N-terminal cysteines affect oligomer stability of the allosterically regulated ammonium transporter LeAMT1;1.
AMMONIUM TRANSPORTER (AMT) proteins are conserved in all domains of life and mediate the transport of ammonium or ammonia across cell membranes. AMTs form trimers and use intermolecular interaction between subunits to regulate activity. So far, binding forces that stabilize AMT protein complexes are not well characterized. High temperature or reducing agents released mono- and dimeric forms from trimeric complexes formed by AMT1;1 from Arabidopsis and tomato. However, in the paralogue LeAMT1;3, trimeric complexes were not detected. LeAMT1;3 differs from the other AMTs by an unusually short N-terminus, suggesting a role for the N-terminus in oligomer stability. Truncation of the N-terminus in LeAMT1;1 destabilized the trimer and led to loss of functionality when expressed in yeast. Swapping of the N-terminus between LeAMT1;1 and LeAMT1;3 showed that sequences in the N-terminus of LeAMT1;1 are necessary and sufficient for stabilization of the interaction among the subunits. Two N-terminal cysteine residues are highly conserved among AMT1 transporters in plants but are lacking in LeAMT1;3. C3S or C27S variants of LeAMT1;1 showed reduced complex stability, which coincided with lower transport capacity for the substrate analogue methylammonium. Both cysteine-substituted LeAMT1;1 variants showed weaker interactions with the wildtype as determined by a quantitative analysis of the complex stability using the mating-based split-ubiquitin assay. These data indicate that the binding affinity of AMT1 subunits is stabilized by cysteines in the N-terminus and suggest a role for disulphide bridge formation via apoplastic N-terminal cysteine residues.
Institute for Plant Nutrition, University of Hohenheim, D-70593 Stuttgart, Germany.
Lucile
L
Graff
Petr
P
Obrdlik
Lixing
L
Yuan
Dominique
D
Loqué
Wolf B
WB
Frommer
Nicolaus
N
von Wirén
D001692:Biological Transport; D027682:Cation Transport Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D017124:Conserved Sequence; D003545:Cysteine / Q000737:chemistry*; D018551:Solanum lycopersicum / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D025941:Protein Interaction Mapping; D055550:Protein Stability; D000644:Quaternary Ammonium Compounds / Q000378:metabolism
D027682:Cation Transport Proteins; D010940:Plant Proteins; D000644:Quaternary Ammonium Compounds; C088952:ammonium transporters, plant; D003545:Cysteine