Abstract
Ribosomes can stall during translation due to defects in the mRNA template or translation machinery, leading to the production of incomplete proteins. The Ribosome-associated Quality control Complex (RQC) engages stalled ribosomes and targets nascent polypeptides for proteasomal degradation. However, how each RQC component contributes to this process remains unclear. Here we demonstrate that key RQC activities-Ltn1p-dependent ubiquitination and Rqc2p-mediated Carboxy-terminal Alanine and Threonine (CAT) tail elongation-can be recapitulated in vitro with a yeast cell-free system. Using this approach, we determined that CAT tailing is mechanistically distinct from canonical translation, that Ltn1p-mediated ubiquitination depends on the poorly characterized RQC component Rqc1p, and that the process of CAT tailing enables robust ubiquitination of the nascent polypeptide. These findings establish a novel system to study the RQC and provide a framework for understanding how RQC factors coordinate their activities to facilitate clearance of incompletely synthesized proteins.
Highlights
Eukaryotic cells contain several cotranslational quality-control pathways that limit the production of aberrant proteins and thereby maintain protein homeostasis
We have shown that establishing a cell-free system that recapitulates both Carboxy-terminal Alanine and Threonine (CAT) tailing and ubiquitination opens new opportunities to explore how the fully functional Ribosome-associated Quality control Complex (RQC) promotes clearance of aberrant translation products
Our analyses reveal that Rqc2p-mediated nascent-chain elongation is mechanistically distinct from canonical translation, that ubiquitination of the nascent polypeptide requires both Ltn1p and Rqc1p, and that the ubiquitination and CAT-tailing activities of the RQC are coupled through a mutual requirement for active Rqc2p (Figure 5)
Summary
Eukaryotic cells contain several cotranslational quality-control pathways that limit the production of aberrant proteins and thereby maintain protein homeostasis. One such pathway is activated when a ribosome fails to complete translation, leading to the recruitment of specialized factors that disassemble the stalled ribosome and facilitate degradation of the nascent protein (Brandman and Hegde, 2016; Shoemaker and Green, 2012). A key effector of this process is the highly conserved Ribosome-associated Quality control Complex (RQC), which in budding yeast comprises the E3 ubiquitin ligase Ltn1p, the ATPase Cdc48p, and the poorly characterized proteins Rqc1p and Rqc2p (Brandman et al, 2012; Defenouillere et al, 2013; Verma et al, 2013)—the human homologs of which are Listerin, VCP/p97, TCF25, and NEMF, respectively. Ltn1p facilitates ubiquitination of the nascent chain while on the 60S subunit, marking the incompletely synthesized protein for proteasomal degradation (Bengtson and Joazeiro, 2010; Shao et al, 2013)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
