Identification of a conserved F-box protein 6 interactor essential for endocytosis and cytokinesis in fission yeast.
Identification of a conserved F-box protein 6 interactor essential for endocytosis and cytokinesis in fission yeast.
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DOI：
10.1042/bj20081659
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发表时间：
2009-05-13
期刊：
影响因子：
--
通讯作者：
Toda T
中科院分区：
文献类型：
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作者：
Jourdain I;Spielewoy N;Thompson J;Dhut S;Yates JR;Toda T
为了鉴定Pof 6的相互作用物，我们从大规模细胞（40 μ L）中分两步进行了非严格的Pof 6-TAP纯化，并将其与MudPIT分析（多维蛋白质鉴定技术）结合。因此，我们决定根据该特定功能：Sip 1来命名pofSix Interactor Protein 1。结构域分析揭示了HEAT重复序列的长氨基末端延伸的存在，这使其成为支架亚基的候选者。因此，我们提出，三个必需蛋白Skp 1，Pof 6和Sip 1形成一个三元复合物，由一种新型的非SCF F-box复合物组成。重要的是，大量的工作放在解剖的行动所需的正确转移的遗传物质的子细胞，很少有人知道的后期分隔导致物理细胞分离。在这项研究中，我们通过TAP纯化和MudPIT分析分离出一种新的Pof 6相互作用物。这个未表征的和必需的ORF被命名为PofSix相互作用蛋白1的Sip 1。Pof 6、Sip 1和Skp 1之间的免疫共沉淀实验和Sip 1-TAP的天然纯化证实了这三种蛋白之间的相互作用。sip 1功能丧失突变体的分离揭示了胞质分裂的致命性，而GFP-Pof 6在后期在赤道区短暂积累。我们认为Sip 1，Pof 6和Skp 1在CAR的组装/收缩和随后的细胞分离中共同发挥重要作用。
To identify Pof6’s interactors, we have performed a non-stringent Pof6-TAP purification in two steps from a large scale of cells (40 L) and coupled it to MudPIT analysis (Multidimentional Protein Identification Technology). We, consequently, decided to name the protein according to that specific feature: Sip1 for pofSix Interactor Protein 1. The domain analysis reveals the presence of a long amino-terminal stretch of HEAT repeats which makes it a candidate as a scaffolding subunit. Consequently, we propose that the three essential proteins Skp1, Pof6 and Sip1 form a ternary complex consisting of a novel type of the non-SCF F-box complex. Important as the amount of work put on dissecting the actions required for a correct transfer of genetic material to the daughter cells, very little is known about the late phase of septation leading to the physical cells separation. In this study we isolated by TAP purification and MudPIT analysis a novel interactor of Pof6. This uncharacterised and essential ORF was named Sip1 for PofSix interactor protein 1. Coimmunoprecipitation experiments between Pof6, Sip1 and Skp1 and native purification of Sip1-TAP confirmed the interactions between the three proteins. The isolation of a sip1 loss-of-function mutant reveals lethality in cytokinesis while GFP-Pof6 transiently accumulated at the equatorial zone in late anaphase. We suggest that Sip1, Pof6 and Skp1 play together an essential role in assembly/constriction of the CAR and subsequent cell separation.