Kar2p availability defines distinct forms of endoplasmic reticulum stress in living cells.
Kar2p availability defines distinct forms of endoplasmic reticulum stress in living cells.
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DOI：
10.1091/mbc.e11-12-0995
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发表时间：
2012-03
影响因子：
3.3
通讯作者：
Snapp EL
中科院分区：
文献类型：
--
作者：
Lajoie P;Moir RD;Willis IM;Snapp EL
内质网（ER）未折叠蛋白反应（UPR）与未折叠分泌水平的变化相关。一种新型荧光生物传感器现在可以报告未折叠蛋白质负荷的变化。本报告揭示了一种 ER 应激形式（肌醇撤退），它可以刺激 UPR，但未折叠蛋白水平不会发生变化。内质网 (ER) 中错误折叠的分泌蛋白的积累会激活未折叠蛋白反应 (UPR) 应激途径。为了增强分泌蛋白折叠并促进对压力的适应，UPR 上调 ER 伴侣水平，包括 BiP。在这里，我们描述了 KAR2（BiP 的酵母同源物）与超级文件夹绿色荧光蛋白 (sfGFP) 的染色体标记，以创建 ER 折叠环境的多功能内源报告基因。 Kar2p-sfGFP 荧光水平的变化与 UPR 活性直接相关，是高通量分析的强大报告基因。该报告器的第二个新颖特征是 Kar2p-sfGFP 迁移率的光漂白显微镜（光漂白后的荧光恢复）报告单个细胞中未折叠分泌蛋白的水平，与 UPR 状态无关。用衣霉素或二硫苏糖醇处理后，Kar2p-sfGFP 的迁移率降低，这与未折叠蛋白水平的增加以及 Kar2p-sfGFP 掺入较慢扩散的复合物一致。在适应过程中，我们观察到 UPR 的下调和未折叠蛋白质负荷的解决之间存在显着的滞后。最后，我们发现 Kar2p-sfGFP 迁移率在肌醇撤除后显着增加，这也激活了 UPR，显然与未折叠蛋白水平无关。因此，Kar2p 迁移率代表了一种强大的新工具，能够区分导致活细胞中 UPR 激活的不同机制。
The endoplasmic reticulum (ER) unfolded protein response (UPR) is correlated with changes in unfolded secretory levels. A novel fluorescence biosensor now reports changes in the unfolded protein burden. This reporter reveals a form of ER stress—inositol withdrawal—that stimulates the UPR without changes in unfolded protein levels. Accumulation of misfolded secretory proteins in the endoplasmic reticulum (ER) activates the unfolded protein response (UPR) stress pathway. To enhance secretory protein folding and promote adaptation to stress, the UPR upregulates ER chaperone levels, including BiP. Here we describe chromosomal tagging of KAR2, the yeast homologue of BiP, with superfolder green fluorescent protein (sfGFP) to create a multifunctional endogenous reporter of the ER folding environment. Changes in Kar2p-sfGFP fluorescence levels directly correlate with UPR activity and represent a robust reporter for high-throughput analysis. A novel second feature of this reporter is that photobleaching microscopy (fluorescence recovery after photobleaching) of Kar2p-sfGFP mobility reports on the levels of unfolded secretory proteins in individual cells, independent of UPR status. Kar2p-sfGFP mobility decreases upon treatment with tunicamycin or dithiothreitol, consistent with increased levels of unfolded proteins and the incorporation of Kar2p-sfGFP into slower-diffusing complexes. During adaptation, we observe a significant lag between down-regulation of the UPR and resolution of the unfolded protein burden. Finally, we find that Kar2p-sfGFP mobility significantly increases upon inositol withdrawal, which also activates the UPR, apparently independent of unfolded protein levels. Thus Kar2p mobility represents a powerful new tool capable of distinguishing between the different mechanisms leading to UPR activation in living cells.