Characterization of the SPOC regulatory function of BUD14

Saccharomyces cerevisiae, also known as the budding yeast, is a commonly used model organism to study the orientation of mitotic spindle and its effects on asymmetric cell division. The mitotic spindle of budding yeast needs to be oriented in the mother-to-daughter polarity axis in order to segregate one copy of its genomic DNA to the daughter cell during the cell cycle. Spindle Position Checkpoint (SPOC) is a surveillance mechanism that inhibits the progression of cell cycle upon spindle mispositioning. Cells with deficient SPOC lose their genomic integrity, eventually resulting in multiploidy and aneuploidy. Therefore, SPOC is necessary for the maintenance of ploidy in budding yeast. However, so far it has not been completely enlightened how the SPOC mechanism functions. In this thesis, we showed that Bud14 is a novel component of SPOC. We showed that deletion of BUD14 promotes the growth of cells with impaired mitotic exit, indicating Bud14 is an inhibitor of mitotic exit. We demonstrated that bud14Δ cells are SPOC deficient, accumulating multinucleated cell populations in the presence of spindle misalignment. Our data showed that SPOC regulatory function of Bud14 is not dependent on its role in formin-dependent actin polymerization regulation. We identified that Bud14-Glc7 interaction is required for the mitotic exit inhibitory function of Bud14 because a temperature sensitive mutant version of Glc7 (glc7-12) that cannot bind to Bud14 resulted in SPOC deficiency in the presence of BUD14 upon spindle misalignment. Our data indicate that Bud14-Glc7 limits the SPB-bound levels of Bfa1-Bub2 complex in metaphase and anaphase. We identified that Bud14 falls off from the bud cortex, spreading through the cell during late anaphase. We showed that phosphatase Bud14-Glc7 and the SPOC kinase Kin4 have different and additive molecular roles in SPOC. We proposed a model in which Bud14-Glc7 complex acts on Bfa1 with a parallel pathway to Kin4 in order to block the cell cycle progression in response to spindle misorientation.