Membrane topology of the human dipeptide transporter, hPEPT1, determined by epitope insertions.
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.
Covitz
K M
KM
Department of Biopharmaceutical Sciences, University of California, San Francisco 94143-0446, USA.
Amidon
G L
GL
Sadée
W
W
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001483:Base Sequence; D016466:CHO Cells; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002463:Cell Membrane Permeability; D006224:Cricetinae; D004151:Dipeptides / Q000378:metabolism*; D000939:Epitopes / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005110:Extracellular Space / Q000378:metabolism; D019085:Fluorescent Antibody Technique, Direct; D006801:Humans; D007106:Immune Sera / Q000378:metabolism; D007424:Intracellular Fluid / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000378:metabolism; D000074011:Peptide Transporter 1; D027981:Symporters; D014162:Transfection
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D004151:Dipeptides; D000939:Epitopes; D007106:Immune Sera; D008565:Membrane Proteins; D010446:Peptide Fragments; D000074011:Peptide Transporter 1; C082186:SLC15A1 protein, human; D027981:Symporters