Identification of a disulfide bridge essential for transport function of the human proton-coupled amino acid transporter hPAT1.
The proton-coupled amino acid transporter 1 (PAT1, SLC36A1) mediates the uptake of small neutral amino acids at the apical membrane of intestinal epithelial cells after protein digestion. The transporter is currently under intense investigation, because it is a possible vehicle for oral drug delivery. Structural features of the protein such as the number of transmembrane domains, the substrate binding site, or essential amino acids are still unknown. In the present study we use mutagenesis experiments and biochemical approaches to determine the role of the three putative extracellular cysteine residues on transport function and their possible involvement in the formation of a disulfide bridge. As treatment with the reducing reagent dithiothreitol impaired transport function of hPAT1 wild type protein, substitution of putative extracellular cysteine residues Cys-180, Cys-329, and Cys-473 by alanine or serine was performed. Replacement of the two highly conserved cysteine residues Cys-180 and Cys-329 abolished the transport function of hPAT1 in Xenopus laevis oocytes. Studies of wild type and mutant transporters expressed in human retinal pigment epithelial (HRPE) cells suggested that the binding of the substrate was inhibited in these mutants. Substitution of the third putative extracellular nonconserved cysteine residue Cys-473 did not affect transport function. All mutants were expressed at the plasma membrane. Biotinylation of free sulfhydryl groups using maleimide-PEG(11)-biotin and SDS-PAGE analysis under reducing and nonreducing conditions provided direct evidence for the existence of an essential disulfide bond between Cys-180 and Cys-329. This disulfide bridge is very likely involved in forming or stabilizing the substrate binding site.
Dorn
Madlen
M
Institute of Biochemistry/Biotechnology, Faculty of Science I, D-06120 Halle; Biozentrum, Martin-Luther-University Halle-Wittenberg, D-06120 Halle.
Weiwad
Matthias
M
Max-Planck Research Unit-Enzymology of Protein Folding, Weinbergweg 22, D-06120 Halle.
Markwardt
Fritz
F
Julius-Bernstein-Institute for Physiology, Faculty of Medicine, Martin-Luther-University Halle-Wittenberg, D-06097 Halle, Germany.
Laug
Linda
L
Biozentrum, Martin-Luther-University Halle-Wittenberg, D-06120 Halle.
Rudolph
Rainer
R
Institute of Biochemistry/Biotechnology, Faculty of Science I, D-06120 Halle.
Brandsch
Matthias
M
Biozentrum, Martin-Luther-University Halle-Wittenberg, D-06120 Halle.
Bosse-Doenecke
Eva
E
Institute of Biochemistry/Biotechnology, Faculty of Science I, D-06120 Halle.
D000595:Amino Acid Sequence; D026905:Amino Acid Transport Systems / Q000737:chemistry* / Q000502:physiology; D000818:Animals; D001665:Binding Sites; D004220:Disulfides; D004229:Dithiothreitol / Q000494:pharmacology; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000378:metabolism; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D027981:Symporters / Q000737:chemistry* / Q000502:physiology; D014982:Xenopus laevis
D026905:Amino Acid Transport Systems; D004220:Disulfides; C475905:SLC36A1 protein, human; D027981:Symporters; D004229:Dithiothreitol