Regulation of the Spindle Checkpoint Protein Bub1p

Baylor College Of Medicine, Houston TX

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Abstract

The Spindle Assembly Checkpoint (SAC) arrests the cell cycle during metaphase, if the chromosomes are not properly bound to the spindle microtubules, and thereby helps to prevent loss of genetic material and aneuploidy. How he Mad and Bub proteins regulate the SAC is not fully understood, but it has been demonstrated that they accumulate at unattached kinetochores during SAC activation and bring about the metaphase arrest. Interestingly, our lab has identified a mutation in the Mad3-like region (bub1-A78V) of the essential protein kinase Bublp that disrupts the nuclear and kinetochore localization of Bublp, BubSp and Mad3p, but not Madlp. When a Nuclear Localization Signal (NLS] was fused to bub1-A78V, it failed to restore the nuclear wild type levels of Bublp. I propose that Bublp has a NLS and a Nuclear Export Signal (NES) and that it shuttles between the cytoplasm and the nucleus. To identify the NLSanc NES in Bublp, I will examine the intracellular localization of GFP fused with different fragments of Bublp and GFP fused with three putative NESs. Strains carrying mutations in importins, which are components of the nuclear protein transport machinery, will be usedto identify which is/are necessary for the regulated transport of Bub1 p into the nucleus. Finally, to identify factors that regulate the sub-cellular localization and/or function of Bublp, I will isolate high copy suppressors of the sensitivity of bub1-A78V to miccotubule destabilization. FRAP (Fluorescence Recovery After 3hotobleaching) studies, which measure protein mobility, will determine if these factors regulate the transport of soluble 3ub1p into the nucleus or the anchoring of Bublp withinthe nucleus. Taken together, these studies will provide insight in how the intracellular localization and function of Bublp is regulated.