Abstract
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, [3H]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 </=25% Gly-Sar uptake when compared with WT-hPepT1. P182C-hPepT1 showed approximately 40% 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 ac...Continue Reading
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