The characterization of separase suppressors

鉴于我们对APC/C及其下游靶标的兴趣，我们一直将研究重点放在APC/C的一个间接靶标上。分离酶是一种在减数分裂时裂解粘连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，Securin被APC/C泛素化，并被26S蛋白酶体降解。 我们采用了一种遗传方法来确定分离酶的调节因子和底物。我们有Sep-1的三个突变等位基因，最近通过与Joshua Bembenek博士的合作表明，这些突变都在皮质颗粒胞吐(CGE)方面存在缺陷。CGE对于蛋壳形成所需的成分的分泌是重要的，而蛋壳形成是正确的极体挤出所间接需要的。为了确定在分离酶途径中发挥作用的其他基因，我们进行了Sep-1的温度敏感等位基因e2406ts的抑制筛选。我们已经确定了三种抑制物，它们可以在不允许的温度下恢复Sep-1突变体的活性。其中一个突变体是基因内抑制子，另外两个是基因外抑制子。我们最近确定，其中一个抑制子突变存在于一种名为PPH-5的磷酸酶基因中。这个磷酸酶突变体，在其他野生型背景下，本身没有明显的表型。我们的PPH-5等位基因av101抑制了我们三个Sep-1等位基因中的两个的胚胎致死性，PPH-5基因的一个缺失等位基因也是如此。PPH-5的RNAi也抑制了我们三个Sep-1等位基因中的两个的胚胎致死性。RNAi缺失或PPH-5的基因突变使CGE在受抑制的Sep-1背景中恢复到野生型水平。为了进一步了解PPH-5如何在分离酶途径中发挥作用，我们采用了蛋白质组学的方法，并产生了表达TAP标记版本的PPH-5的转基因动物。对标记的磷酸酶及其相关蛋白进行纯化，然后进行质谱分析，发现了两种相互作用的蛋白质。这些相互作用的蛋白质之一是DAF-21，它是线虫HSP90的同源基因。此前已有研究表明，daf-21的RNAi可导致线虫不育，其表型类似于CDK-1的抑制剂WEE-1.3的RNAi。我们目前的模型是PPH-5影响DAF-21伴侣的活性，而DAF-21伴侣又调节WEE-1.3和CDK-1。由于CDK-1是SEP-1活性的已知调节因子，它可能是PPH-5突变抑制Sep-1突变体胚胎致死性的间接机制。或者，在其他系统中，PPH-5在许多其他信号途径中起作用，我们正在确定PPH-5是否可能通过这些其他途径之一发挥作用。鉴于分离酶途径中的成员已知受磷酸化调控，确定PPH-5的磷酸蛋白底物(S)现在是我们的目标。

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.