Characterization of Bud14 function in yeast centrosome size maintenance

The cell cycle is an extensively conserved process that governs DNA duplication and chromosome segregation. Eukaryotic cells distribute their chromosomes into daughter cells by the mitotic spindle, formed between spindle poles named centrosome. The Spindle Pole Body (SPB) is a functional equivalent of mammalian centrosome in S. cerevisiae. SPB consists of distinct structural layers and is embedded into a nuclear envelope throughout its life cycle, thereby nucleating cytoplasmic and nuclear microtubules. The yeast centrosome size changes throughout the cell cycle. In addition, cell cycle arrest conditions have various effects on SPB size. Recent studies illustrated that SPB undergoes growth and exchange cycles, thus portraying a dynamic yeast centrosome to an extent that was not anticipated previously. In this study, we have identified a novel role for Bud14 in SPB size maintenance. Through microscopy-based quantification of relative and exact amounts of SPB structural proteins, we showed that cells lacking Bud14 had more Spc72, Nud1, Spc97, Spc42, Spc29, and Spc110 at their SPBs. Bud14 is also a regulatory subunit of the Protein Phosphatase 1 Glc7. We indicated that Bud14 function in Spc29, Spc97, Spc110, and Spc42 size maintenance depends on its interaction with Glc7. We elucidated Spc110 and Spc72 dynamics by measuring their molecule numbers throughout the cell cycle. This dynamic nature might be integral to the mitotic spindle formation and presents a mechanism for reacting to changes in cellular conditions. Furthermore, this research established that functional Cdc14 phosphatase is required for Spc72 and Spc110 size maintenance in cells lacking Bud14. Forceful localization of Cdc28 to SPB outer plaque disclosed that phosphorylation of SPB structural proteins causes their enlargement at the SPB. This research determined that Bud14 regulates SPB size primarily via its interaction with Glc7. Cdc14 also functions in the SPB size maintenance network.