In vitro analysis of RQC activities provides insights into the mechanism and function of CAT tailing

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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.

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Beatriz A Osuna

Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States

Competing interests
The authors declare that no competing interests exist.
Conor J Howard

Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States

Competing interests
The authors declare that no competing interests exist.
Subheksha KC

Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States

Competing interests
The authors declare that no competing interests exist.
Adam Frost

Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States

For correspondence
adam.frost@ucsf.edu

Competing interests
The authors declare that no competing interests exist.
David E Weinberg

Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States

For correspondence
david.weinberg@ucsf.edu

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-9348-1709

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.

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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.