Department of Biochemistry, College of Biotechnology and Life Science, Yonsei University, Seoul, Korea.
PLoS Genet. 2012 Jan;8(1):e1002450. doi: 10.1371/journal.pgen.1002450. Epub 2012 Jan 12.
In budding yeast, the major regulator of the mitotic exit network (MEN) is Tem1, a GTPase, which is inhibited by the GTPase-activating protein (GAP), Bfa1/Bub2. Asymmetric Bfa1 localization to the bud-directed spindle pole body (SPB) during metaphase also controls mitotic exit, but the molecular mechanism and function of this localization are not well understood, particularly in unperturbed cells. We identified four novel Cdc5 target residues within the Bfa1 C-terminus: (452)S, (453)S, (454)S, and (559)S. A Bfa1 mutant in which all of these residues had been changed to alanine (Bfa1(4A)) persisted on both SPBs at anaphase and was hypo-phosphorylated, despite retaining its GAP activity for Tem1. A Bfa1 phospho-mimetic mutant in which all of these residues were switched to aspartate (Bfa1(4D)) always localized asymmetrically to the SPB. These observations demonstrate that asymmetric localization of Bfa1 is tightly linked to its Cdc5-dependent phosphorylation, but not to its GAP activity. Consistent with this, in kinase-defective cdc5-2 cells Bfa1 was not phosphorylated and localized to both SPBs, whereas Bfa1(4D) was asymmetrically localized. BFA1(4A) cells progressed through anaphase normally but displayed delayed mitotic exit in unperturbed cell cycles, while BFA1(4D) cells underwent mitotic exit with the same kinetics as wild-type cells. We suggest that Cdc5 induces the asymmetric distribution of Bfa1 to the bud-directed SPB independently of Bfa1 GAP activity at anaphase and that Bfa1 asymmetry fine-tunes the timing of MEN activation in unperturbed cell cycles.
在芽殖酵母中，有丝分裂退出网络（MEN）的主要调节因子是 Tem1，它是一种 GTPase，被 GTPase 激活蛋白（GAP）Bfa1/Bub2 抑制。在中期，Bfa1 不对称地定位于朝向芽的纺锤体极体（SPB）也控制着有丝分裂的退出，但这种定位的分子机制和功能还不是很清楚，特别是在未受干扰的细胞中。我们在 Bfa1 的 C 端发现了四个新的 Cdc5 靶位残基：（452）S、（453）S、（454）S 和（559）S。一个 Bfa1 突变体，其所有这些残基都突变为丙氨酸（Bfa1(4A)），在后期仍然存在于两个 SPB 上，并且磷酸化程度较低，尽管它保留了对 Tem1 的 GAP 活性。一个 Bfa1 磷酸模拟突变体，其所有这些残基都突变为天冬氨酸（Bfa1(4D)），总是不对称地定位于 SPB。这些观察结果表明，Bfa1 的不对称定位与 Cdc5 依赖性磷酸化紧密相关，但与 GAP 活性无关。与此一致的是，在激酶缺陷型 cdc5-2 细胞中，Bfa1 没有被磷酸化，并且定位在两个 SPB 上，而 Bfa1(4D)则是不对称定位的。BFA1(4A)细胞正常进入后期，但在未受干扰的细胞周期中，有丝分裂退出延迟，而 BFA1(4D)细胞的有丝分裂退出与野生型细胞的动力学相同。我们认为，Cdc5 在后期独立于 Bfa1 GAP 活性诱导 Bfa1 不对称分布到朝向芽的 SPB，而 Bfa1 不对称性精细调整了 MEN 在未受干扰的细胞周期中激活的时机。
