Extended Data Fig. 2: Subcellular localization, domain structures, interaction and ubiquitination assays of PUB30/31.

(a) PUB30/31-YFP signals could be detected at the PM in both meristemoids and pavement cells. Confocal microscopy of 6-d-old abaxial cotyledon epidermis of proPUB30::PUB30-YFP; pub30 pub31 or proPUB31::PUB31-YFP; pub30 pub31 transgenic lines. Scale bar, 10 µm. (b) Schematic diagram of the domains of PUB30 and PUB31. PUB30 and PUB31 contain a U-box domain, a linker domain, and an ARMADILLO (ARM) repeat domain. The light blue, orange and pink color rectangles indicate the U-box domain, the linker domain, and the ARM repeats domain, respectively. Scale bar: 100 aa. (c) PUB30 interacts with ER_CD in vitro. MBP-ER_CD was pulled down (PD) by GST-PUB30 immobilized on Glutathione Sepharose 4B and analyzed by immunoblotting (IB) using an anti-MBP antibody. GST and MBP were used as negative controls. (d) PUB31 interacts with ER_CD in vitro. (e) The alignment of PUB30 and PUB31. Protein sequences above light blue lines, orange lines, and pink lines represent U-box domain, linker domain, and ARM repeats domain, respectively. The amino acid position of PUB30 is labeled on the top. The Tryptophan 97 (W97) site in PUB30 and the conserved W93 site in PUB31 are labeled with #. The Threonine 155 (T155) site in PUB30 and the conserved T151 site in PUB31 are labeled with *. (f) PUB30 ubiquitinates ERECTA in vitro. The ubiquitination of MBP-ER_CD was carried out by using GST-fused PUB30 as the E3 ligase, His-fused AtUBA1 as E1 activating enzyme, and His-fused UBC8 as E2 conjugating enzyme. MBP was used as a control. (g) PUB31 ubiquitinates ERECTA in vitro.