A ribosome-bound quality control complex triggers degradation of nascent peptides and signals translation stress

Abstract

The conserved transcriptional regulator heat shock factor 1 (Hsf1) is a key sensor of proteotoxic and other stress in the eukaryotic cytosol. 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.

Figure 1. A genome-wide screen reveals that Hsf1 senses diverse stress conditions

(A) Schema for fluorescent Hsf1 reporter. An RFP driven by the TEF2 promoter and a GFP driven by a synthetic promoter with multiple Hsf1 binding sites were integrated in the URA3 locus of the yeast genome. (B) Independent crosses of reporter strain into 731 loss-of-function alleles (selected hits from full genome screen) shows allelic variation and reproducibility of the reporter system. (C) Selected categories of annotated functions from genome-wide screen. Red bars indicate number of strains below a standard deviation for selected categories. Genes beyond one standard deviation are labeled as space is available, with full results in Table S1. (D) Hsf1 and Msn2/4 activities with genome-wide library of alleles at steady state (25°C) and after 1 hour heat shock at 37°C. P-values from student’s t-test show enrichment for selected categories in each quadrant (delimited by 1 standard deviation from the median). (E) Effects of constitutively active hsf1 allele (hsf1*) and msn2 allele (msn2*) on Hsf1 and Msn2/4 activities at 25°C. Also see Tables S1 and S2 and Figure S1.

Figure 2. Translation stress identifies Ltn1 and Rqc1

(A) Schematic diagram illustrating strategy for quantifying genetic interactions in double mutant strains. An expected value for the double mutant is first computed based on single mutant reporter levels. The expected value is then compared to the actual double mutant value to arrive at a genetic interaction score. Positive genetic interactions (double mutant is higher than expected) are colored in yellow while negative genetic interactions (double mutant is lower than expected) are colored in blue. (B) Genetic interactions corresponding to a set of translation-related genes that clustered together in a genetic interaction map. (C–D) Hsf1 activity levels in single and double mutant strains. The y-axis of each graph shows double mutant values for a common mutant (rps0aΔ or rqc1Δ) combined with diverse alleles. Single mutant values appear on the x-axis. (E) Comparison of Ltn1 and Rqc1 genetic interaction scores. Inset: Top enriched Gene Ontology (GO) categories of positive interactors with rqc1Δ from full genome screen (see Tables S1 an S3). (F) HSP82 reporter levels showing a positive genetic interaction between rqc1Δ and rps0aΔ (red line denotes expected value of double mutant). Translation and RNA related genes are marked in red in (D–E).

Figure 3. Rqc1/Ltn1 organize a larger cotranslational quality control complex

(A) Immunoprecipation (IP) of endogenous Rqc1 3xFLAG fusion protein viewed with Coomassie staining. Selected non-background bands identified by mass spectrometry are labeled (all identified non-background bands available in Figure S2) (B) Silver staining of Rqc1 and Tae2 IPs in selected deletion backgrounds and with cycloheximide (CHX) (100 ug/ml, added 2 min before harvesting). Below, western blot for Cdc48 in IPs along with quantified amounts (Cdc48/FLAG). (C) RNA absorbance (260 nm) of 10–50% sucrose gradient for input and output of Rqc1 IP. Each trace is independently scaled. (D) Class averages of particles selected from electron micrographs of negatively stained Rqc1-FLAG IP in WT, tae2Δ and ltn1Δ strain backgrounds. (E) Western blot of a cotranslationally degraded model substrate containing a polybasic region in selected genetic backgrounds. (F) GFP levels in samples from (E) measured using a flow cytometer and normalized to control. (G) Western blot for mono-ubiquitin in IP samples. Also see Figures S2–4.

Figure 4. Rqc1 levels are autoregulated by a conserved negative feedback loop

(A) GFP levels in strains expressing a contranslationally degraded polybasic reporter subject to 10 hours cycloheximide treatment at the indicated concentration. (B) Ribosome footprint density at endogenous polybasic stretches (6 or greater K or R per 10 residues, N=103). (C) Conservation of Rqc1, with polybasic and TCF25 (Bateman et al., 2004) domains highlighted. (D) Assay showing the ability of Rqc1 alleles (WT, rqc1-FLAG, rqc1_ala-FLAG, rqc1Δ) to act upon a model cotranslationally degraded substrate. (E) rqc1-FLAG and rqc1_ala-FLAG protein levels in deletion strains. (F) Results of screen for regulators model polybasic substrate (full results in Table S1). tae2Δ and the four strongest hits labeled. (G) GFP and RFP levels of model polybasic substrate in selected hits from full-genome screen. Also see Table S4 and Figure S5.

Figure 5. Tae2 is responsible for a distinct Hsf1 branch that monitors translation stress

(A) Hsf1 activity in deletions strains for the RQC and a ribosomal subunit causing synergistic activation of Hsf1. (B) GFP levels of the cotranslationally degraded reporter construct in selected strain backgrounds from (A). (C) Effect of TAE2 deletion on Hsf1 activity arising from non-translation stresses. Also see Figure S6.

Figure 6. Hsf1 senses translation stress distinctly from other cellular stresses

(A) Schematic diagram illustrating genetic interactions between loss-of-function alleles and mutant Hsf1 alleles. Procedure is identical to that of Figure 2A except interactions are computed between hsf1 mutants and loss of function alleles (or pairs of alleles, as are shown in (B)). (B) Genetic interactions between hsf1 mutants and double mutant alleles activating translation stress signaling. Each point represents one hsf1 mutant. Strong positive interactors (1) and negative interactors (2) are circled. (C) Genetic interactions between hsf1 mutants circled in (B) and loss-of-function alleles having strong effect on Hsf1 activity. The hierarchical clustering tree shown was calculated from the full set of 290 hsf1 mutants, not just the six shown. (D) Hsf1 reporter levels showing genetic interactions between mutations to a region altered in each member of the positive interacting group (I246N and G244V) and genetic backgrounds inducing translation stress. Red marks denote expected values and deviations from these reveal genetic interactions.

Figure 7. Schematic model for RQC-mediated degradation of nascent chains

The 80S ribosome stalls during translation (left panel) and, for polybasic substrates (++++ symbol), this is recognized by Asc1 and Hel2 leading to translation termination and possibly RNA cleavage. Ltn1, Rqc1, and Tae2 are then recruited and the 40S subunit disassociates (the order of these events remains to be determined). Ltn1 then ubiquitylates the nascent chain (second panel) leading to recruitment of Cdc48 and its cofactors Npl4 and Ufd1 (third panel). In addition, to its role in substrate ubiquitylation, Tae2 signals translation stress to Hsf1. Levels of Rqc1 are downregulated by the activity of the RQC leading to a negative feedback loop controlling overall activity of the pathway.