Recognition and removal of organelle damage at the endoplasmic reticulum membrane

细胞采用许多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。细胞器膜为检测和消除受损蛋白质提供了屏障，但这些屏障需要始终保持。内质网（ER）是一个中心的蛋白质折叠区室，容易积累错误折叠的蛋白质。在早期的工作中，我们集中在两个相关的质量控制机制，酵母响应ER压力。首先，我们发现ER的选择性微自噬（micro-ER-phagy）涉及在溶酶体中降解的多层ER螺旋的形成，可能是为了消除ER损伤。我们最近发现，ESCRT蛋白是必不可少的micro-ER-phagy，因为它们介导溶酶体膜分裂完成microautophagy的螺纹。我们还发现，控制蛋白质进入螺旋可能是关键的选择性micro-ER-phagy。其次，我们发现ER应激触发了ESCRT蛋白向形态异常的ER亚结构域的显著募集。我们称这种机制为REMED（将ESCRT机器招募到ER损伤部位）。我们推测，REMED修复ER膜损伤，并与微ER-吞噬合作。在此，我们拟研究微ER-吞噬和REMED的机制，并探讨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ER螺旋的蛋白质含量，从而定义货物选择性。目的二是通过酵母基因筛选和细胞生物学实验阐明REMED的作用机制，并探讨REMED是否存在于人体细胞中。在目标3中，我们将使用酵母中的遗传相互作用研究和功能测定来理解微ER-吞噬和REMED的生理作用。该项目将产生新的洞察ER损伤识别和去除。此外，鉴于操纵ER质量控制承诺健康益处，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.