Structural involvement in substrate recognition of an essential aspartate residue conserved in Mep/Amt and Rh-type ammonium transporters.
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 aspartate(186) 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 Mep2(D186E) variant to be particularly affected at the level of substrate affinity, suggesting an involvement of aspartate(186) 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.
Laboratoire de Physiologie Moléculaire de la Cellule, Institut de Biologie et de Médecine Moléculaires, Université Libre de Bruxelles CP300, Rue des Professeurs Jeener et Brachet, 12, 6041, Gosselies, Belgium. ammarini@dbm.ulb.ac.be
Anna Maria
AM
Marini
Mélanie
M
Boeckstaens
Fatine
F
Benjelloun
Baya
B
Chérif-Zahar
Bruno
B
André
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism; D001798:Blood Proteins / Q000378:metabolism*; D027682:Cation Transport Proteins / Q000235:genetics / Q000378:metabolism*; D017124:Conserved Sequence; D006801:Humans; D017136:Ion Transport / Q000235:genetics / Q000502:physiology; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D000644:Quaternary Ammonium Compounds / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D013379:Substrate Specificity / Q000235:genetics
D001798:Blood Proteins; D027682:Cation Transport Proteins; C107270:MEP2 protein, S cerevisiae; D008562:Membrane Glycoproteins; D000644:Quaternary Ammonium Compounds; C498523:RHAG protein, human; C413213:RHCG protein, human; D029701:Saccharomyces cerevisiae Proteins; D001224:Aspartic Acid