Fig. 4. Characterization and verification of nuclear export and nucleolar localization sequences by point mutations in full-length NIK. (A) To verify the functionality of the NES at position 795-805 that was previously identified with truncation mutants, point mutations of the conserved leucine or isoleucine residues were generated. Mutation of leucines at positions 803 and 805 to alanine induced a significant shift from the cytosol into the nucleus and accumulation in nucleoli. The same pattern was observed for variants in which isoleucine-800 or all hydrophobic amino acids of the predicted NES (NESmut) were replaced by alanine. (B) The NES mutant of full-length NIK accumulates in nucleoli and colocalizes with nucleolar CFP-L23 chimera. (C) Replacement of the basic amino acids RKKR at positions 143-146 with alanines in the NES-mutant form of NIK leads to nuclear localization with exclusion of nucleoli. (D) Mutation of all seven basic amino acids RKKRKKK at positions 143-149 of the NES mutant results in a predominant cytosolic localization.