Analysis of Transmembrane Segment 7 of the Dipeptide Transporter hPepT1 by Cysteine-scanning Mutagenesis

Abstract

To investigate the involvement of transmembrane segment 7 (TMS7) of hPepT1 in forming the putative central aqueous channel through which the substrate traverses, we individually mutated each of the 21 amino acids in TMS7 to a cysteine and analyzed the mutated transporters using the scanning cysteine accessibility method. Y287C-and M292C-hPepT1 did not express at the plasma membrane. Out of the remaining 19 transporters, three (F293C-, L296C-, and F297C-hPepT1) showed negligible glycyl-sarcosine (gly-sar) uptake activity and may play an important role in defining the overall hPepT1 structure. K278C-hPepT1 showed ϳ40% activity and the 15 other transporters exhibited more than 50% gly-sar uptake when compared with wild type (WT)-hPepT1. Gly-sar uptake for the 16 active transporters containing cysteine mutations was then measured in the presence of 2.5 mM 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA) or 1 mM [2-(trimethylammonium) ethyl] methanethiosulfonate bromide (MTSET). Gly-sar uptake was significantly inhibited for each of the 16 single cysteine mutants in the presence of 2.5 mM MTSEA. In contrast, significant inhibition of uptake was only observed for K278C-, M279C-, V280C-, T281C-, M284C-, L286C-, P291C-, and D298C-hPepT1 in the presence of 1 mM MTSET. MTSET modification of R282C-hPepT1 resulted in a significant increase in gly-sar uptake. To investigate this further, we mutated WT-hPepT1 to R282A-, R282E-, and R282K-hPepT1. R282E-hPepT1 showed a 43% reduction in uptake activity, whereas R282A-and R282K-hPepT1 had activities comparable with WT-hPepT1, suggesting a role for the Arg-282 positive charge in substrate translocation. Most of the amino acids that were MTSET-sensitive upon cysteine mutation, including R282C, are located toward the intracellular end of TMS7. Hence, our results suggest that TMS7 of hPepT1 is relatively solvent-accessible along most of its length but that the intracellular end of the transmembrane domain is particularly so. From a structure-function perspective, we speculate that the extracellular end of TMS7 may shift following substrate binding, providing the basis for channel opening and substrate translocation.The human dipeptide transporter, hPepT1, is a 708-amino acid protein containing 12 ␣-helical transmembrane segments.

Genetic Variants of the Human Dipeptide Transporter PEPT1

“…Previous studies have shown that tyrosine residues in positions 54, 65, and 167 seem to be involved in proton binding (Yeung et al, 1998;Chen et al, 2000). For position 282, a substitution of arginine to lysine did not alter PEPT1 transport activity, whereas substitution to glutamine resulted in the uncoupling of proton dependence (Kulkarni et al, 2003). Because the three-dimensional structure of PEPT1 is currently unknown, no one knows how these residues interact.…”

Association of PepT1 with lipid rafts differently modulates its transport activity in polarized and nonpolarized cells

“…Recently, it has been demonstrated that the transmembrane helix 7 of hPepT1 is important for hPepT1 transport activity (19). Interestingly, it was found that mutation at F293, F297, and L296 in transmembrane segment 7 of hPepT1 dramatically decreased the transport activity without affecting the delivery to membrane of this transporter (19). In contrast, substitution of T281 did not decrease hPepT1 transport activity (19).…”

“…Interestingly, it was found that mutation at F293, F297, and L296 in transmembrane segment 7 of hPepT1 dramatically decreased the transport activity without affecting the delivery to membrane of this transporter (19). In contrast, substitution of T281 did not decrease hPepT1 transport activity (19). It has also been hypothesized that some hydrophobic amino acid residues such as phenylalanine in transmembrane domains (TMDs) of transmembrane proteins may be important for their interaction with lipid rafts (41).…”

“…An important unanswered question in lipid raft research is how transmembrane proteins are targeted to lipid rafts. Recently, it has been demonstrated that the transmembrane helix 7 of hPepT1 is important for hPepT1 transport activity (19). Interestingly, it was found that mutation at F293, F297, and L296 in transmembrane segment 7 of hPepT1 dramatically decreased the transport activity without affecting the delivery to membrane of this transporter (19).…”

Functional and structural determinants of reverse operation in the pH-dependent oligopeptide transporter PepT1

“…The tyrosine residue Y167 has an important function as well, since its mutation to alanine completely abolished transport via PepT1 [15]. By using the cysteine scanning method, Kulkarni and coworkers [16] identified several residues important for the transport function in transmembrane segments 5 and 7 and suggested that these segments line the putative aqueous channel. Finally, the same authors identified two oppositely charged residues (R282 and D341) in transmembrane segments 7 and 8, respectively, that form a salt bridge and play an important role in the substrate translocation via PepT1 [17].…”