Recognition and removal of organelle damage at the endoplasmic reticulum membrane

细胞采用多种质量控制机制来确保细胞器的完整性。与细胞质中的质量控制相比，膜封闭细胞器的质量控制提出了独特的挑战。细胞器膜是检测和消除受损蛋白质的障碍，但这些障碍需要始终保持。内质网(ER)是蛋白质折叠的中心区域，容易导致错误折叠的蛋白质堆积。在早期的工作中，我们重点研究了酵母中响应内质网压力的两种相关质量控制机制。首先，我们证明了选择性的内质网微自噬(Micro-ER-phagy)涉及形成多层内质网螺旋，这些内质网在溶酶体中被降解，推测是为了消除内质网损伤。我们最近发现ESCRT蛋白对于内质网的微噬是必不可少的，因为它们介导了溶酶体膜的分裂，从而完成了螺旋体的微自噬。我们还发现，蛋白质进入轮生的控制可能对微内质网吞噬的选择性至关重要。第二，我们发现内质网应激触发ESCRT蛋白显著地募集到形态异常的内质网亚域。我们称这种机制为REMEM(将ESCRT机器招募到ER损坏的位置)。我们推测，Remed修复ER膜损伤，并与内质网微噬协同作用。在这里，我们建议研究内质网微吞噬和Remed的机制，并探索它们之间的功能联系。在目标1中，我们将应用蛋白质组学方法来确定内质网轮纹的蛋白质含量，从而确定货物的选择性。在目标2中，我们将通过酵母遗传筛选和细胞生物学实验来阐明RMERE的机制，并询问RMERE是否存在于人类细胞中。在目标3中，我们将利用酵母中的遗传相互作用研究和功能分析来了解微内质网吞噬和Remed的生理作用。这个项目将对ER损害的识别和移除产生新的见解。此外，考虑到操纵ER质量控制承诺对健康有益，我们的研究可能会发现新的治疗方法。

Cells employ numerous quality control mechanisms to ensure organelle integrity. Compared to quality control in the cytosol, quality control of membrane-enclosed organelles poses unique challenges. Organelle membranes present barriers for the detection and elimination of damaged proteins, yet these barriers need to be maintained at all times. The endoplasmic reticulum (ER) is a central protein folding compartment liable to the accumulation of misfolded proteins. In earlier work, we focused on two related quality control mechanisms in yeast that respond to ER stress. First, we showed that selective microautophagy of ER (micro-ER-phagy) involves the formation of multi-lamellar ER whorls that are degraded in lysosomes, presumably to remove ER damage. We recently found that ESCRT proteins are essential for micro-ER-phagy because they mediate lysosomal membrane fission to complete microautophagy of whorls. We also found that the control of protein entry into whorls likely is critical for the selectivity of micro-ER-phagy. Second, we discovered that ER stress triggers a striking recruitment of ESCRT proteins to morphologically aberrant ER subdomains. We call this mechanism REMED (Recruitment of ESCRT Machinery to sites of ER Damage). We hypothesize that REMED repairs ER membrane damage and cooperates with micro-ER-phagy. Here, we propose to investigate the mechanisms of micro-ER-phagy and REMED, and explore the functional links between them. In Aim 1, we will apply proteomic approaches to determine the protein contents of ER whorls and thus define cargo selectivity. In Aim 2, we will elucidate the mechanism of REMED through yeast genetic screens and cell biological experiments, and ask whether REMED exists in human cells. In Aim 3, we will use genetic interaction studies and functional assays in yeast to understand the physiological roles of micro-ER-phagy and REMED. This project will yield new insight into ER damage recognition and removal. Moreover, given that manipulating ER quality control promises health benefits, our research may identify new therapeutic approaches.