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. 2017 Jul 28;357(6349):414-417.
doi: 10.1126/science.aam7787.

CAT-tailing as a Fail-Safe Mechanism for Efficient Degradation of Stalled Nascent Polypeptides

Free PMC article

CAT-tailing as a Fail-Safe Mechanism for Efficient Degradation of Stalled Nascent Polypeptides

Kamena K Kostova et al. Science. .
Free PMC article


Ribosome stalling leads to recruitment of the ribosome quality control complex (RQC), which targets the partially synthesized polypeptide for proteasomal degradation through the action of the ubiquitin ligase Ltn1p. A second core RQC component, Rqc2p, modifies the nascent polypeptide by adding a carboxyl-terminal alanine and threonine (CAT) tail through a noncanonical elongation reaction. Here we examined the role of CAT-tailing in nascent-chain degradation in budding yeast. We found that Ltn1p efficiently accessed only nascent-chain lysines immediately proximal to the ribosome exit tunnel. For substrates without Ltn1p-accessible lysines, CAT-tailing enabled degradation by exposing lysines sequestered in the ribosome exit tunnel. Thus, CAT-tails do not serve as a degron, but rather provide a fail-safe mechanism that expands the range of RQC-degradable substrates.


Fig. 1
Fig. 1. Degradation of RQC substrates requires lysines
(A) Model for RQC-mediated degradation of nascent polypeptides. (B, C) Immunoblots (IBs) of stalling reporters with or without lysines in RQC deletion strains. (D) Proteasome inhibition time course for stalling constructs expressed in pdr5Δ cells. The relative amount of stalling substrate (mean ± SD, N=3) accumulating over time was visualized by IB (left) and quantified by densitometry (right).
Fig. 2
Fig. 2. Lysine positioning is critical for Ltn1p-mediated ubiquitination and proteasomal degradation
(A) IB of stalling constructs with variable number of amino acids (AA) between the most C-terminal lysine of GFP and the first ariginine of the stall site. The relative amount of substrate remaining in wt cells relative to rqc2Δ cells was quantified and normalized to a loading control (bar graph) (mean ± SD, N=3). (B) IB of TEV-treated XTEN constructs. (C, D) Denaturing immunoprecipitation (Flag IP) of XTEN 0, 10, and 80 stalling constructs from cells expressing Myc-tagged ubiquitin.
Fig. 3
Fig. 3. The ability of Ltn1p to access lysines outside the exit tunnel is primarily determined by the distance of the lysines from the C-terminus
(A, C) IB of TEV-treated GFPLys-free stalling constructs with XTEN linkers of the indicated length inserted after the lyisines. (B) IB of a stalling construct with a lysine positioned between two unstructured sequences. (D) Quantification of protein levels from the stalling constructs in (C) (left). Shown is the percent stalling construct remaining in wt or rqc2mut cells relative to rqc2Δ cells as a function of the distance between the lysines and the R12 stalling site (mean ± SD, N=3). Schematic of Ltn1p’s accessible region (shaded area) (right). (E) IB of non-stop GFPLys-free construct.
Fig. 4
Fig. 4. CAT-tail-dependent degradation of endogenous RQC substrates
(A) Fraction of stalling positions leading to a RQC-degradable nascent polypeptide in the presence (solid line) or absence (dashed line) of CAT-tails, graphed as a function of the number of amino acids accessible to Ltn1p and assuming a fixed exit tunnel length of 35 amino acids. The arrow shows the increase in RQC-degradable substrates in the presence of CAT-tails at the estimated Ltn1p reach of 12 amino acids. (B) Growth phenotypes of RQC mutant strains in the absence or presence of cycloheximide (CHX). (C) Model for the function of CAT-tails in vivo.

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