Figure 5.

TaMAB2 likely accumulates in the cytoplasm in active dimeric E3 ligase complexes. This is indicated by dimerization as well as interaction with CUL3 and presence of ubiquitin. (A) Y2H protein interaction assay of TaMAB2 with AtCUL3 and cytoskeletal proteins AtKAT, AtACT11, AtTUBG1, AtTUBG2, AtTUA6, and AtTUB8 in His prototrophy and β-galactosidase assays. For each experiment six individual colonies were analyzed. Specificity of the bait construct was confirmed by co-transformation with empty prey vectors (negative control). DMS3-RDM1 interaction served as a positive control. (B) TaMAB2-green fluorescent protein (GFP) and ubiquitin co-localize to cytoplasmic complexes in transgenic Arabidopsis protoplasts as shown by Duolink In Situ proximity ligation assay (PLA). Primary antibodies against ubiquitin and GFP were combined with secondary antibodies emitting a red fluorescent signal [Texas (TX) red] when both antibodies are in close proximity. Protoplast nuclei were stained with 4′,6-diamidino-2-phenylindole and visualized under UV light (B, top) and merged with TX red signals (B, bottom). A minimum of 30 protoplasts emitting a PLA signal was analyzed in three independent experiments. Protoplasts of transgenic plants overexpressing TaMAB2-GFP (line 82) are shown. Scale bar = 20 µm.