Yeast Securin is trafficked through the endomembrane system to regulate cell cycle arrest during the DNA damage response

The yeast securin protein, Pds1, belongs to a class of highly conserved eukaryotic proteins that regulate the timing of chromatid segregation during mitosis by inhibiting separase, Esp1. During the metaphase to anaphase transition Pds1 is degraded by the ubiquitin-proteasome system in the nucleus, unleashing Esp1's protease activity to cleave cohesin surrounding sister chromatids. In response to DNA damage Pds1 is phosphorylated and stabilized, stalling mitotic progression to preserve genomic integrity. In addition, during the DNA damage checkpoint response, securin and separase are partially localized in the vacuole. Here we genetically dissect the requirements for securin's vacuolar localization and find that it is dependent on the Alkaline Phosphatase (ALP) endosomal transport pathway but not autophagy. Blocking retrograde traffic between the Golgi and the endoplasmic reticulum, by inhibiting COPI vesicle transport, drives Pds1 into the vacuole, whereas inhibiting antegrade transport by disrupting COPII-mediated traffic, results in the unexpected loss of Pds1 and the extinction of DNA damage-induced cell cycle arrest. We report that the induction of ER stress, either genetically or by treating cells with dithiothreitol, is sufficient to extinguish the DNA damage checkpoint, suggesting crosstalk between these two pathways. These data highlight new ways in which Pds1 and Esp1 are regulated during a DNA damage induced G2/M arrest and its requirement in the maintenance of the DNA damage checkpoint response.