Ubiquitylation of the ER-Shaping Protein Lunapark via the CRL3KLHL12 Ubiquitin Ligase Complex

Abstract

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 CRL3KLHL12 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.