Mapping the mammalian ribosome quality control complex interactome using proximity labeling approaches

Abstract

Previous genetic and biochemical studies from Saccharomyces cerevisiae have identified a critical ribosome-associated quality control complex (RQC) that facilitates resolution of stalled ribosomal complexes. While components of the mammalian RQC have been examined in vitro, a systematic characterization of RQC protein interactions in mammalian cells has yet to be described. Here we utilize both proximity-labeling proteomic approaches, BioID and APEX, and traditional affinity-based strategies to both identify interacting proteins of mammalian RQC members and putative substrates for the RQC resident E3 ligase, Ltn1. Surprisingly, validation studies revealed that a subset of substrates are ubiquitylated by Ltn1 in a regulatory manner that does not result in subsequent substrate degradation. We demonstrate that Ltn1 catalyzes the regulatory ubiquitylation of ribosomal protein S6 kinase 1 and 2 (RPS6KA1, RPS6KA3). Further, loss of Ltn1 function results in hyperactivation of RSK1/2 signaling without impacting RSK1/2 protein turnover. These results suggest that Ltn1-mediated RSK1/2 ubiquitylation is inhibitory and establishes a new role for Ltn1 in regulating mitogen-activated kinase signaling via regulatory RSK1/2 ubiquitylation. Taken together, our results suggest that mammalian RQC interactions are difficult to observe and may be more transient than the homologous complex in S. cerevisiae and that Ltn1 has RQC-independent functions.

Rqc1 and other yeast proteins containing highly positively charged sequences are not targets of the RQC complex

“…Taking our data into account, we speculate that the K-rich basic sequence can function as the ubiquitylation site involved in Rqc1 regulation. Since it was previously reported that Ltn1 could ubiquitylate substrates independently of the RQC complex [80,81], we speculate that Ltn1 might regulate Rqc1 in a process that is uncoupled from Rqc1 translation.…”

EDF1 coordinates cellular responses to ribosome collisions

“…In light of its critical binding site on colliding ribosomes and its general impact on reactions critical to cellular signaling, we wondered whether EDF1 might act as a molecular scaffold to recruit other QC factors to the disome interface. To define the interactome of EDF1 under basal growth conditions and under conditions that induce ribotoxic stress, we performed both traditional immunoaffinity purification and proximity-based labeling (BioID) approaches ( Roux et al, 2013 ; Zuzow et al, 2018 ). To avoid artefacts associated with protein overexpression, and in light of the natural high abundance of EDF1 (~10 5 copies per cell, Wiśniewski et al, 2014 ), we first immunoaffinity purified endogenous EDF1 from untreated (UT) HEK293T cells or those treated with low dose emetine (EL; 1.8 µM, 15 min) using Protein A coupled EDF1-antibody ( Figure 5A and Figure 5—figure supplement 1A ).…”

“…For example, genome wide screens in yeast identified the endonuclease Cue2 (N4BP2 in mammals) that degrades problematic mRNAs through the No-Go decay pathway ( D'Orazio et al, 2019 ), QC factors such as Hel2, Asc1, and Slh1 (ZNF598, RACK1, and ASCC3 in mammals, respectively) involved in the recognition and resolution of stalled ribosomes ( Brandman et al, 2012 ; Kuroha et al, 2010 ; Letzring et al, 2013 ), and downstream peptide targeting factors such as Rqc2, Ltn1, and Cdc48 (NEMF, Listerin, and VCP in mammals, respectively) ( Bengtson and Joazeiro, 2010 ; Brandman et al, 2012 ). Proteomic approaches have also been employed in yeast and mammals to identify ribosome-mediated QC factors, though these have generally relied on candidate-based screens involving affinity purification of known factors ( Garzia et al, 2017 ; Matsuo et al, 2017 ; Sitron et al, 2017 ; Zuzow et al, 2018 ).…”

Proximity Dependent Biotinylation: Key Enzymes and Adaptation to Proteomics Approaches

“…The list of enzymes for PDB is still growing, but because most studies use a single enzyme, systematic comparisons are still largely lacking. A notable exception, that performed both APEX (APEX2) and BioID (BirA*) on baits within the ribosome quality control pathway, revealed little overlap between the approaches (and a better recall of known interactors with BioID) (144). While it is not clear whether this is a widespread phenomenon, it raises interesting questions regarding the degree of complementarity between these different methods.…”