Liya Xu1, Ian S Haworth, Ashutosh A Kulkarni, Michael B Bolger, Daryl L Davies. 1. Alcohol and Brain Research Laboratory, Titus Family Department of Clinical Pharmacy and Pharmaceutical Economics & Policy, School of Pharmacy, University of Southern California, Los Angeles, California 90033, USA.
Abstract
PURPOSE: The human dipeptide transporter (hPEPT1) facilitates transport of dipeptides and drugs from the intestine into the circulation. The role of transmembrane domain 10 (TMD10) of hPEPT1 in substrate translocation was investigated using cysteine-scanning mutagenesis with 2-Trimethylammonioethyl methanethiosulfonate (MTSET). METHODS: Each amino acid in TMD10 was mutated individually to cysteine, and transport of [(3)H]Gly-Sar was evaluated with and without MTSET following transfection of each mutant in HEK293 cells. Similar localization and expression levels of wild type (WT) hPEPT1 and all mutants were confirmed by immunostaining and biotinylation followed by western blot analysis. RESULTS: E595C- and G594C-hPEPT1 showed negligible Gly-Sar uptake. E595D-hPEPT1 showed similar uptake to WT-hPEPT1, but E595K- and E595R-hPEPT1 did not transport Gly-Sar. Double mutations E595K/R282E and E595R/R282E did not restore uptake. G594A-hPEPT1 showed similar uptake to WT-hPEPT1, but G594V-hPEPT1 eliminated uptake. Y588C-hPEPT1 showed uptake of 20% that of WT-hPEPT1. MTSET modification supported a model of TMD10 with an amphipathic helix from I585 to V600 and increased solvent accessibility from T601 to F605. CONCLUSIONS: Our results suggest that G594 and E595 in TMD10 of hPEPT1 have key roles in substrate transport and that Y588 may have an important secondary mechanistic role.
PURPOSE: The humandipeptide transporter (hPEPT1) facilitates transport of dipeptides and drugs from the intestine into the circulation. The role of transmembrane domain 10 (TMD10) of hPEPT1 in substrate translocation was investigated using cysteine-scanning mutagenesis with 2-Trimethylammonioethyl methanethiosulfonate (MTSET). METHODS: Each amino acid in TMD10 was mutated individually to cysteine, and transport of [(3)H]Gly-Sar was evaluated with and without MTSET following transfection of each mutant in HEK293 cells. Similar localization and expression levels of wild type (WT) hPEPT1 and all mutants were confirmed by immunostaining and biotinylation followed by western blot analysis. RESULTS: E595C- and G594C-hPEPT1 showed negligible Gly-Sar uptake. E595D-hPEPT1 showed similar uptake to WT-hPEPT1, but E595K- and E595R-hPEPT1 did not transport Gly-Sar. Double mutations E595K/R282E and E595R/R282E did not restore uptake. G594A-hPEPT1 showed similar uptake to WT-hPEPT1, but G594V-hPEPT1 eliminated uptake. Y588C-hPEPT1 showed uptake of 20% that of WT-hPEPT1. MTSET modification supported a model of TMD10 with an amphipathic helix from I585 to V600 and increased solvent accessibility from T601 to F605. CONCLUSIONS: Our results suggest that G594 and E595 in TMD10 of hPEPT1 have key roles in substrate transport and that Y588 may have an important secondary mechanistic role.
Authors: A K Yeung; S K Basu; S K Wu; C Chu; C T Okamoto; S F Hamm-Alvarez; H von Grafenstein; W C Shen; K J Kim; M B Bolger; I S Haworth; D K Ann; V H Lee Journal: Biochem Biophys Res Commun Date: 1998-09-08 Impact factor: 3.575
Authors: R Liang; Y J Fei; P D Prasad; S Ramamoorthy; H Han; T L Yang-Feng; M A Hediger; V Ganapathy; F H Leibach Journal: J Biol Chem Date: 1995-03-24 Impact factor: 5.157
Authors: Fatma Guettou; Esben M Quistgaard; Lionel Trésaugues; Per Moberg; Caroline Jegerschöld; Lin Zhu; Agnes Jin Oi Jong; Pär Nordlund; Christian Löw Journal: EMBO Rep Date: 2013-07-19 Impact factor: 8.807
Authors: Paola Pisani; Maria Daniela Renna; Francesco Conversano; Ernesto Casciaro; Marco Di Paola; Eugenio Quarta; Maurizio Muratore; Sergio Casciaro Journal: World J Orthop Date: 2016-03-18
Authors: Simon Newstead; David Drew; Alexander D Cameron; Vincent L G Postis; Xiaobing Xia; Philip W Fowler; Jean C Ingram; Elisabeth P Carpenter; Mark S P Sansom; Michael J McPherson; Stephen A Baldwin; So Iwata Journal: EMBO J Date: 2010-12-03 Impact factor: 11.598
Authors: Nicolae Solcan; Jane Kwok; Philip W Fowler; Alexander D Cameron; David Drew; So Iwata; Simon Newstead Journal: EMBO J Date: 2012-06-01 Impact factor: 11.598