Homo- and hetero-oligomerization of ammonium transporter-1 NH4 uniporters.
In most organisms, high affinity ammonium uptake is catalyzed by members of the ammonium transporter family (AMT/MEP/Rh). A single point mutation (G458D) in the cytosolic C terminus of the plasma membrane transporter LeAMT1;1 from tomato leads to loss of function, although mutant and wild type proteins show similar localization when expressed in yeast or plant protoplasts. Co-expression of LeAMT1;1 and mutant in Xenopus oocytes inhibited ammonium transport in a dominant negative manner, suggesting homo-oligomerization. In vivo interaction between LeAMT1;1 proteins was confirmed by the split ubiquitin yeast two-hybrid system. LeAMT1;1 is isolated from root membranes as a high molecular mass oligomer, converted to a approximately 35-kDa polypeptide by denaturation. To investigate interactions with the LeAMT1;2 paralog, co-localizing with LeAMT1;1 in root hairs, LeAMT1;2 was characterized as a lower affinity NH4+ uniporter. Co-expression of wild types with the respective G458D/G465D mutants inhibited ammonium transport in a dominant negative manner, supporting the formation of heteromeric complexes in oocytes. Thus, in yeast, oocytes, and plants, ammonium transporters are able to oligomerize, which may be relevant for regulation of ammonium uptake.
Ludewig
Uwe
U
Zentrum für Molekularbiologie der Pflanzen, Pflanzenphysiologie, Universität Tübingen, Auf der Morgenstelle 1, 72076 Tübingen, Germany.
Wilken
Stephanie
S
Wu
Binghua
B
Jost
Wolfgang
W
Obrdlik
Petr
P
El Bakkoury
Mohamed
M
Marini
Anne-Marie
AM
André
Bruno
B
Hamacher
Tanja
T
Boles
Eckhard
E
von Wirén
Nicolaus
N
Frommer
Wolf B
WB
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry*; D027682:Cation Transport Proteins; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004594:Electrophysiology; D005260:Female; D005799:Genes, Dominant; D049452:Green Fluorescent Proteins; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D018551:Solanum lycopersicum; D008744:Methylamines / Q000737:chemistry; D008969:Molecular Sequence Data; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry*; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D000644:Quaternary Ammonium Compounds; D018075:RNA, Complementary / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D014981:Xenopus
D002352:Carrier Proteins; D027682:Cation Transport Proteins; D008164:Luminescent Proteins; D008744:Methylamines; D010455:Peptides; D010940:Plant Proteins; D000644:Quaternary Ammonium Compounds; D018075:RNA, Complementary; C088952:ammonium transporters, plant; D049452:Green Fluorescent Proteins; C027451:methylamine