Shugoshin Prevents Dissociation of Cohesin from Centromeres During Mitosis in Vertebrate Cells

Cohesion between sister chromatids is essential for their bi-orientation on mitotic spindles. It is mediated by a multisubunit complex called cohesin. In yeast, proteolytic cleavage of cohesin's α kleisin subunit at the onset of anaphase removes cohesin from both centromeres and chromosome arms and thus triggers sister chromatid separation. In animal cells, most cohesin is removed from chromosome arms during prophase via a separase-independent pathway involving phosphorylation of its Scc3-SA1/2 subunits. Cohesin at centromeres is refractory to this process and persists until metaphase, whereupon its α kleisin subunit is cleaved by separase, which is thought to trigger anaphase. What protects centromeric cohesin from the prophase pathway? Potential candidates are proteins, known as shugoshins, that are homologous to Drosophila MEI-S332 and yeast Sgo1 proteins, which prevent removal of meiotic cohesin complexes from centromeres at the first meiotic division. A vertebrate shugoshin-like protein associates with centromeres during prophase and disappears at the onset of anaphase. Its depletion by RNA interference causes HeLa cells to arrest in mitosis. Most chromosomes bi-orient on a metaphase plate, but precocious loss of centromeric cohesin from chromosomes is accompanied by loss of all sister chromatid cohesion, the departure of individual chromatids from the metaphase plate, and a permanent cell cycle arrest, presumably due to activation of the spindle checkpoint. Remarkably, expression of a version of Scc3-SA2 whose mitotic phosphorylation sites have been mutated to alanine alleviates the precocious loss of sister chromatid cohesion and the mitotic arrest of cells lacking shugoshin. These data suggest that shugoshin prevents phosphorylation of cohesin's Scc3-SA2 subunit at centromeres during mitosis. This ensures that cohesin persists at centromeres until activation of separase causes cleavage of its α kleisin subunit. Centromeric cohesion is one of the hallmarks of mitotic chromosomes. Our results imply that it is not an intrinsically stable property, because it can easily be destroyed by mitotic kinases, which are kept in check by shugoshin.

姐妹染色单体（sister chromatids）之间的黏连对于其在有丝分裂纺锤体上的双向定向至关重要。该黏连由名为黏连蛋白（cohesin）的多亚基复合物所介导。在酵母中，于分裂后期起始时对黏连蛋白的α kleisin亚基进行蛋白水解切割，可将黏连蛋白从着丝粒与染色体臂上移除，进而触发姐妹染色单体分离。在动物细胞中，绝大多数黏连蛋白会在前期通过一条不依赖分离酶的通路从染色体臂上移除，该通路涉及黏连蛋白Scc3-SA1/2亚基的磷酸化修饰。着丝粒处的黏连蛋白对此过程具有抗性，会持续存在直至中期，此时其α kleisin亚基会被分离酶切割，这一过程被认为是触发分裂后期的关键步骤。
是什么保护着丝粒处的黏连蛋白免受前期通路的影响？潜在的候选蛋白是一类被称为守护蛋白（shugoshins）的分子，它们与果蝇MEI-S332及酵母Sgo1蛋白同源，可在第一次减数分裂时阻止减数分裂黏连蛋白复合物从着丝粒处被移除。一种脊椎动物来源的类守护蛋白会在前期结合于着丝粒，并在分裂后期起始时消失。通过RNA干扰（RNA interference）将其敲低后，海拉（HeLa）细胞会出现有丝分裂阻滞。多数染色体可在赤道板上完成双向定向，但染色体上过早丢失的着丝粒黏连蛋白会伴随所有姐妹染色单体黏连的丧失、单个染色单体脱离赤道板，以及永久性的细胞周期阻滞——这一现象推测是由纺锤体检验点的激活所导致。
值得注意的是，将Scc3-SA2的有丝分裂磷酸化位点突变为丙氨酸后表达该突变体，可缓解缺失守护蛋白的细胞中姐妹染色单体黏连的过早丢失以及有丝分裂阻滞。上述实验结果表明，守护蛋白可在有丝分裂过程中阻止着丝粒处黏连蛋白的Scc3-SA2亚基发生磷酸化。这一机制确保了黏连蛋白会持续存在于着丝粒处，直至分离酶被激活并切割其α kleisin亚基。着丝粒黏连是有丝分裂染色体的标志性特征之一。我们的研究结果暗示，着丝粒黏连并非一种内在稳定的属性，因为它可轻易被有丝分裂激酶破坏，而这类激酶的活性会受到守护蛋白的抑制。