Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States; Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States
Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States; California Institute for Quantitative Biomedical Research, San Francisco, United States; Chan Zuckerberg Biohub, San Francisco, United States
Subheksha KC
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States
Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States; California Institute for Quantitative Biomedical Research, San Francisco, United States; Chan Zuckerberg Biohub, San Francisco, United States; Department of Biochemistry, University of Utah, Salt Lake City, United States
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States; Sandler Faculty Fellows Program, University of California, San Francisco, San Francisco, United States
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.
