Abstract
AtDUR3 represents the major transporter for high-affinity urea transport across the plasma membrane of nitrogen-deficient Arabidopsis roots. Although the physiological function of AtDUR3 in Arabidopsis root is well characterized, its biochemical property needs to be elucidated. Plasmalemma phosphoproteome analysis showed that AtDUR3 may have two potential phosphorylated amino acid residues; Tyr 566 and Ser 568 (Hem et al., 2007), suggesting a post-translational regulation of AtDUR3 activity by phosphorylation.
We substituted Asp and Ala for Tyr 566 and Ser 568 in AtDUR3 by site directed mutagenesis to mimic phosphorylated and dephosphorylated AtDUR3. When growth of transformed yeast strains were compared on the medium containing urea as sole nitrogen, a yeast strain harboring Y566D grew faster than a strain harboring wild-type while S568D grew slower. Furthermore we screened proteins that interact with AtDUR3 by mating based split-ubiquitin system (mbSUS) (Petr et al., 2004). We purchased a commercial Arabidopsis cDNA plasmid library, and then transferred the library into mbSUS vectors by Gateway reaction. Around 150 positive clones were isolated for further study.
