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(和PPH-5基因的缺失等位基因)抑制了我们三个Sep-1等位基因中的两个对胚胎的致死性。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。 与哈罗德·史密斯(NIDDK)合作，我们进行了深度测序，以确定av102抑制株的突变。在一个编码羧甲基酶的基因中发现了突变。众所周知，酵母中该基因的同源基因可以促进蛋白磷酸酶2A复合体的形成。因此，我们在线虫中的研究表明，两种不同的磷酸酶可能调节分离酶的功能。由于分离酶途径的许多成分都是已知的激酶或磷酸蛋白，因此特定磷酸基团的去除必须在分离酶活性中发挥重要的调节作用。我们试图确定这两种磷酸酶的底物以及突变抑制Sep-1致死性的机制。

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 (and a deletion allele of the pph-5 gene), suppresses the embryonic lethality of two of our three sep-1 alleles. 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. In collaboration with Harold Smith (NIDDK), we have carried out deep sequencing to determine the mutation in the av102 suppressor strain. A mutation was found in a gene that encodes a carboxyl methylase. Orthologs of this gene in yeast are known to promote the formation of the protein phosphatase 2A complex. Thus our studies in C. elegans suggest that two distinct phosphatases may regulate separase function. Since numerous components of the separase pathway are known kinases or phosphoproteins, the removal of specific phosphate groups must play an important regulatory role in separase activity. We seek to determine the substrates of these two phosphatases and the mechanisms by which mutations suppress sep-1 lethality.