Given our interest in the APC/C and its downstream targets, we have been focusing our studies on an indirect target of the APC/C. Separase is the protease that cleaves the cohesin complex that holds homologs together at meiosis. Securin inhibits separase from carrying out this role until the metaphase to anaphase transition, at which time securin is ubiquitinated by the APC/C and degraded by the 26S proteasome.? ? We have taken a similar genetic approach to identify regulators and substrates of separase. We have four mutant alleles of sep-1 and have carried out a suppression screen with one of these alleles. which is temperature-sensitive. We have identified three suppressors that restore viability to sep-1 mutants at the non-permissive temperature. One of these mutants is an intragenic suppressor; the other two are extragenic. We have recently determined that one of these mutations is in a phosphatase gene. The mutation in this gene in an otherwise wild-type background has no obvious phenotypes. RNAi of this phosphatase gene and a deletion allele of this gene also suppresses the embryonic lethality of sep-1 alleles. To further understand how this phosphatase gene works in the separase pathway, we have undertaken a proteomics approach and have genertaed transgenic animals that express a TAP-tagged version of this phosphatase. Purification of the tagged phosphatase and its associated proteins, followed by mass spectometry, has revealed two interacting proteins. The biochemical and genetic characterization of these interactions is underway.? ? We are currently mapping the third suppressor so that we can molecularly identify it; we have it narrowed down to a small genetic interval.
| Bembenek, Joshua N; Richie, Christopher T; Squirrell, Jayne M et al. (2007) Cortical granule exocytosis in C. elegans is regulated by cell cycle components including separase. Development 134:3837-48 |
