Role of a ribosome-associated E3 ubiquitin ligase in protein quality control

Abstract

Messenger RNA lacking stop codons ('non-stop mRNA') can arise from errors in gene expression, and encode aberrant proteins whose accumulation could be deleterious to cellular function. In bacteria, these 'non-stop proteins' become co-translationally tagged with a peptide encoded by ssrA/tmRNA (transfer-messenger RNA), which signals their degradation by energy-dependent proteases. How eukaryotic cells eliminate non-stop proteins has remained unknown. Here we show that the Saccharomyces cerevisiae Ltn1 RING-domain-type E3 ubiquitin ligase acts in the quality control of non-stop proteins, in a process that is mechanistically distinct but conceptually analogous to that performed by ssrA: Ltn1 is predominantly associated with ribosomes, and it marks nascent non-stop proteins with ubiquitin to signal their proteasomal degradation. Ltn1-mediated ubiquitylation of non-stop proteins seems to be triggered by their stalling in ribosomes on translation through the poly(A) tail. The biological relevance of this process is underscored by the finding that loss of Ltn1 function confers sensitivity to stress caused by increased non-stop protein production. We speculate that defective protein quality control may underlie the neurodegenerative phenotype that results from mutation of the mouse Ltn1 homologue Listerin.

Figure 1. The yeast Listerin/Ltn1 E3 ligase functions in quality control of nonstop proteins

a, mRNA encoding GFP-Flag-HIS3 (K0), a nonstop (NS) protein and a protein fused to 12 lysines (K12). b, Regulation of NS protein levels is Ltn1 RING domain-dependent. Top, Ltn1 structure. Conserved regions are shaded. Bottom, K0 and NS protein expression in a wild type (WT) strain, an LTN1 deletion strain (ltn1Δ) and a strain whose endogenous Ltn1 lacks the RING domain. Rpl3 immunoblot controls for loading. Below, relative levels of the corresponding mRNAs (from Fig.S3b; nd, not determined). c, Ski7 and Ltn1 independently control NS protein expression. Immunoblot of K0 and NS in various strains. d, RING domain point mutations impaired Ltn1’s ability to downregulate NS expression. NS levels in a ltn1Δ strain expressing plasmidborne HA-Ltn1 wildtype or Trp1542 mutants. e, Ubiquitin (Ub) blot, LTN1 deletion does not exert a general effect on ubiquitylation; Flag blot, proteasomal degradation of NS proteins is Ltn1-dependent. Immunoblots of whole cell extracts (WCE). Cells were treated (+) or not (−) with the proteasome inhibitor MG132. f, Ltn1 and NS specifically co-IP. Strains expressing endogenous HALtn1 and K0 or NS were used for anti-Flag IP, followed by anti-HA or Flag blot. g, NS proteins are ubiquitylated, and this depends on Ltn1. SDS-boiled lysates of WT or or ltn1Δ strains expressing K0 or NS were used for Flag IP and immunoblots. The WT strain expressing no Flag-tagged proteins was a negative control. The corresponding WCEs are shown in “e”. Asterisk, cross-reacting faint band. h, Ltn1 is not required for VHL degradation. VHL immunoblot in WT or ltn1Δ strains at steady state (t=0; 22°C) or 90 min following cycloheximide addition and switch to 30°C or 37°C.

Figure 2. Ltn1 targets newly-synthesized nonstop proteins

a, NS expression during a 1-min labeling with ³⁵S-Met was increased in response to LTN1 deletion. K0 and NS expression in WT and ltn1Δ strains, normalized to K0 expression in WT cells. The inset shows similar labeling efficiency of total cellular proteins (cpm/µg WCE) in WT and ltn1Δ strains expressing NS protein. Below, corresponding mRNA levels (from Fig.S3b). b, Degradation of newly-synthesized NS protein is Ltn1-dependent. Cells labelled with a 1-min pulse (panel “a”) were chased with cold Met and cycloheximide before lysis and Flag IP. Labels: K0, squares; NS, diamonds; WT strain, black; ltn1Δ, white. Error bars shown below the labels for the WT and above for the ltn1Δ train. Each curve was normalized to chase time=0. a–b, The data is an average of two experiments (n=2) performed in duplicates each, and are representative of four independent experiments. Error bars show s.d.

Figure 3. Nascent poly(Lys) peptides stall in ribosomes, cause translational arrest and trigger Ltn1-mediated ubiquitylation

a, K12 levels are regulated in a Ltn1 RING-dependent manner. As in Fig.1b. b, Ltn1 and K12 specifically co-IP. These results are part of the set in Fig.1f. c, Proteasomal degradation of K12 is Ltn1-dependent. Immunoblot of WCEs. d, K12 ubiquitylation and degradation is Ltn1-dependent. K12 expressed in WT or ltn1Δ strains was Flag IP’ed and used for immunoblots, as in Fig.1g. e, A nascent Lys tract located at various distances from the C-terminus mediates translational arrest and Ltn1-dependent degradation. Expression of K0 and K12 constructs with 0–4 C-terminal HA tags, in WT and ltn1Δ strains. f, Nascent NS protein stalls in 80S ribosomes and is cleared by Ltn1. Sucrose gradient fractions of lysates expressing K0 or NS proteins were analyzed by anti-Flag immunoblot. Exposures were adjusted to facilitate comparison of the proteins’ distribution. The sedimentation of ribosomal particles was inferred from the A₂₅₄ profile (e.g., Figs.4c and S8c) and confirmed by reprobing blots for the 60S component, Rpl3. Lower 4 panels, NS-expressing ltn1Δ/ski7Δ cell lysate was treated or not with EDTA prior to centrifugation. EDTA dissociates 80S ribosomes and promotes loss of certain ribosomal components, slowing down sedimentation of 40S and 60S subunits (indicated by S’). Rpl3 co-fractionates mostly with 80S ribosomes in the absence, and 60S’ subunits in the presence, of EDTA (see also Fig.S8c). g, NS and K12, but not K0, co-IP with the 60S protein, Rpl3. Lysates of ltn1Δ strains expressing the reporters were Flag IP’ed, followed by anti-Rpl3 blot.

Figure 4. Ltn1 is predominantly associated with ribosomes

Strains expressing C-terminally Flag-tagged endogenous Ltn1 (a-c) or Ltn1 ΔRING (a) were used in this figure. a, Ltn1 specifically co-IP’s with Rpl3. Indicated lysates were Flag IP’ed, followed by anti-Rpl3 blot. K0 and the untagged WT strain were negative controls. Arrowhead, Ltn1 and Ltn1 ΔRING; arrow, K0. b, Ltn1 is predominantly cytoplasmic. Pellet (P) and supernatant (S) samples were taken following centrifugation of lysate at 300, 13K and 100K × g. Blots were probed for Flag, Lys4-di-methylated histone H3, Pgk1, and Rpl3. c, Ltn1 is predominantly 60S-bound in steady-state. Ltn1’s distribution in sucrose gradient fractions analyzed by immunoblot. Line tracing, A₂₅₄ profile.

Figure 5. Ltn1 confers resistance to stress caused by nonstop protein production

Cultures of the indicated strains normalized to equal cell density were spotted in 5-fold serial dilutions onto plates with rich media (YPD), containing or not the indicated drugs.