A Ribosome-Bound Quality Control Complex Triggers Degradation of Nascent Peptides and Signals Translation Stress

Abstract

Summary The conserved transcriptional regulator Heat Shock Factor 1 (Hsf1) is a key sensor of proteotoxic and other stress in the eukaryotic cytosol, yet its regulation is poorly understood. We surveyed Hsf1 activity in a genome-wide loss-of-function library in Saccaromyces cerevisiae as well as ~78,000 double mutants and found Hsf1 activity to be modulated by highly diverse stresses. These included disruption of a ribosome-bound complex we named the Ribosome Quality Control Complex (RQC) comprising the Ltn1 E3 ubiquitin ligase, two highly conserved but poorly characterized proteins (Tae2 and Rqc1), and Cdc48 and its cofactors. Electron microscopy and biochemical analyses revealed that the RQC forms a stable complex with 60S ribosomal subunits containing stalled polypeptides and triggers their degradation. A negative feedback loop regulates the RQC and Hsf1 senses an RQC-mediated translation stress signal distinctly from other stresses. Our work reveals the range of stresses Hsf1 monitors and elucidates a conserved cotranslational protein quality control mechanism.

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.…”

“…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).…”

Single-particle EM reveals extensive conformational variability of the Ltn1 E3 ligase

“…Currently, the regions involved in the Ltn1-ribosome association, and whether this association occurs directly or indirectly, are not understood. Drawing on the parallels with CRLs, as well as on the observations that Ltn1 associates with the 60S ribosomal subunit (7,27) and that its N terminus is conserved in evolution, we suggest that this portion of the protein may contain a site that confers specificity in ribosome attachment (Fig. 6A).…”

PATACSDB—the database of polyA translational attenuators in coding sequences

“…Over the years, we have got to know that certain sequence features can trigger ribosome stalling. These are damaged bases (Cruz-Vera et al, 2004), stable stem-loop structures (Doma & Parker, 2006), rare codons (Letzring, Dean & Grayhack, 2010), mRNAs lacking stop codons (so called non-stop mRNAs) (Dimitrova et al, 2009), runs of codons that encode consecutive basic aminoacids (Kuroha et al, 2010;Brandman et al, 2012), or finally, runs of adenines encoding poly-lysine tracks Arthur et al, 2015).…”