Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232-8240, USA.
Mol Biol Cell. 2010 Mar 15;21(6):1072-87. doi: 10.1091/mbc.e09-07-0615. Epub 2010 Jan 28.
The assembly, distribution, and functional integrity of nuclear pore complexes (NPCs) in the nuclear envelope (NE) are key determinants in the nuclear periphery architecture. However, the mechanisms controlling proper NPC and NE structure are not fully defined. We used two different genetic screening approaches to identify Saccharomyces cerevisiae mutants with defects in NPC localization. The first approach examined green fluorescent protein (GFP)-Nic96 in 531 strains from the yeast Tet-promoters Hughes Collection with individual essential genes expressed from a doxycycline-regulated promoter (TetO(7)-orf). Under repressive conditions, depletion of the protein encoded by 44 TetO(7)-orf strains resulted in mislocalized GFP-Nic96. These included STH1, RSC4, RSC8, RSC9, RSC58, ARP7, and ARP9, each encoding components of the RSC chromatin remodeling complex. Second, a temperature-sensitive sth1-F793S (npa18-1) mutant was identified in an independent genetic screen for NPC assembly (npa) mutants. NPC mislocalization in the RSC mutants required new protein synthesis and ongoing transcription, confirming that lack of global transcription did not underlie the phenotypes. Electron microscopy studies showed significantly altered NEs and nuclear morphology, with coincident cytoplasmic membrane sheet accumulation. Strikingly, increasing membrane fluidity with benzyl alcohol treatment prevented the sth1-F793S NE structural defects and NPC mislocalization. We speculate that NE structure is functionally linked to proper chromatin architecture.
核孔复合物（NPC）在核膜（NE）中的组装、分布和功能完整性是核周区结构的关键决定因素。然而，控制 NPC 和 NE 结构的机制尚未完全确定。我们使用两种不同的遗传筛选方法来鉴定 NPC 定位缺陷的酿酒酵母突变体。第一种方法检测了来自酵母 Tet 启动子 Hughes 集合的 531 个菌株中的绿色荧光蛋白（GFP）-Nic96，这些菌株中的单个必需基因由四环素调控的启动子（TetO(7)-orf）表达。在抑制条件下，44 个 TetO(7)-orf 菌株编码的蛋白耗竭导致 GFP-Nic96 定位错误。这些蛋白包括 STH1、RSC4、RSC8、RSC9、RSC58、ARP7 和 ARP9，它们分别编码 RSC 染色质重塑复合物的成分。其次，在 NPC 组装（npa）突变体的独立遗传筛选中，鉴定出温度敏感的 sth1-F793S（npa18-1）突变体。RSC 突变体中的 NPC 定位错误需要新的蛋白质合成和持续的转录，这证实了缺乏全局转录并不是这些表型的基础。电子显微镜研究表明，内质网和核形态发生了明显改变，同时细胞质膜片层积累。引人注目的是，用苯甲醇处理增加膜流动性可以防止 sth1-F793S 的 NE 结构缺陷和 NPC 定位错误。我们推测，NE 结构与适当的染色质结构在功能上是相关的。
