLO(链球菌溶血素O)结合使用。我们发现,放置在TMD 7和8(位置301),9和10(360),11和12(429)之间的预测细胞外环中的半胱氨酸残基可以被200 microM生物素马来酰亚胺生物素化,并且标记可以被SM。但是,仅在用SLO透化细胞后,才能检测到位于TMD 8和9(位置332)与TMD 10和11(位置388)之间的预测细胞内环中的半胱氨酸的生物素化作用,并且通过N-乙基马来酰亚胺预处理消除了半胱氨酸的生物素化作用。这些结果强烈支持hRFC蛋白的12-TMD拓扑结构。

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