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Proper chromosome segregation is crucial for maintaining genomic stability, and dependent on separase, a conserved and essential cohesin protease. Securins are key regulators of separases, but remain elusive in many organisms due to sequence divergence. Here, we demonstrate that the separase homologue Esp1p in the ascomycete , an important pathogen of humans, is essential for chromosome segregation. However, lacks a sequence homologue of securins found in model ascomycetes. We sought a functional homologue through identifying Esp1p interacting factors. Affinity purification of Esp1p and mass spectrometry revealed Eip1p/Orf19.955p, an uncharacterized protein specific to species. Functional analyses demonstrated that Eip1p is important for chromosome segregation but not essential, and modulated in an APC-dependent manner, similar to securins. Eip1p is strongly enriched in response to MMS or HU treatment, and its depletion partially suppresses an MMS or HU-induced metaphase block. Further, Eip1p depletion reduces Mcd1p/Scc1p, a cohesin subunit and separase target. Thus, Eip1p may function as a securin. However, other defects in Eip1p-depleted cells suggest additional roles. Overall, the results introduce a candidate new securin, provide an approach for identifying these divergent proteins, reveal a putative anti-fungal therapeutic target, and highlight variations in mitotic regulation in eukaryotes. Movie S1 Movie S1 Time-lapse imaging of Htb1p-GFP demonstrating normal chromosome segregation in cells expressing . Strain SS44() was incubated in inducing medium for 5 h, followed by mounting on an 8-well μSlide in inducing medium. Images were captured every 5 min for 180 min. Movie S2 Movie S2 Time-lapse imaging of Htb1p-GFP in an Eip1p-depleted cell demonstrating uneven segregation of chromosomes within the mother cell. Strain SS44 () wasincubated in repressing medium for 5 h, followed by mounting on an 8-well μSlide in repressing medium. Images were captured every 5 min for 180 min. Movie S3 Movie S3 Time-lapse imaging of Htb1p-GFP in Eip1p-depleted cells demonstrating abnormal segregation of chromosomes or abnormal nuclear translocation between the mother and daughter cells. Strain SS44 () wasincubated in repressing medium for 5 h, followed by mounting on an 8-well μSlide in repressing medium. Images were captured every 5 min for 180 min. Movie S4 Movie S4 Time-lapse imaging of Tub2p-GFP demonstrating normal spindle assembly and disassembly during mitosis in cells expressing . Strain SS38 () was incubated in inducing medium for 5 h, followed by mounting on an 8-well μSlide in inducing medium. Images were captured every 5 min for 180 min. Movie S5 Movie S5 Time-lapse imaging of Tub2p-GFP in an Eip1p-depleted cell demonstrating maintenance and oscillations of an elongated spindle. Strain SS38 () wasincubated in repressing medium for 5 h, followed by mounting on an 8-well μSlide in repressing medium. Images were captured every 5 min for 180 min. Movie S6 Movie S6 Time-lapse imaging of Tub2p-GFP in an Eip1p-depleted cell demonstrating oscillations of spindle pole bodies between the mother and daughter cells. Strain SS38 () wasincubated in repressing medium for 5 h, followed by mounting on an 8-well μSlide in repressing medium. Images were captured every 5 min for 180 min.
This article was published in the following journal.
Name: Molecular biology of the cell
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Securin is involved in the control of the metaphase-anaphase transition during MITOSIS. It promotes the onset of anaphase by blocking SEPARASE function and preventing proteolysis of cohesin and separation of sister CHROMATIDS. Overexpression of securin is associated with NEOPLASTIC CELL TRANSFORMATION and tumor formation.
Separase is a caspase-like cysteine protease, which plays a central role in triggering ANAPHASE by cleaving the SCC1/RAD21 subunit of the cohesin complex. Cohesin holds the sister CHROMATIDS together during METAPHASE and its cleavage results in chromosome segregation.
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