Mutational analysis of histidine residues in the human proton-coupled amino acid transporter PAT1.
The proton-coupled amino acid transporter 1 (PAT1) represents a major route by which small neutral amino acids are absorbed after intestinal protein digestion. The system also serves as a novel route for oral drug delivery. Having shown that H+ affects affinity constants but not maximal velocity of transport, we investigated which histidine residues are obligatory for PAT1 function. Three histidine residues are conserved among the H+-coupled amino acid transporters PAT1 to 4 from different animal species. We individually mutated each of these histidine residues and compared the catalytic function of the mutants with that of the wild type transporter after expression in HRPE cells. His-55 was found to be essential for the catalytic activity of hPAT1 because the corresponding mutants H55A, H55N and H55E had no detectable l-proline transport activity. His-93 and His-135 are less important for transport function since H93N and H135N mutations did not impair transport function. The loss of transport function of His-55 mutants was not due to alterations in protein expression as shown both by cell surface biotinylation immunoblot analyses and by confocal microscopy. We conclude that His-55 might be responsible for binding and translocation of H+ in the course of cellular amino acid uptake by PAT1.
Metzner
Linda
L
Membrane Transport Group, Biozentrum, Martin-Luther-University Halle-Wittenberg, D-06120 Halle, Germany.
Natho
Kristin
K
Zebisch
Katja
K
Dorn
Madlen
M
Bosse-Doenecke
Eva
E
Ganapathy
Vadivel
V
Brandsch
Matthias
M
D000595:Amino Acid Sequence; D026905:Amino Acid Transport Systems / Q000737:chemistry* / Q000378:metabolism*; D001692:Biological Transport; D002478:Cells, Cultured; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D018076:DNA, Complementary; D005455:Fluorescent Antibody Technique; D006639:Histidine / Q000235:genetics*; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D011392:Proline / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D027981:Symporters / Q000737:chemistry* / Q000378:metabolism*
D026905:Amino Acid Transport Systems; D018076:DNA, Complementary; D050505:Mutant Proteins; D011993:Recombinant Fusion Proteins; C475905:SLC36A1 protein, human; D027981:Symporters; D006639:Histidine; D011392:Proline