Fig. 7
OsAIRP2 exhibits E3 ubiquitin ligase activity and ubiquitinates OsPT4. A OsPT4 interacts with OsAIPR2, as proved by split-ubiquitin Y2H assays. Interaction between the bait APP with Fe65 as a positive control. The coexpression of TSU2-APP and PR3-N regarded as the negative controls. B OsPT4 interacts with OsAIPR2 on the plasma membrane, as proved by BiFC analysis. N- and C-terminal fragments of YFP (YFPN and YFPC) were fused to the N terminus of OsPT4 and OsAIPR2, respectively. The yellow signals indicate YFP, the red signal indicates the plasma membrane specifically stained with dye FM4-64, and the orange signals indicate the overlapping part of YFP signal and FM4-64 staining. Bars = 20 μm. NubG, the mutated N-terminal fragment of ubiquitin; X-gal, a substrate of the bacterial enzyme β-galactosidase encoded by the color reporter gene lacZ; FM4-64, BF, bright field. C E3 Ub ligase activity of OsAIRP2. D In vitro ubiquitination of OsPT4 protein. The numbers on the left indicate the molecular masses of marker proteins in kD