Mutational analysis of histidine residues in the human proton-coupled amino acid transporter PAT1
| Autor: | Vadivel Ganapathy, Kristin Natho, Matthias Brandsch, Madlen Dorn, Katja Zebisch, Linda Metzner, Eva Bosse-Doenecke |
|---|---|
| Rok vydání: | 2007 |
| Předmět: |
DNA
Complementary Amino Acid Transport Systems Proline Protein digestion Recombinant Fusion Proteins Mutant Amino acid transport DNA Mutational Analysis Immunoblotting Molecular Sequence Data Biophysics Fluorescent Antibody Technique Biology Biochemistry Substrate Specificity Humans Histidine Amino acid transporter Amino Acid Sequence Cells Cultured Conserved Sequence chemistry.chemical_classification PAT1 Symporters Wild type Transporter Biological Transport Cell Biology Histidine residues Amino acid chemistry Biotinylation Drug delivery Mutant Proteins Sequence Alignment Site directed mutagenesis |
| Zdroj: | Biochimica et biophysica acta. 1778(4) |
| ISSN: | 0006-3002 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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