Esc1, a nuclear periphery protein required for Sir4-based plasmid anchoring and partitioning
Esc1, a nuclear periphery protein required for Sir4-based plasmid anchoring and partitioning
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DOI：
10.1128/mcb.22.23.8292-8301.2002
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发表时间：
2002-12-01
影响因子：
5.3
通讯作者：
Sternglanz, R
中科院分区：
文献类型：
--
作者：
Andrulis, ED;Zappulla, DC;Sternglanz, R
进行靶向沉默筛选以鉴定酵母蛋白质，当其与端粒连接时，抑制由Rap 1截短引起的端粒沉默缺陷。一个以前未表征的蛋白质，EscI（建立沉默染色质），回收，除了良好的表征蛋白质Rap 1，Sir 1，和Rad 7。端粒沉默略有减少Deltaesc 1突变体，但沉默的HM位点不受影响。另一方面，在Deltaesc 1突变体中，各种束缚蛋白的靶向沉默被大大削弱。双杂交分析表明，Esc 1和Sir 4通过Esc 1的34个氨基酸部分（残基1440至1473）和Sir 4的羧基末端结构域（称为PAD 4）（残基950至1262）相互作用。当与DNA连接时，该Sir 4结构域赋予对否则不稳定的质粒的有效分配，并阻断结合的DNA片段在体内自由旋转的能力。在这里，这两种现象被证明需要ESC 1。Sir蛋白介导的基于端粒的质粒的分配也需要ESC 1。表达绿色荧光蛋白（GFP）-Esc 1的细胞的荧光显微镜显示，该蛋白质定位于细胞核周边，已知细胞核的一个区域在功能上对沉默很重要。然而，GFP-Esc 1定位与端粒、核仁或核孔复合体并不完全一致。我们的数据表明，Esc 1是一个冗余的途径，功能定位沉默复合物的核周边的一个组成部分。
A targeted silencing screen was performed to identify yeast proteins that, when tethered to a telomere, suppress a telomeric silencing defect caused by truncation of Rap1. A previously uncharacterized protein, EscI (establishes silent chromatin), was recovered, in addition to well-characterized proteins Rap1, Sir1, and Rad7. Telomeric silencing was slightly decreased in Deltaesc1 mutants, but silencing of the HM loci was unaffected. On the other hand, targeted silencing by various tethered proteins was greatly weakened in Deltaesc1 mutants. Two-hybrid analysis revealed that Esc1 and Sir4 interact via a 34-amino-acid portion of Esc1 (residues 1440 to 1473) and a carboxyl-terminal domain of Sir4 known as PAD4 (residues 950 to 1262). When tethered to DNA, this Sir4 domain confers efficient partitioning to otherwise unstable plasmids and blocks the ability of bound DNA segments to rotate freely in vivo. Here, both phenomena were shown to require ESC1. Sir protein-mediated partitioning of a telomere-based plasmid also required ESC1. Fluorescence microscopy of cells expressing green fluorescent protein (GFP)-Esc1 showed that the protein localized to the nuclear periphery, a region of the nucleus known to be functionally important for silencing. GFP-Esc1 localization, however, was not entirely coincident with telomeres, the nucleolus, or nuclear pore complexes. Our data suggest that Esc1 is a component of a redundant pathway that functions to localize silencing complexes to the nuclear periphery.