Cdc48-associated complex bound to 60S particles is required for the clearance of aberrant translation products

Abstract

Ribosome stalling on eukaryotic mRNAs triggers cotranslational RNA and protein degradation through conserved mechanisms. For example, mRNAs lacking a stop codon are degraded by the exosome in association with its cofactor, the SKI complex, whereas the corresponding aberrant nascent polypeptides are ubiquitinated by the E3 ligases Ltn1 and Not4 and become proteasome substrates. How translation arrest is linked with polypeptide degradation is still unclear. Genetic screens with SKI and LTN1 mutants allowed us to identify translation-associated element 2 (Tae2) and ribosome quality control 1 (Rqc1), two factors that we found associated, together with Ltn1 and the AAA-ATPase Cdc48, to 60S ribosomal subunits. Translation-associated element 2 (Tae2), Rqc1, and Cdc48 were all required for degradation of polypeptides synthesized from NonStop mRNAs (Non-Stop protein decay; NSPD). Both Ltn1 and Rqc1 were essential for the recruitment of Cdc48 to 60S particles. Polysome gradient analyses of mutant strains revealed unique intermediates of this pathway, showing that the polyubiquitination of Non-Stop peptides is a progressive process. We propose that ubiquitination of the nascent peptide starts on the 80S and continues on the 60S, on which Cdc48 is recruited to escort the substrate for proteasomal degradation.quality control | Saccharomyces cerevisiae

Structural basis for translational surveillance by the large ribosomal subunit-associated protein quality control complex

“…Ltn1 was found only in association with 60S ribosomal subunits in the samples analyzed, and there was no indication that it can bind to 80S ribosomes. This observation supports previous data showing that Ltn1 is predominantly found in the 60S fraction of sucrose gradients (4), copurifies with 60S subunits (4,7,9), and relies on ribosome splitting for function (17). To understand the structural elements underlying this selectivity, the segmented Ltn1 density from RQC ΔR was docked onto an 80S ribosome.…”

“…4 F-I), Tae2 and the 40S subunit cannot bind simultaneously to the 60S subunit. This observation explains Tae2's reported 60S subunit binding selectivity (7,9), as is also the case with Ltn1 (Figs. 3 and 4).…”

“…4 A and B and Fig. S6 also confirms that Ltn1 is able to bind to ribosomes in cells deficient for Tae2 (7,9), although Ltn1 binding is less stable (9) and the ubiquitylation and degradation of nonstop substrates is reduced in Tae2's absence (7,9).…”

“…Mutation of the Ltn1 mouse ortholog, listerin, causes neurodegeneration (6), suggesting an important function for this process. Ltn1 works together with several cofactors as part of the ribosome-associated quality control complex (RQC) (7)(8)(9) and appears to first associate with nascent chain-stalled 60S subunits together with two proteins of unknown function, Tae2 and Rqc1 (7,9). Ltn1-mediated ubiquitylation of the stalled polypeptide then results in the recruitment of the AAA ATPase Cdc48/p97/ VCP.…”

“…Ltn1-mediated ubiquitylation of the stalled polypeptide then results in the recruitment of the AAA ATPase Cdc48/p97/ VCP. This recruitment also requires Rqc1 and Tae2, and is followed by Cdc48-mediated nascent chain extraction and delivery to the proteasome for degradation (7)(8)(9).…”