Abstract
Ubiquitin-dependent proteolysis can initiate at ribosomes for myriad reasons including misfolding of a nascent chain or stalling of the ribosome during translation of mRNA. Clearance of a stalled complex is required to recycle the ribosome for future use. Here we show that the ubiquitin (Ub) pathway segregase Cdc48/p97 and its adaptors Ufd1-Npl4 participate in ribosome-associated degradation (RAD) by mediating the clearance of ubiquitinated, tRNA-linked nascent peptides from ribosomes. Through characterization of both endogenously-generated and heterologous model substrates for the RAD pathway, we conclude that budding yeast Cdc48 functions downstream of the Ub ligases Ltn1 and Ubr1 to release nascent proteins from the ribosome so that they can be degraded by the proteasome. Defective RAD could contribute to the pathophysiology of human diseases caused by mutations in p97.DOI:http://dx.doi.org/10.7554/eLife.00308.001.
Highlights
Maintaining protein homeostasis is key for the healthy lifespan of the organism (Koga et al, 2011) and cells have evolved sophisticated mechanisms to control the quality of newly synthesized proteins (Bukau et al, 2006; Smith et al, 2011)
Deletion of LTN1 leads to hygromycin-sensitivity and since Ltn1 is involved in quality control (QC) of non-stop decay (NSD) pathway substrates (Bengtson and Joazeiro, 2010), we explored the possibility that Cdc48–Ufd1–Npl4 and one or more Ubx proteins function in this pathway
Coomassie blue staining of purified ribosomes confirmed that Cdc48 activity was not required for their assembly (Figure 1, Figure 1—figure supplement 2A, lane 3), a conclusion that was further validated by sucrose gradient fractionation of ribosomes from wildtype, cdc48-3, and ufd1-2 whole cell lysates (Figure 1—figure supplement 2B)
Summary
Maintaining protein homeostasis is key for the healthy lifespan of the organism (Koga et al, 2011) and cells have evolved sophisticated mechanisms to control the quality of newly synthesized proteins (Bukau et al, 2006; Smith et al, 2011). Depending upon the relative rate at which malfolded sequences that comprise degrons form and bind their Ub ligase receptors vs the rate at which translation is completed, ubiquitination and degradation of misfolding proteins may initiate on the ribosome. It has been shown, for instance, that the acetylated N-terminus recognized by the Ub ligase Doa (Hwang et al, 2010) is generated co-translationally (Gautschi et al, 2003; Polevoda et al, 2008), and that the Ub ligase Ubr can initiate degradation of an N-end rule substrate prior to completion of its translation (Turner and Varshavsky, 2000)
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