Once secretory proteins have been targeted to the endoplasmic reticulum (ER) lumen, the proteins typically remain partitioned from the cytosol. If the secretory proteins misfold, they can be unfolded and retrotranslocated into the cytosol for destruction by the proteasome by ER-Associated protein Degradation (ERAD). Here, we report that correctly folded and targeted luminal ER fluorescent protein reporters accumulate in the cytosol during acute misfolded secretory protein stress in yeast. Photoactivation fluorescence microscopy experiments reveal that luminal reporters already localized to the ER relocalize to the cytosol, even in the absence of essential ERAD machinery. We named this process "ER reflux." Reflux appears to be regulated in a size-dependent manner for reporters. Interestingly, prior heat shock stress also prevents ER stress-induced reflux. Together, our findings establish a new ER stress-regulated pathway for relocalization of small luminal secretory proteins into the cytosol, distinct from the ERAD and preemptive quality control pathways. Importantly, our results highlight the value of fully characterizing the cell biology of reporters and describe a simple modification to maintain luminal ER reporters in the ER during acute ER stress.

中文翻译：

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大小依赖性分泌蛋白回流到细胞质中与急性内质网应激相关。

一旦分泌蛋白被靶向内质网 (ER) 腔，这些蛋白通常会与细胞质保持分离。如果分泌蛋白错误折叠，它们可以展开并逆转位到胞质溶胶中，通过内质网相关蛋白降解 (ERAD) 被蛋白酶体破坏。在这里，我们报告了在酵母的急性错误折叠分泌蛋白应激期间，正确折叠和靶向的管腔内质网荧光蛋白报告基因在胞质溶胶中积累。光活化荧光显微镜实验表明，即使在没有必要的 ERAD 机制的情况下，已经定位到 ER 的管腔报告基因也会重新定位到细胞质中。我们将这个过程命名为“ER 反流”。对于记者来说，回流似乎是按照大小相关的方式进行调节的。有趣的是，先前的热休克应激也可以防止内质网应激引起的反流。总之，我们的研究结果建立了一条新的 ER 应激调节途径，用于将小管腔分泌蛋白重新定位到细胞质中，这与 ERAD 和先发制人的质量控制途径不同。重要的是，我们的结果强调了充分表征记者细胞生物学特征的价值，并描述了在急性 ER 应激期间维持 ER 内腔 ER 记者的简单修改。