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CAT tails drive degradation of stalled polypeptides on and off the ribosome

Nature Structural & Molecular Biology volume 26, pages 450–459 (2019)Cite this article

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Abstract

Stalled translation produces incomplete, ribosome-tethered polypeptides that the ribosome-associated quality control (RQC) pathway targets for degradation via the E3 ubiquitin ligase Ltn1. During this process, the protein Rqc2 and the large ribosomal subunit elongate stalled polypeptides with carboxy-terminal alanine and threonine residues (CAT tails). Failure to degrade CAT-tailed proteins disrupts global protein homeostasis, as CAT-tailed proteins can aggregate and sequester chaperones. Why cells employ such a potentially toxic process during RQC is unclear. Here, we developed quantitative techniques to assess how CAT tails affect stalled polypeptide degradation in Saccharomyces cerevisiae. We found that CAT tails enhance the efficiency of Ltn1 in targeting structured polypeptides, which are otherwise poor Ltn1 substrates. If Ltn1 fails to ubiquitylate those stalled polypeptides or becomes limiting, CAT tails act as degrons, marking proteins for proteasomal degradation off the ribosome. Thus, CAT tails functionalize the carboxy termini of stalled polypeptides to drive their degradation on and off the ribosome.

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Fig. 1: Loss of CAT tails stabilizes specific RQC substrates.
Fig. 2: Conditions that favor RQC substrate folding increase CAT tail dependence.
Fig. 3: CATylated RQC substrates are degraded independently of Ltn1.
Fig. 4: CAT tails are degrons.
Fig. 5: Decomposition of the contribution of CAT tails to RQC substrate degradation.

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Data availability

The datasets that inform the conclusions of this study are available from the corresponding author upon request.

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Acknowledgements

We thank S. Marqusee, J. Frydman, R. Hegde and B. Lu for helpful discussions. We thank L. Steinman, R. Kopito, E.P. Geiduschek and members of the Brandman and Kopito Labs for comments on the manuscript. We acknowledge J. Work (Stanford University, Stanford, CA, USA) for his gift of the 10–31 degron and control plasmids. J. Schulze (University of California at Davis, Davis, CA, USA) performed the amino acid analysis at the UC Davis Genome Center Molecular Structure Facility. Stanford University (O.B.), the US National Institutes of Health (grant No. R01GM115968 to O.B.), and the National Institute of General Medical Sciences of the US National Institutes of Health (grant No. T32GM007276 to C.S.S.) supported this work.

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  1. Department of Biochemistry, Stanford University, Stanford, CA, USA

    Cole S. Sitron & Onn Brandman

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Contributions

O.B. and C.S.S. conceived of the study and designed the experiments. C.S.S. performed the experiments. O.B. and C.S.S. analyzed and interpreted the data. O.B. and C.S.S. wrote the manuscript. O.B. supervised the study.

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Correspondence to Onn Brandman.

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Integrated supplementary information

Supplementary Fig. 1 Controlling for substrate expression to enable more faithful measurement of RQC substrate degradation.

a, Mean RFP measurements from an internal expression control for a model RQC substrate. GST (glutathione S-transferase) was used in place of GFP to eliminate GFP bleed-through into the RFP channel when measuring the levels of the RFP expression control in this experiment. b, Comparison of mean expression-controlled (GFP:RFP) and bulk (GFP) measurements of a non-stalling two-color construct. c, Normalized stability measurements for two model RQC substrates (schematic above) from Fig. 1e with additional ltn1∆ background controls displayed (n = 5 independent cultures). Data are normalized as in Fig. 1. d, Quantification of a stalling reporter similar to that developed by the Hegde and Bennett laboratories (schematic above)9,11. The 12xArg stalling sequence matches that from other model RQC substrates used. hel2∆ and slh1∆ strains have previously been shown to be deficient in stalling11,12,13,17. e, Non-normalized stability measurements from a model RQC substrate, RQCsubLONG (schematic above), expressed from the moderate MET17 promoter and the strong TDH3 promoter. f, Additional ltn1∆ background controls displayed for stability data in Fig. 1f. For all bar plots, error bars indicate s.e.m. from n = 3 independent cultures unless otherwise indicated.

Supplementary Fig. 2 Additional ltn1∆ background controls for substrates in Fig. 2.

a–c, Normalized stability measurements of two model RQC substrates (schematic above) from Fig. 2c–e with additional ltn1∆ background controls displayed. Cartoons of structural features of the substrates shown emerging from the ribosome exit tunnel below. For all bar plots, data is normalized as in Fig. 1f and error bars indicate s.e.m. from n = 3 independent cultures.

