Hubrecht Institute-KNAW and University Medical Center Utrecht, 3584 CT Utrecht, the Netherlands.
Department of Biotechnology, University of Verona, 37134 Verona, Italy.
Cell Rep. 2020 May 19;31(7):107664. doi: 10.1016/j.celrep.2020.107664.
Cullin-RING ligases (CRLs) control key cellular processes by promoting ubiquitylation of a multitude of soluble cytosolic and nuclear proteins. Subsets of CRL complexes are recruited and activated locally at cellular membranes; however, few CRL functions and substrates at these distinct cellular compartments are known. Here, we use a proteomic screen to identify proteins that are ubiquitylated at cellular membranes and found that Lunapark, an endoplasmic reticulum (ER)-shaping protein localized to ER three-way junctions, is ubiquitylated by the CRL3 ubiquitin ligase. We demonstrate that Lunapark interacts with mechanistic target of rapamycin complex-1 (mTORC1), a central cellular regulator that coordinates growth and metabolism with environmental conditions. We show that mTORC1 binds Lunapark specifically at three-way junctions, and lysosomes, where mTORC1 is activated, make contact with three-way junctions where Lunapark resides. Inhibition of Lunapark ubiquitylation results in neurodevelopmental defects indicating that KLHL12-dependent ubiquitylation of Lunapark is required for normal growth and development.
Cullin-RING 连接酶（CRLs）通过促进大量可溶性细胞质和核蛋白的泛素化来控制关键的细胞过程。CRL 复合物的亚基在细胞膜上被招募并局部激活；然而，在这些不同的细胞隔室中，只有少数 CRL 功能和底物是已知的。在这里，我们使用蛋白质组学筛选来鉴定在细胞膜上泛素化的蛋白质，发现 Lunapark，一种定位于内质网三通接头的内质网（ER）成形蛋白，被 CRL3 泛素连接酶泛素化。我们证明 Lunapark 与机械靶标雷帕霉素复合物-1（mTORC1）相互作用，mTORC1 是一种中央细胞调节剂，可协调生长和代谢与环境条件。我们表明，mTORC1 特异性地在三通接头和溶酶体上结合 Lunapark，其中 mTORC1 被激活，溶酶体与 Lunapark 所在的三通接头接触。抑制 Lunapark 泛素化会导致神经发育缺陷，表明 KLHL12 依赖性 Lunapark 泛素化对于正常生长和发育是必需的。
