Effect of Bud3 phosphorylation site mutations on bud site selection and cell cycle progression in Saccharomyces cerevisiae

Abstract

In Saccharomyces cerevisiae, the protein Bud3 is important in bud site selection. It localizes to the mother-bud neck, where it promotes the axial pattern of budding, wherein a new bud forms next to the bud scar left by the previous cell division. It also localizes the cyclin protein Clb2 to the bud neck, which is important for normal cell cycle progression, including mitotic exit and cytokinesis. We studied the effects of Bud3 phosphorylation site mutations on bud site selection and cell cycle progression. Our study included both alanine mutants, which are nonphosphorylatable, and aspartate/glutamate mutants, which constitutively mimic phosphorylation. To observe bud site selection, we used calcofluor white staining and fluorescence microscopy to visualize bud scars for single, double, and triple mutants. For cell cycle progression, we arrested the cells in G1 using alpha-factor mating pheromone, released them from the arrest, and analyzed budding state over time from 0-180 min using brightfield microscopy. Our results indicate that phosphorylation of Bud3 has a small, but statistically significant effect on bud site selection. The triple alanine mutation combination resulted in decreased axial budding, while aspartate/glutamate mutants showed no significant change in budding pattern. Additionally, we found that phosphorylation does affect cell cycle progression: the triple alanine mutant strain had a delay in budding after release from G1 arrest that persisted through the following cell cycle; in contrast, the triple aspartate/glutamate phosphomimetic mutant strain budded with normal timing and actually progressed more quickly through the cell cycle than the wildtype (WT) strain, possibly indicating division at a smaller-than-normal cell size.