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

Laurensia Yuniati; Angela Lauriola; Manouk Gerritsen; Susana Abreu; Eric Ni; Chiara Tesoriero; Jacob O Onireti; Teck Yew Low; Albert J R Heck; Andrea Vettori; Timothy Cardozo; Daniele Guardavaccaro

doi:10.1016/j.celrep.2020.107664

Ubiquitylation of the ER-Shaping Protein Lunapark via the CRL3^KLHL12 Ubiquitin Ligase Complex

Cell Rep. 2020 May 19;31(7):107664. doi: 10.1016/j.celrep.2020.107664.

Affiliations

¹ Hubrecht Institute-KNAW and University Medical Center Utrecht, 3584 CT Utrecht, the Netherlands.
² Department of Biotechnology, University of Verona, 37134 Verona, Italy.
³ Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, NYU Langone Health, New York, NY 10016, USA.
⁴ UKM Medical Molecular Biology Institute (UMBI), Universiti Kebangsaan Malaysia, 56000 Kuala Lumpur, Malaysia.
⁵ Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, 3584 CH Utrecht, the Netherlands; The Netherlands Proteomics Center, 3584 CH Utrecht, the Netherlands.
⁶ Hubrecht Institute-KNAW and University Medical Center Utrecht, 3584 CT Utrecht, the Netherlands; Department of Biotechnology, University of Verona, 37134 Verona, Italy. Electronic address: daniele.guardavaccaro@univr.it.

PMID: 32433973
DOI: 10.1016/j.celrep.2020.107664

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 CRL3^KLHL12 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.

Keywords: ER three-way junction; Lunapark; cullin-RING ligases; endoplasmic reticulum; lysosome; mTORC1; ubiquitin.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Adaptor Proteins, Signal Transducing / metabolism*
Animals
Animals, Genetically Modified
Cell Line, Tumor
Cullin Proteins / metabolism*
Endoplasmic Reticulum / metabolism*
HEK293 Cells
Humans
Mechanistic Target of Rapamycin Complex 1 / metabolism
Membrane Proteins / metabolism*
Ubiquitin-Protein Ligases / metabolism*
Ubiquitination
Zebrafish

Substances

Adaptor Proteins, Signal Transducing
Cullin Proteins
KLHL12 protein, human
LNPK protein, human
Membrane Proteins
Ubiquitin-Protein Ligases
Mechanistic Target of Rapamycin Complex 1