Drosophila PIM and THR are required for sister chromatid separation in mitosis and associate in vivo. Neither of these two proteins shares significant sequence similarity with known proteins. However, PIM has functional similarities with securin proteins. Like securin, PIM is degraded at the metaphase-to-anaphase transition and this degradation is required for sister chromatid separation. Securin binds and inhibits separase, a conserved cysteine endoprotease. Proteolysis of securin at the metaphase-to-anaphase transition activates separase, which degrades a conserved cohesin subunit, thereby allowing sister chromatid separation. To address whether PIM regulates separase activity or functions with THR in a distinct pathway, we have characterized a Drosophila separase homolog (SSE). SSE is an unusual member of the separase family. SSE is only about one-third the size of other separases and has a diverged endoprotease domain. However, our genetic analyses show that SSE is essential and required for sister chromatid separation during mitosis. Moreover, we show that SSE associates with both PIM and THR. Although our work shows that separase is required for sister chromatid separation in higher eukaryotes, in addition, it also indicates that the regulatory proteins have diverged to a surprising degree, particularly in Drosophila. A distinct hallmark of eukaryotes is their use of a microtubule-based spindle to segregate their genetic information onto two daughter cells during cell division. This mechanism necessitates regulated sister chromatid cohesion. Sister chromatids have to remain in association after DNA replication so that they can be recognized as such and oriented in the mitotic spindle during prometaphase. However, after their correct bipolar orientation in the mitotic spindle, cohesion has to be resolved so that sister chromatids can be segregated to opposite poles during anaphase.Because regulated sister chromatid cohesion is an essential element of eukaryotic cell divisions, its molecular basis is expected to be conserved. Most of our current mechanistic understanding of how sister chromatid cohesion is established during S phase, maintained until the end of metaphase, and resolved at the onset of anaphase, has been obtained with yeast (for recent reviews, see Dej and Orr
The centromere-specific histone H3 variant CENP-A plays a crucial role in kinetochore specification and assembly. We chose a genetic approach to identify interactors of the Drosophila CENP-A homolog CID. Overexpression of cid in the proliferating eye imaginal disk results in a rough eye phenotype, which is dependent on the ability of the overexpressed protein to localize to the kinetochore. A screen for modifiers of the rough eye phenotype identified mutations in the Drosophila condensin subunit gene Cap-G as interactors. Yeast two-hybrid experiments also reveal an interaction between CID and Cap-G. While chromosome condensation in Cap-G mutant embryos appears largely unaffected, massive defects in sister chromatid segregation occur during mitosis. Taken together, our results suggest a link between the chromatin condensation machinery and kinetochore structure.
We thank D. Tautz, R. Blackman and D. Maier for providing libraries and flies, J.-M. Peters and I. Waizenegger for human separase and securin cDNAs, and K. Neugebauer for technical help. We gratefully acknowledge support from the Deutsche Forschungsgemeinschaft (Le 987/2-1 and 3-3) and the FCI.
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