Abstract
Human BUB3 is a key mitotic checkpoint factor that recognizes centromeric components and recruits other mitotic checkpoint molecules to the unattached kinetochore. The key amino acid residues responsible for its localization are not yet defined. In this study, we identified a motif from Lys(216) to Lys(222) in BUB3 as its nuclear localization signal. A BUB3 mutant with deletion of this motif (Del216-222) was found to localize to both the cytoplasm and the nucleus, distinct from the exclusively nuclear distribution of wild-type BUB3. Further analysis revealed that residues Glu(213), Lys(216), Lys(217), Lys(218), Tyr(219), and Phe(221), but not Lys(222), contribute to nuclear localization. Interestingly, the nuclear localization signal was also critical for the kinetochore localization of BUB3. The deletion mutant Del216-222 and a subtle mutant with four residue changes in this region (E213Q/K216E/K217E/K218E (QE)) did not localize to the kinetochore efficiently or mediate mitotic checkpoint arrest. Protein interaction data suggested that the QE mutant was able to interact with BUB1, MAD2, and BubR1 but that its association with the centromeric components CENP-A and KNL1 was impaired. A motif from Leu(61) to Leu(65) in CENP-A was found to be involved in the association of BUB3 and CENP-A in cells; however, further assays suggested that CENP-A does not physically interact with BUB3 and does not affect BUB3 localization. Our findings help to dissect the mechanisms of BUB3 in mitotic checkpoint signaling.