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J Mol Biol. 2019 Jan 18;431(2):123-141. doi: 10.1016/j.jmb.2018.10.009. Epub 2018 Oct 24.

BiP/GRP78 Mediates ERAD Targeting of Proteins Produced by Membrane-Bound Ribosomes Stalled at the STOP-Codon.

Author information

1
Laboratory of Molecular Immunology, International Centre for Genetic Engineering and Biotechnology, ICGEB, Padriciano 99, 34149 Trieste, Italy.
2
Laboratory of Protein Networks, International Centre for Genetic Engineering and Biotechnology, ICGEB, Padriciano 99, 34149 Trieste, Italy.
3
Centre for Integrative Biology (CIBIO), University of Trento, Via Sommarive 9, 38123 Trento, Italy; Center for Sustainable Future Technologies, Fondazione Istituto Italiano di Tecnologia, C.so Trento 21, 10129 Torino, Italy; Center for Sustainable Future Technologies, Fondazione Istituto Italiano di Tecnologia, C.so Trento 21, 10129 Torino, Italy.
4
Centre for Integrative Biology (CIBIO), University of Trento, Via Sommarive 9, 38123 Trento, Italy. Electronic address: gianluca.petris@unitn.it.
5
Laboratory of Molecular Immunology, International Centre for Genetic Engineering and Biotechnology, ICGEB, Padriciano 99, 34149 Trieste, Italy. Electronic address: burrone@icgeb.org.

Abstract

Translational stalling of ribosome bound to endoplasmic reticulum (ER) membrane requires an accurate clearance of the associated polypeptides, which is not completely understood in mammals. We characterized in mammalian cells the model of ribosomal stalling at the STOP-codon based on proteins tagged at the C-terminus with the picornavirus 2A peptide followed by a termination codon instead of the Proline (2A*). We exploited the 2A* stalling model to characterize the pathway of degradation of ER-targeted polypeptides. We report that the ER chaperone BiP/GRP78 is a new main factor involved. Moreover, degradation of the ER-stalled polypeptides required the activities of the AAA-ATPase VCP/p97, its associated deubiquitinylase YOD1, the ribosome-associated ubiquitin ligase Listerin and the proteasome. In human proteome, we found two human C-terminal amino acid sequences that cause similar stalling at the STOP-codon. Our data suggest that translational stalling at the ER membrane activates protein degradation at the interface of ribosomal- and ER-associated quality control systems.

KEYWORDS:

2A peptide; BiP; ER-associated degradation; STOP-codon; ribosomal stalling

PMID:
30367842
DOI:
10.1016/j.jmb.2018.10.009

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