Identification of protein complexes required for efficient sister chromatid cohesion
Identification of protein complexes required for efficient sister chromatid cohesion
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DOI：
10.1091/mbc.e03-08-0619
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发表时间：
2004-04-01
影响因子：
3.3
通讯作者：
Hieter, P
中科院分区：
文献类型：
--
作者：
Mayer, ML;Pot, I;Hieter, P
Ctf8p是Ctf18-RFC的一个组成部分，Ctf18-RFC是酿酒酵母姐妹染色单体高效凝聚所必需的一种类似复制因子C的替代性复合体。我们进行了合成遗传阵列(SGA)分析，以ctf8缺失菌株作为初步筛选，以确定有效的姐妹染色单体凝聚力所需的其他非必需基因。然后，我们评估了含有ctf8 SGA筛查中确定的基因缺失的菌株在三个染色体座位上的凝聚力熟练程度。7个基因(CHL1、CSM3、BIM1、KAR3、TOF1、CTF4和VIK1)的缺失导致姐妹染色单体凝聚力缺陷。免疫沉淀复合物的质谱分析证实了Kar3p和Vik1p之间的物理结合以及Csm3p和Tof1p之间的相互作用，我们通过细胞提取物的免疫共沉淀证实了这一点。这些数据表明，结合特定二级筛选的合成基因阵列分析可以有效地识别与参考基因功能相关的蛋白质复合体。此外，我们发现参与有丝分裂纺锤体完整性和定位的基因在姐妹染色单体凝聚力中具有以前未被认识到的作用。
Ctf8p is a component of Ctf18-RFC, an alternative replication factor C-like complex required for efficient sister chromatid cohesion in Saccharomyces cerevisiae. We performed synthetic genetic array (SGA) analysis with a ctf8 deletion strain as a primary screen to identify other nonessential genes required for efficient sister chromatid cohesion. We then assessed proficiency of cohesion at three chromosomal loci in strains containing deletions of the genes identified in the ctf8 SGA screen. Deletion of seven genes (CHL1, CSM3, BIM1, KAR3, TOF1, CTF4, and VIK1) resulted in defective sister chromatid cohesion. Mass spectrometric analysis of immunoprecipitated complexes identified a physical association between Kar3p and Vik1p and an interaction between Csm3p and Tof1p that we confirmed by coimmunoprecipitation from cell extracts. These data indicate that synthetic genetic array analysis coupled with specific secondary screens can effectively identify protein complexes functionally related to a reference gene. Furthermore, we find that genes involved in mitotic spindle integrity and positioning have a previously unrecognized role in sister chromatid cohesion.