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

Area of Science:

• Cellular biology
• Molecular mechanisms of protein quality control
• Ubiquitination and signaling pathways

Background:

• The ribosome-associated quality control (RQC) complex resolves stalled ribosomal complexes.
• Mammalian RQC components have been studied in vitro, but cellular interactions remain poorly characterized.
• Ltn1 is an E3 ligase resident in the RQC, implicated in substrate ubiquitylation.

Purpose of the Study:

• To systematically characterize protein interactions of mammalian RQC members in cells.
• To identify substrates of the RQC-resident E3 ligase, Ltn1.
• To elucidate the regulatory role of Ltn1 in mammalian cells.

Main Methods:

• Proximity-labeling proteomic approaches (BioID and APEX).
• Traditional affinity-based protein interaction strategies.
• Validation studies including ubiquitylation assays and functional analysis of Ltn1 loss-of-function.

Main Results:

• Identified interacting proteins of mammalian RQC members and putative Ltn1 substrates.
• Discovered that Ltn1 ubiquitylates a subset of substrates, including RSK1/2, in a regulatory, non-degradative manner.
• Demonstrated that loss of Ltn1 leads to RSK1/2 hyperactivation without affecting protein turnover, indicating inhibitory ubiquitylation.

Conclusions:

• Mammalian RQC interactions are transient and difficult to observe compared to yeast.
• Ltn1 regulates mitogen-activated kinase signaling through inhibitory ubiquitylation of RSK1/2.
• Ltn1 possesses RQC-independent functions in cellular signaling regulation.