FIGURE 6.
Delineating the region of ABI5 required for KEG-mediated ubiquitination. A, amino acid sequence of ABI5 C-terminal region showing the positions of the deleted regions, NLS1 (bold and underlined), NLS2 (bold and italics), and NLS3 (bold). B–F, subcellular localization of the CFP-ABI5 deletion mutants in transiently transformed tobacco leaf epidermal cells. Fluorescence images are a combined series of Z-stack. G–P, BiFC assay in tobacco leaf epidermal cells. KEG-YN or KEGAA-YN was coexpressed with the indicated ABI5-YC deletion mutant. Numbers indicate the deleted (Δ) amino acids for each ABI5 mutant. Bars = 100 μm. Q, levels of each ABI5-YC deletion mutant following coexpression with KEG-YN. Analysis was done using protein extracts derived from the same tobacco leaf samples assayed for BiFC fluorescence shown in G–K. Note: ABI5-YC also contains a FLAG tag. Arrows indicate ABI5. Coomassie staining was used to confirm equal loading.