THE CARBOXY TERMINI OF SIR4 AND RAP1 AFFECT SIR3 LOCALIZATION - EVIDENCE FOR A MULTICOMPONENT COMPLEX REQUIRED FOR YEAST TELOMERIC SILENCING
THE CARBOXY TERMINI OF SIR4 AND RAP1 AFFECT SIR3 LOCALIZATION - EVIDENCE FOR A MULTICOMPONENT COMPLEX REQUIRED FOR YEAST TELOMERIC SILENCING
复制标题
DOI：
10.1083/jcb.129.4.909
复制
发表时间：
1995-05-01
影响因子：
7.8
通讯作者：
GASSER, SM
中科院分区：
文献类型：
--
作者：
COCKELL, M;PALLADINO, F;GASSER, SM
在酵母端粒区域观察到的染色质介导的基因抑制需要沉默信息调节蛋白 Sir3 和 Sir4 以及端粒重复结合蛋白 RAP1。所有三种蛋白质均通过免疫荧光染色定位到核周边附近的病灶，表明亚核定位和沉默之间的关系。我们提出了一些免疫学和生化证据，证明 Sir3、Sir4 和 RAP1 在完整酵母细胞中相互作用。首先，在携带 RAP1 末端 28 或 165 个氨基酸缺失的 rap1 突变体中，Sir3 对酵母核外围焦点的免疫定位丢失。其次，Sir3 和 RAP1 的核周定位因 Sir4 COOH 末端的过量产生而受到干扰。第三，Sir4 COOH 末端的过量产生改变了 Sir3 和全长 Sir4 的溶解度特性。最后，我们使用抗 RAP1 或抗 Sir4 抗体证明 RAP1 和 Sir4 在免疫复合物中共沉淀。我们认为 Sir4、Sir3 和 RAP1 之间的三级复合体的完整性涉及端粒抑制的维持和核外围附近病灶中端粒的聚集。
The Silent Information Regulatory proteins, Sir3 and Sir4, and the telomeric repeat-binding protein RAP1 are required for the chromatin-mediated gene repression observed at yeast telomeric regions. All three proteins are localized by immunofluorescence staining to foci near the nuclear periphery suggesting a relationship between subnuclear localization and silencing. We present several lines of immunological and biochemical evidence that Sir3, Sir4, and RAP1 interact in intact yeast cells. First, immunolocalization of Sir3 to foci at the yeast nuclear periphery is lost in rap1 mutants carrying deletions for either the terminal 28 or 165 amino acids of RAP1. Second, the perinuclear localization of both Sir3 and RAP1 is disrupted by overproduction of the COOH terminus of Sir4. Third, overproduction of the Sir4 COOH terminus alters the solubility properties of both Sir3 and full-length Sir4. Finally, we demonstrate that RAP1 and Sir4 coprecipitate in immune complexes using either anti-RAP1 or anti-Sir4 antibodies. We propose that the integrity of a tertiary complex between Sir4, Sir3, and RAP1 is involved in both the maintenance of telomeric repression and the clustering of telomeres in foci near the nuclear periphery.