Separase Protease Activity is Required for Cytokinesis in addition to Chromosome Segregation

Abstract Chromosomal segregation and cytokinesis are tightly regulated processes required for successful cell division. The cysteine protease separase cleaves a subunit of the cohesin complex to allow chromosome segregation at anaphase onset. Separase also regulates meiotic cortical granule exocytosis and vesicle trafficking during cytokinesis, both of which involve RAB-11. Separase has non-proteolytic signaling functions in addition to its role in substrate cleavage, and its mechanism in exocytosis is unknown. We sought to determine whether separase regulates RAB-11 vesicle exocytosis through a proteolytic or non-proteolytic mechanism. To address this question, we generated a protease-dead separase, SEP-1 PD ::GFP, and unexpectedly found that it is dominant negative. Consistent with its role in cohesin cleavage, SEP-1 PD ::GFP causes chromosome segregation defects. Depletion of the substrate subunit of cohesin rescues this defect, suggesting that SEP-1 PD ::GFP impairs cohesin cleavage by a substrate trapping mechanism. We investigated whether SEP-1 PD ::GFP also impairs RAB-11 vesicle trafficking. SEP-1 PD ::GFP causes a low rate of cytokinesis failure that is synergistically exacerbated by depletion of the core exocytic t-SNARE protein SYX-4. Interestingly, SEP-1 PD ::GFP causes an accumulation of RAB-11 vesicles at the cleavage furrow site and delayed the exocytosis of cortical granules during anaphase I. Depletion of syx-4 further enhanced RAB-11::mCherry and SEP-1 PD ::GFP plasma membrane accumulation during cytokinesis. These findings suggest that the protease activity of separase is required for the exocytosis of RAB-11 vesicles during cortical granule exocytosis and mitotic cytokinesis. Author Summary The defining event of cell division is the equal distribution of the genetic material to daughter cells. Once sister chromatids align on the metaphase plate, the cell releases the brakes to enter anaphase by activating the protease separase. Separase cleaves the cohesin glue holding duplicated sister chromatids together allowing chromosome segregation. Subsequently, the cell must orchestrate a complex series of anaphase events to equally partition the chromatids and the rest of the cellular components into two distinct daughter cells during cytokinesis. Separase has multiple functions during anaphase to help regulate several key events, including promoting vesicle exocytosis required for cytokinesis. Previous studies have shown that separase can exert control over different events either through substrate cleavage, or by triggering signaling pathways. Here we analyze the cellular functions of separase that are impacted by protease inactive separase. Our results show that separase cleaves cohesin to promote chromosome segregation and also cleaves another independent substrate to promote exocytosis. These findings provide a foundation for understanding the molecular control of separase in exocytosis and indicate that separase has multiple independent substrates that it must cleave to execute various functions. This mechanism may enable the cell to coordinate multiple anaphase events with chromosome segregation.