CRISPR screening using an expanded toolkit of autophagy reporters identifies TMEM41B as a novel autophagy factor
CRISPR screening using an expanded toolkit of autophagy reporters identifies TMEM41B as a novel autophagy factor
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DOI：
10.1371/journal.pbio.2007044
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发表时间：
2019-04-01
期刊：
影响因子：
9.8
通讯作者：
Denic, Vladimir
中科院分区：
文献类型：
--
作者：
Shoemaker, Christopher J.;Huang, Tina Q.;Denic, Vladimir
酵母正向遗传学的力量是自噬研究领域站稳脚跟的基础。在高等真核生物中关于自噬的补充工作揭示了这一过程的深层保守性，以及在发育、免疫和神经元动态平衡的背景下调节自噬的新机制。最近出现的新的簇状规则间隔回文重复序列/CRISPR相关蛋白9(CRISPR/Cas9)技术已经开始促进在哺乳动物细胞中通过正向遗传筛选来定义新的自噬因子和途径的努力。在这里，我们着手开发一个适用于CRISPR/CAS9筛查的自噬记者的扩展工具包。对我们的记者在哺乳动物细胞中进行的全基因组筛选发现，几乎所有已知的自噬相关(ATG)因子以及以前未描述的因子，包括空泡蛋白分选37同源A(VPS37A)、跨膜蛋白251(TMEM251)、肌萎缩侧索硬化症2(ALS2)和TMEM41B。为了验证这个数据集，我们使用了定量显微镜和生化分析来证明1个新的HIT，TMEM41B，是吞噬菌体成熟所必需的。TMEM41B是一种完整的内质网(ER)膜蛋白，与已建立的自噬因子液泡膜蛋白1(VMP1)有较远的亲缘关系，我们的数据表明这两个因子在自噬小体的生物发生中发挥着相关的作用，尽管不是完全重叠。总之，我们的工作发现了新的自噬因子，揭示了自噬受体遗传相互作用的可塑性网络，并为进一步挖掘新的自噬机制提供了有价值的资源(http://crispr.deniclab.com))。
The power of forward genetics in yeast is the foundation on which the field of autophagy research firmly stands. Complementary work on autophagy in higher eukaryotes has revealed both the deep conservation of this process, as well as novel mechanisms by which autophagy is regulated in the context of development, immunity, and neuronal homeostasis. The recent emergence of new clustered regularly interspaced palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-based technologies has begun facilitating efforts to define novel autophagy factors and pathways by forward genetic screening in mammalian cells. Here, we set out to develop an expanded toolkit of autophagy reporters amenable to CRISPR/Cas9 screening. Genome-wide screening of our reporters in mammalian cells recovered virtually all known autophagy-related (ATG) factors as well as previously uncharacterized factors, including vacuolar protein sorting 37 homolog A (VPS37A), transmembrane protein 251 (TMEM251), amyotrophic lateral sclerosis 2 (ALS2), and TMEM41B. To validate this data set, we used quantitative microscopy and biochemical analyses to show that 1 novel hit, TMEM41B, is required for phagophore maturation. TMEM41B is an integral endoplasmic reticulum (ER) membrane protein distantly related to the established autophagy factor vacuole membrane protein 1 (VMP1), and our data show that these two factors play related, albeit not fully overlapping, roles in autophagosome biogenesis. In sum, our work uncovers new ATG factors, reveals a malleable network of autophagy receptor genetic interactions, and provides a valuable resource (http://crispr.deniclab.com) for further mining of novel autophagy mechanisms.