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 the 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 Bub1p that disrupts the nuclear and kinetochore localization of Bub1p, Bub1p and Mad3p, but not Mad1p. 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 Bub1p has a NLS and a Nuclear Export Signal (NES) and that it shuttles between the cytoplasm and the nucleus. To identify the NLS anc NES in Bub1p, will examine the intracellular localization of GFP fused with different fragments of Bub1p and GFP fused with three putative NESs. Strains carrying mutations in importins, which are components of the nuclear protein transport machinery, will be used to identify which is/are necessary for the regulated transport of Bub1p into the nucleus. Finally, to identify factors that regulate the sub-cellular localization and/or function of Bub1p, I will isolate high copy suppressors of the sensitivity of bub1-A78V to miccotubule destabilization. FRAP (Fluorescence Recovery After photobleaching) studies, which measure protein mobility, will determine if these factors regulate the transport of soluble bub1p into the nucleus or the anchoring of Bub1p within the nucleus. Taken together, these studies will provide insight in how the intracellular localization and function of Bub1p is regulated. ? ?
|Gonzalez, Yanira; Saito, Akira; Sazer, Shelley (2012) Fission yeast Lem2 and Man1 perform fundamental functions of the animal cell nuclear lamina. Nucleus 3:60-76|
|Gonzalez, Yanira; Meerbrey, Kristen; Chong, Jennifer et al. (2009) Nuclear shape, growth and integrity in the closed mitosis of fission yeast depend on the Ran-GTPase system, the spindle pole body and the endoplasmic reticulum. J Cell Sci 122:2464-72|