The characterization of separase suppressors

鉴于我们对APC/C及其下游目标的兴趣，我们一直将研究重点放在APC/C的间接目标上。分离酶是在减数分裂过程中分裂将同源物结合在一起的内聚蛋白复合体的蛋白酶。Securin抑制分离酶发挥这一作用，直到中期到后期转变，此时Securin被APC/C泛素化，并被26S蛋白酶体降解。

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 genetic approach to identify regulators and substrates of separase. We have three mutant alleles of sep-1 and have recently shown, through a collaboration with Dr. Joshua Bembenek, that these mutants all have defects in cortical granule exocytosis (CGE). CGE is important for the secretion of components necessary for eggshell formation, which is indirectly required for proper polar body extrusion. In order to identify other genes that function in the separase pathway, we carried out a suppression screen with a temperature-sensitive allele of sep-1, e2406ts. 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 suppressor mutations is in a phosphatase gene called pph-5. This phosphatase mutant, in an otherwise wild-type background, has no obvious phenotypes on its own. Our pph-5 allele, av101, suppresses the embryonic lethality of two of our three sep-1 alleles, as does a deletion allele of the pph-5 gene. RNAi of pph-5 also suppresses the embryonic lethality of two of our three sep-1 alleles. RNAi depletion or genetic mutation of pph-5 restores CGE to wildtype levels in the suppressed sep-1 backgrounds. To further understand how PPH-5 works in the separase pathway, we have undertaken a proteomics approach and have generated transgenic animals that express a TAP-tagged version of PPH-5. Purification of the tagged phosphatase and its associated proteins, followed by mass spectrometry, has revealed two interacting proteins. One of these interacting proteins is DAF-21, a C. elegans HSP90 ortholog. RNAi of daf-21 has previously been shown to cause sterility in C. elegans, a phenotype similar to that of RNAi of wee-1.3, an inhibitor of CDK-1. Our current model is that PPH-5 influences the activity of the DAF-21 chaperone, which in turn regulates WEE-1.3 and CDK-1. Since CDK-1 is a known regulator of SEP-1 activity, it may be the indirect mechanism by which pph-5 mutations suppress the embryonic lethality of sep-1 mutants. Alternatively, PPH-5 in other systems works in a number of other signaling pathway and we are in the process of determining whether pph-5 might be functioning through one of these other pathways. Given that the players in the separase pathway are known to be regulated by phosphorylation, identifying the phosphoprotein substrate(s) of pph-5 is now a goal of ours.