Transmembrane segment 5 of the dipeptide transporter hPepT1 forms a part of the substrate translocation pathway
Transmembrane segment 5 of the dipeptide transporter hPepT1 forms a part of the substrate translocation pathway
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DOI：
10.1016/s0006-291x(03)00926-4
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发表时间：
2003-06-20
影响因子：
3.1
通讯作者：
Lee, VHL
中科院分区：
文献类型：
--
作者：
Kulkarni, AA;Haworth, IS;Lee, VHL
这项研究首次系统地研究了hPepT1跨膜片段5在形成水底物转运途径中的作用，这是不同物种中最保守的片段。我们将半胱氨酸扫描诱变与巯基特异性试剂MTSEA和MTSET结合使用。这两种试剂都不能降低HEK293细胞和爪蟾卵母细胞中野生型hpept1的转运活性。通过用半胱氨酸替换TMS5中的每个残基，在hPepT1中产生了21个单半胱氨酸突变。然后用每种突变蛋白转染HEK293细胞，测量稳态蛋白水平、[H-3]Gly-Sar摄取活性和对MTS试剂的敏感性。质膜上未表达S164C-、L168C-、G173C-和I179C-hPepT1。与WT-hPepT1相比，Y167C-， N171C-和S174C-hPepT1显示小于或等于25%的Gly-Sar摄取。与WT-hPepT1相比，P182C-hPepT1表现出类似于40%的特异性活性，而所有其他转运蛋白虽然对单一半胱氨酸突变仍然敏感，但表现出超过50%的特异性活性。2.5 mM MTSEA对F166C-、L176C-、S177C-、T178C-、I180C-、T181C-、P182C-hPepT1活性有部分抑制作用，对F163C-、I170C-hPepT1活性有完全抑制作用。F163C、I165C、F166C、A169C、I170C、S177C、T181C和P182C可以清晰地接触到1mm MTSET。总的来说，这些结果表明，TMS5排列在假定的水通道上，并且与通道的垂直轴略有倾斜，表面的一半形成，一个经典的两亲α -螺旋，细胞质的一半是高度溶剂可接近的。(C) 2003 Elsevier Science（美国）版权所有。
This study is the first systematic attempt to investigate the role of transmembrane segment 5 of hPepT1, the most conserved segment across different species, in forming a part of the aqueous substrate translocation pathway. We used cysteine-scanning mutagenesis in conjunction with the sulfhydryl-specific reagents, MTSEA and MTSET. Neither of these reagents reduced wild-type-hPepT1 transport activity in HEK293 cells and Xenopus oocytes. Twenty-one single cysteine mutations in hPepT1 were created by replacing each residue within TMS5 with a cysteine. HEK293 cells were then transfected with each mutated protein and the steady-state protein level, [H-3]Gly-Sar uptake activity, and sensitivity to the MTS reagents were measured. S164C-, L168C-, G173C-, and I179C-hPepT1 were not expressed on the plasma membrane. Y167C-, N171C-, and S174C-hPepT1 showed less than or equal to25% Gly-Sar uptake when compared with WT-hPepT1. P182C-hPepT1 showed similar to40% specific activity whereas all the remaining transporters, although still sensitive to single cysteine mutations, exhibited more than 50% specific activity when compared to WT-hPepT1. The activity of F166C-, L176C-, S177C-, T178C-, I180C-, T181C-, and P182C-hPepT1 was partially inhibited, while the activity of F163C- and I170C-hPepT1 was completely inhibited by 2.5 mM MTSEA. F163C, I165C, F166C, A169C, I170C, S177C, T181C, and P182C were clearly accessible to 1 mM MTSET. Overall, these results suggest that TMS5 lines the putative aqueous channel and is slightly tilted from the vertical axis of the channel, with the exofacial half forming, a classical amphipathic alpha-helix and the cytoplasmic half being highly solvent accessible. (C) 2003 Elsevier Science (USA). All rights reserved.