Mechanistic insight into the role of AUXIN RESISTANCE4 in trafficking of AUXIN1 and LIKE AUX1-2.

AUXIN RESISTANCE4 (AXR4) regulates trafficking of auxin influx carrier AUXIN1 (AUX1), a plasma-membrane protein that predominantly localises to the endoplasmic reticulum (ER) in the absence of AXR4. In Arabidopsis (Arabidopsis thaliana), AUX1 is a member of a small multigene family comprising four highly conserved genes - AUX1, LIKE-AUX1 (LAX1), LAX2 and LAX3. We report here that LAX2 also requires AXR4 for correct localization to the plasma membrane. AXR4 is a plant-specific protein and contains a weakly conserved α/β hydrolase fold domain that is found in several classes of lipid hydrolases and transferases. We have previously proposed that AXR4 may either act as 1) a post translational modifying enzyme through its α/β hydrolase fold domain or 2) an ER accessory protein, which is a special class of ER protein that regulates targeting of their cognate partner proteins. Here, we show that AXR4 is unlikely to act as a post-translational modifying enzyme as mutations in several highly conserved amino acids in the α/β hydrolase fold domain can be tolerated and active site residues are missing. We also show that AUX1 and AXR4 physically interact with each other and that AXR4 reduces aggregation of AUX1 in a dose-dependent fashion. Our results suggest that AXR4 acts as an ER accessory protein. Better understanding of AXR4 mediated trafficking of auxin transporters in crop plants will be crucial for improving root traits (designer roots) for better acquisition of water and nutrients for sustainable and resilient agriculture.