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

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 Ltn1’s efficiency 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.

Aggregation of CAT tails blocks their degradation and causes proteotoxicity in S. cerevisiae

“…Single-color model RQC substrates like RQCsub, while useful for visualizing CAT tails by SDS-PAGE and microscopy, have limited utility in degradation measurements due to noisy expression (Sitron and Brandman, 2019). To assess how aggregation affects degradation of CATylated RQC substrates, we utilized the two-color model RQC substrate RQCsub LONG (Sitron and Brandman, 2019) (Figure 1D,E). RQCsub LONG includes an internal expression control, RFP followed by viral T2A peptides, at the N-terminus of GFP-linker-arginine (Figure 1E).…”

“…The ratio of GFP:RFP at the single-cell level serves as an internal, expression-controlled readout of RQCsub LONG stability (Figure 1E). CAT tail-mediated degradation of RQCsub LONG in the absence of Ltn1 requires the proteasome-associated E3 ubiquitin ligase Hul5 and the proteasome (Sitron and Brandman, 2019). Therefore, comparing stability before and after HUL5 deletion quantifies CAT tail-mediated degradation (Figure 1E).…”

“…To quantify CAT tail degradation, we measured RQCsub LONG stability in ROLD and ROLD bud27 Δ cells (Figure 3D). The difference in RQCsub LONG stability (GFP:RFP) in each of these backgrounds with Hul5 (degradation intact) and without Hul5 (degradation blocked) defined the degree of degradation (Sitron and Brandman, 2019). We validated this approach by demonstrating that the proteasome inhibitor bortezomib did not significantly stabilize RQCsub LONG in ROLD hul5 Δ and ROLD bud27 Δ hul5 Δ strains (Figure 3-figure supplement 1E), indicating that HUL5 deletion blocked degradation in the ROLD and ROLD bud27 Δ backgrounds.…”

“…A conserved back-up degradation pathway mediated by Rqc2 and its prokaryotic homologs mitigates some of the toxicity associated with loss of tmRNA or Ltn1 function (Lytvynenko et al, 2019; Sitron and Brandman, 2019). Rqc2 homologs bind the large ribosomal subunit and direct it to elongate the incomplete polypeptide’s C-terminus with either alanine (“Ala tails” in prokaryotes) or both alanine and threonine residues (“CAT tails” in yeast) (Lytvynenko et al, 2019; Osuna et al, 2017; Shen et al, 2015).…”

“…Metazoan CAT tails may include a more diverse repertoire of amino acids (Wu Z, Tantray I, Lim J, Chen S, Li Y, Davis Z, Sitron C, Dong J, Gispert S, Auburger G, Brandman O, Bi X, Snyder M, Lu B., 2019). These extensions, made without a small subunit or mRNA, act as “degrons” to mark incomplete polypeptides for degradation by the bacterial protease ClpXP or the eukaryotic proteasome (Lytvynenko et al, 2019; Sitron and Brandman, 2019).…”