Supplementary Fig. 3 hul5∆ stabilizes an RQC substrate in the ltn1∆ background comparably to loss of CATylation.

a, Mean stability of RQCsubLONG in the ltn1∆ background with or without deletion of the vacuolar proteinase maturation factor PEP4. Error bars are s.e.m. from n = 3 independent cultures. The results of a one-way, one-tailed ANOVA test with 4 degrees of freedom are indicated above bars. b, IB of cells expressing RQCsubLONG (schematic above) with and without TEV protease co-expression. c, Normalized stability of RQCsubLONG in the ltn1∆ background with additional deletions of ubiquitin proteasome system factors. Data are presented as mean, normalized against ltn1∆ (normalized to 0) and rqc2-d98a ltn1∆ (normalized to 1). d, RQCsubLONG stability after cycloheximide (200 μg/ml) pulse in the ltn1∆ background with or without perturbations to CATylation (rqc2-d98a) or HUL5. Data are normalized to the value measured just before cycloheximide addition and error bars indicate s.e.m. from n = 3 independent cultures. e, Amino acid analysis of RQCsubLONG IPed from indicated strains competent in CATylation (ltn1∆), with impaired CATylation (rqc2-d98a ltn1∆), or with impaired HUL5 (hul5∆ ltn1∆). f, Mean stability measurements of a two color construct with or without a non-stalling C-terminal degron, called “10–31.” Results from one-way, one-tailed ANOVA tests with 4 degrees of freedom indicated above bars. Error bars indicate s.e.m. from n = 3 independent cultures.

Supplementary Fig. 4 Effects of Ltn1-independent degradation blockade on substrate size and solubility.

a, IB replicates of ltn1∆ whole cell extracts containing RQCsubLONG with or without HUL5 deletion. b, IB of RQCsubLONG from whole cell extracts of ltn1∆ with indicated RQC2 alleles that produce CAT tails of varying size. c, Stability measurements from ltn1∆ cells carrying different RQC2 alleles, expressing RQCsubLONG. Data are normalized as in Supplementary Fig. 3b and error bars indicate s.e.m. from n = 3 independent cultures. d, IB of fractions from supernatant-pellet fraction of ltn1∆ lysates expressing RQCsubLONG.

Supplementary Fig. 5 Additional data for decomposition of CAT tail function.

a, Stability measurements of RQCsubLONG and RQCsubLONG with the C-terminal GFP lysine residue mutated (RQCsubLONG-KlastR, as in Fig. 1f) with additional perturbations of HUL5 deletion, TEV co-expression, and RQC2 mutation. Data are presented as mean, normalized against the no perturbation condition (normalized to 0) and rqc2-d98a (normalized to 1). Error bars indicate s.e.m. from n = 3 independent cultures and results of indicated one-way, one-tailed ANOVA tests appear above the bars (4 d.o.f.). Because the value in the hul5∆ + in vivo TEV condition was slightly greater with RQC2-WT than rqc2-d98a, we considered all CAT tail-mediated degradation to be abolished in the former condition (RQC2-WT hul5∆ + in vivo TEV).

Supplementary Fig. 6 Example workflow for flow cytometry analysis.

a, Example log-scale scatter plot of flow cytometry results from single cultures of indicated strains expressing RQCsubLONG (for schematic see Fig. 1e) or RFP-(T2A)2-GST-stall (an RFP-only control to assess RFP bleed-through into GFP channel, for schematic see Supplementary Fig. 1a). Each set of points represents a single independent culture that contributed towards the measurements in Supplementary Fig. 1c. GFP fluorescence was measured through the FL1-A channel and RFP was measured through the FL4-A channel. Cells were gated based on RFP fluorescence to determine cells for which the GFP:RFP ratio (slope) was linear. The upper and lower bounds of this gate are indicated by the dotted vertical lines. b, Mean GFP:RFP from cultures in a. While a is on a log scale, b is on a linear scale. c, GFP:RFP from cultures expressing RQCsubLONG in b after subtraction of RFP-only background from RFP-(T2A)2-GST-stall. d, GFP:RFP from c normalized to rqc2-d98a ltn1∆. Plots derived in a similar fashion to c and d appear throughout the figures.

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Sitron, C.S., Brandman, O. CAT tails drive degradation of stalled polypeptides on and off the ribosome. Nat Struct Mol Biol 26, 450–459 (2019). https://doi.org/10.1038/s41594-019-0230-1

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