In vitro analysis of RQC activities provides insights into the mechanism and function of CAT tailing
- University of California, San Francisco, United States
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.
Article and author information
Author details
Funding
National Science Foundation (Graduate Research Fellowship)
- Beatriz A Osuna
UCSF Mortiz-Heyman Discovery Fellowship (Graduate Student Research Fellowship)
- Beatriz A Osuna
UCSF Hillblom Fellowship (Graduate Student Research Fellowship)
- Conor J Howard
Searle Scholars Program (13SSP218)
- Adam Frost
NIH Office of the Director (DP2GM110772)
- Adam Frost
UCSF Program for Breakthrough Biomedical Research funded in part by the Sandler Foundation
- Adam Frost
- David E Weinberg
NIH Office of the Director (DP5OD017895)
- David E Weinberg
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2017, Osuna et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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In vitro analysis of RQC activities provides insights into the mechanism and function of CAT tailing
eLife 6:e27949.
https://doi.org/10.7554/eLife.27949
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