Kinetochore protein interactions and their regulation by the Aurora kinase Ipl1p
Kinetochore protein interactions and their regulation by the Aurora kinase Ipl1p
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DOI：
10.1091/mbc.e02-11-0765
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发表时间：
2003-08-01
影响因子：
3.3
通讯作者：
Barnes, G
中科院分区：
文献类型：
--
作者：
Shang, C;Hazbun, TR;Barnes, G
尽管最近对出芽酵母着丝粒成分的鉴定取得了爆炸性的进展，但构成着丝粒功能的物理相互作用仍然不清楚。为了更好地了解着丝粒如何附着在微管上以及这种附着是如何调节的，我们试图表征着丝粒蛋白之间的相互作用，特别是与微管结合 Dam1 复合物有关的相互作用。 Dam1 复合体在染色体-纺锤体附着中发挥着至关重要的作用，并且是 Aurora 激酶 Ip11p 对该附着进行磷酸化调节的关键靶标。为了确定涉及 Dam1 复合物的蛋白质-蛋白质相互作用，以及 Dam1p 磷酸化状态对这些物理相互作用的影响，我们对 Dam1p 进行了全基因组双杂交筛选和一系列生化结合测定。对 6000 个酵母开放阅读框的文库进行双杂交筛选，鉴定出 9 种着丝粒蛋白作为 Dam1p 相互作用的伙伴。从涉及 Dam1 复合物的所有 9 个亚基和 32 个动粒蛋白的 113 个体外结合反应中，我们发现了 Dam1 复合物内至少有 9 个相互作用以及 Dam1 复合物的 19 个潜在伴侣。引人注目的是，我们发现 Dam1p-Ndc80p 和 Dam1p-Spc34p 相互作用因模仿 Ipl1p 位点磷酸化的突变而减弱，这使我们能够建立一个模型来研究磷酸化对着丝粒功能的影响。
Although there has been a recent explosion in the identification of budding yeast kinetochore components, the physical interactions that underlie kinetochore function remain obscure. To better understand how kinetochores attach to microtubules and how this attachment is regulated, we sought to characterize the interactions among kinetochore proteins, especially with respect to the microtubule-binding Dam1 complex. The Dam1 complex plays a crucial role in the chromosome-spindle attachment and is a key target for phospho-regulation of this attachment by the Aurora kinase Ip11p. To identify protein-protein interactions involving the Dam1 complex, and the effects of Dam1p phosphorylation state on these physical interactions, we conducted both a genome-wide two-hybrid screen and a series of biochemical binding assays for Dam1p. A two-hybrid screen of a library of 6000 yeast open reading frames identified nine kinetochore proteins as Dam1p-interacting partners. From 113 in vitro binding reactions involving all nine subunits of the Dam1 complex and 32 kinetochore proteins, we found at least nine interactions within the Dam1 complex and 19 potential partners for the Dam1 complex. Strikingly, we found that the Dam1p-Ndc80p and Dam1p-Spc34p interactions were weakened by mutations mimicking phosphorylation at Ipl1p sites, allowing us to formulate a model for the effects of phosphoregulation on kinetochore function.