Membrane Topology of the Human Dipeptide Transporter, hPEPT1, Determined by Epitope Insertions

Abstract

We have used epitope insertion to analyze the transmembrane topology of the human H+-dipeptide symporter hPEPT1. An epitope tag, EYMPME (EE), was inserted into different locations at amino acids 39, 78, 106, 412, and 708 of hPEPT1 by site-directed mutagenesis. The functional integrity of the tagged protein was tested by measuring its dipeptide transport activity in transfected Cos7 cells. Further, cells expressing hPEPT1 or EE-tagged hPEPT1 derivatives were labeled with an anti-EE-monoclonal antibody (anti-EE-mAb) or an antiserum raised against the carboxyl terminus of hPEPT1 (anti-hPEPT1) and examined by immunofluorescence confocal microscopy. EE106-, 412-, and 708-hPEPT1 transported the dipeptide tracer as well as wild-type hPEPT1. Tags at position 106 and 412 were shown to be extracellular because they were accessible to anti-epitope antibody in nonpermeabilized cells. In contrast, the carboxyl-terminal domain and EE708 were shown to be intracellular since they were only accessible to the antibodies in permeabilized cells. These results are consistent with a 12-transmembrane domain (TMD) topological model of PEPT1. Epitope insertions at regions linking the putative TMD1 and TMD2, and TMD2 and TMD3 (EE39- and EE78-hPEPT1), abolished the dipeptide transport into the cells. In transfected Cos7 cells, these tagged proteins remained largely intracellular rather than at the plasma membrane. These results suggest that the integrity of these regions is essential for transporter trafficking and/or function. Thus, the topology of the amino-terminal portion, including putative TMD1 and -2, remains to be clarified.

Human proton/oligopeptide transporter (POT) genes: Identification of putative human genes using bioinformatics

“…Most of the POT members share a common structural architecture with ~12 predicted transmembrane domains (TMDs), but among the dipeptide transporters in distant phyla, variations on this theme do occur. We have confirmed the transmembrane topology of at least a portion of the main intestinal transporter, hPepT1, using an epitope tagging approach (12).…”

Catalog of 238 variations among six human genes encoding solute carriers (hSLCs) in the Japanese population

“…Although mutations at the C/EBP sites markedly reduce transcriptional activity (Shiao et al 2000), we found no variations in this region in our test population. Among the four novel nonsynonymous polymorphisms, one, Gly419Ala in SLC15A1, was located in the large extracellular loop that carries potential N-glycosylation sites (Liang et al 1995;Covitz et al 1998). On the other hand, two polymorphisms in SLC22A1, Pro283Leu and Arg287Gly, were located in the intracellular loop that carries potential phosphorylation sites for protein kinase C (Zhang et al 1997).…”

Drug inhibition of Gly-Sar uptake and hPepT1 localization using hPepT1-GFP fusion protein

“…In situ hybridization detects hPepT1 in the tip of mucosal cell villi but not in the crypts. The predicted protein has 708 amino acids with 12 transmembrane domains (7,8). This peptide transporter accepts dipeptides and tripeptides as its substrates, but it cannot transport free amino acids (9).…”