Le Masson Ivan, Saveanu Cosmin, Chevalier Anne, Namane Abdelkader, Gobin Renée, Fromont-Racine Micheline, Jacquier Alain, Mann Carl
Service de Biochimie et de Génétique Moléculaire, Bât. 142, CEA/Saclay, F-91191 Gif-sur-Yvette, France.
Mol Microbiol. 2002 Mar;43(6):1431-43. doi: 10.1046/j.1365-2958.2002.02844.x.
Mps2 (monopolar spindle protein) is a coiled-coil protein found at the spindle pole body (SPB) and at the nuclear envelope that is required for insertion of the SPB into the nuclear envelope. We identified three proteins that interact with Mps2 in a two-hybrid screen: Bbp1, Ynl107w and Spc24. All three proteins contain coiled-coil motifs that appear to be required for their interaction with Mps2. In this work, we verified the Mps2-Spc24 interaction by co-immunoprecipitation in vivo and by the in vitro interaction of recombinant proteins. Previous two-hybrid screens with Spc24 as bait had identified Spc25 and Ndc80 as putative interacting partners, and we verified these interactions in vivo by purification of TAP-tagged derivatives of Spc24 and Ndc80. Finally, we found that spc24 thermosensitive mutants had a chromosome segregation defect, but no apparent defect in SPB duplication. These results are consistent with recently published data showing that Spc24, Spc25 and Ndc80 are peripheral kinetochore com-ponents required for chromosome segregation. The Mps2-Spc24 interaction may contribute to the localization of Spc24 and other kinetochore components to the inner plaque of the SPB.
Mps2（单极纺锤体蛋白）是一种卷曲螺旋蛋白，存在于纺锤体极体（SPB）和核膜处，是将SPB插入核膜所必需的。我们在双杂交筛选中鉴定出三种与Mps2相互作用的蛋白：Bbp1、Ynl107w和Spc24。这三种蛋白均含有卷曲螺旋基序，这似乎是它们与Mps2相互作用所必需的。在这项研究中，我们通过体内共免疫沉淀和重组蛋白的体外相互作用验证了Mps2与Spc24的相互作用。先前以Spc24为诱饵的双杂交筛选已鉴定出Spc25和Ndc80为假定的相互作用伴侣，我们通过纯化带有TAP标签的Spc24和Ndc80衍生物在体内验证了这些相互作用。最后，我们发现spc24温度敏感突变体存在染色体分离缺陷，但在SPB复制方面没有明显缺陷。这些结果与最近发表的数据一致，表明Spc24、Spc25和Ndc80是染色体分离所需的外周动粒成分。Mps2与Spc24的相互作用可能有助于Spc24和其他动粒成分定位于SPB的内斑。
