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Fig. 2. The Kaiso NLS maps to ten amino acids upstream of the zinc-finger domain. (A) Schematic representation showing deletion mutants of the Kaiso-4 construct to delineate further the Kaiso NLS; these deletion mutants were called Kaiso-4A, Kaiso-4B, Kaiso-4A1 and Kaiso-4A2, as indicated. (B,C) Interestingly, the Kaiso-4A and Kaiso-4A2 fusion proteins both localized to nuclei in HeLa cells, whereas Kaiso-4B and Kaiso-4A1 localized to the cytosol. The Kaiso-4A2 construct encoded a stretch of basic amino acids (amino acids 469-478) with the potential to serve as an NLS. (D) BLAST analysis revealed that the putative Kaiso NLS is highly conserved across diverse species such as M. musculus, H. sapiens and X. laevis. (E) Amino acids 469-478 (PPNKRMKVKH) of Kaiso constitute a functional NLS that is capable of targeting a heterologous ß-gal/GFP fusion protein to HeLa-cell nuclei. (F) A point mutation at a key basic residue within the Kaiso NLS (K472A) abolishes its ability to target the ß-gal/GFP fusion protein to nuclei. The images shown are representative of at least 100 cells observed for each construct in three independent trials. Bars, 20 µm.

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