A FRET-based study reveals site-specific regulation of spindle position checkpoint proteins at yeast centrosomes

The spindle position checkpoint (SPOC) is a spindle pole body (SPB, equivalent of mammalian centrosome) associated surveillance mechanism that halts mitotic exit upon spindle mis-orientation. Here, we monitored the interaction between SPB proteins and the SPOC component Bfa1 by FRET microscopy. We show that Bfa1 binds to the scaffold-protein Nud1 and the γ-tubulin receptor Spc72. Spindle misalignment specifically disrupts Bfa1-Spc72 interaction by a mechanism that requires the 14-3-3-family protein Bmh1 and the MARK/PAR-kinase Kin4. Dissociation of Bfa1 from Spc72 prevents the inhibitory phosphorylation of Bfa1 by the polo-like kinase Cdc5. We propose Spc72 as a regulatory hub that coordinates the activity of Kin4 and Cdc5 towards Bfa1. In addition, analysis of spc72∆ cells shows that a mitotic-exit-promoting dominant signal, which is triggered upon elongation of the spindle into the bud, overrides the SPOC. Our data reinforce the importance of daughter-cell-associated factors and centrosome-based regulations in mitotic exit and SPOC control. DOI: eLife digest A cell must duplicate its genetic material and then separate the two copies before it divides. This process is carefully controlled so that each new cell receives an identical set of chromosomes after cell division. In budding yeast, new daughter cells grow as a bud on the side a larger mother cell and are eventually pinched off. A surveillance mechanism in budding yeast monitors the placement of the molecular machine (called the spindle) that separates the copies of the chromosomes. This mechanism then stops the cell from dividing if the spindle is not positioned correctly. Many of the components of this surveillance mechanism -- which is called the spindle position checkpoint -- associate with structures at the ends of the spindle. However, it was not clear how these components do this and how it helps them to check if the spindle is positioned correctly. Now, Gryaznova, Caydasi et al. have used a technique called FRET to answer these questions for an important component of the spindle position checkpoint, a protein called Bfa1. The main advantage of FRET is that it can be used to monitor changes in protein-protein interactions in living cells. This approach identified two proteins that provide sites for Bfa1 to bind to at the ends of the spindle. The experiments also showed that Bfa1 specifically detaches from one of these proteins (called Spc72) when the spindle position checkpoint is activated.