Research Article

Current Genetics

, Volume 49, Issue 6, pp 364-374

Structural involvement in substrate recognition of an essential aspartate residue conserved in Mep/Amt and Rh-type ammonium transporters

  • Anna Maria MariniAffiliated withLaboratoire de Physiologie Moléculaire de la Cellule, Institut de Biologie et de Médecine Moléculaires, Université Libre de Bruxelles CP300 Email author 
  • , Mélanie BoeckstaensAffiliated withLaboratoire de Physiologie Moléculaire de la Cellule, Institut de Biologie et de Médecine Moléculaires, Université Libre de Bruxelles CP300
  • , Fatine BenjellounAffiliated withInserm U 467, Université Paris V, Faculté de Médecine Necker-Enfants Malades
  • , Baya Chérif-ZaharAffiliated withInserm U 467, Université Paris V, Faculté de Médecine Necker-Enfants Malades
  • , Bruno AndréAffiliated withLaboratoire de Physiologie Moléculaire de la Cellule, Institut de Biologie et de Médecine Moléculaires, Université Libre de Bruxelles CP300

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Abstract

Ammonium transport proteins belonging to the Mep/Amt/Rh family are spread throughout all domains of life. A conserved aspartate residue plays a key role in the function of Escherichia coli AmtB. Here, we show that the analogous aspartate residue is critical for the transport function of eukaryotic family members as distant as the yeast transporter/sensor Mep2 and the human RhAG and RhCG proteins. In yeast Mep2, replacement of aspartate186 with asparagine produced an inactive transporter localized at the cell surface, whilst replacement with alanine was accompanied by stacking of the protein in the endoplasmic reticulum. Introduction of an acidic residue, glutamate, produced a partially active protein. A carboxyl group at position 186 of Mep2 therefore appears mandatory for function. Kinetic analysis shows the Mep2D186E variant to be particularly affected at the level of substrate affinity, suggesting an involvement of aspartate186 in ammonium recognition. Our data also put forward that ammonium recognition and/or transport by Mep2 is required for the sensor role played in the development of pseudohyphal growth. Finally, replacement of the conserved aspartate with asparagine in human RhAG and RhCG proteins resulted in the loss of bi-directional transport function. Hence, this aspartate residue might play a preserved functional role in Mep/Amt/Rh proteins.

Keywords

Ammonium Transport Pseudohyphae Yeast