Abstract
AtDUR3 represents the major transporter for high-affinity urea transport across the plasma membrane of nitrogen-deficient roots. Since ammonium derived from urea degradation might cause toxicity, the process of urea transport should be regulated. Although the physiological function of AtDUR3 is well characterized, its biochemical property needs to be elucidated. Plasmalemma phosphoproteome analysis and prediction analysis with topology showed that AtDUR3 may have 11 potential phosphorylated amino acid residues, suggesting a post-translational regulation of AtDUR3 activity by phosphorylation.
We substituted Asp and Ala for Tyr, Ser and Thr in AtDUR3 by site directed mutagenesis to mimic phosphorylation and dephosphorylation. Comparing the growth of transformed yeast strain on the medium containing urea as sole nitrogen, some of yeast strain harboring mutated AtDUR3 grew faster than a strain harboring wild-type while some grew slower. Furthermore we screened 11 candidate proteins that may interact with AtDUR3 by mating based split-ubiquitin system. The effect of amino acid substitution and interacting protein co-expression on isotope labeled urea uptake in yeast will be discussed.
