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

Journal name:
Nature
Volume:
467,
Pages:
470–473
Date published:
DOI:
doi:10.1038/nature09371
Received
Accepted
Published online

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 function1, 2. 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 proteases1, 3. 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.

At a glance

Figures

  1. The yeast Listerin/Ltn1 E3 ligase functions in quality control of non-stop proteins.
    Figure 1: The yeast Listerin/Ltn1 E3 ligase functions in quality control of non-stop proteins.

    a, Schematic diagrams of mRNA encoding GFP–Flag–HIS3 (K0), a non-stop (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 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 Supplementary Fig. 3b). n.d., 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 the ability of Ltn1 to downregulate NS expression. NS levels in an ltn1Δ strain expressing plasmid-borne HA–Ltn1 wild-type 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 (WCEs). Cells were treated (+) or not (−) with the proteasome inhibitor MG132. f, Ltn1 and NS specifically co-immunoprecipitate. Strains expressing endogenous HA–Ltn1 and K0 or NS were used for anti-Flag immunoprecipitation (IP), followed by anti-HA or Flag blotting. g, NS proteins are ubiquitylated, and this depends on Ltn1. SDS-boiled lysates of WT or ltn1Δ strains expressing K0 or NS were used for Flag immunoprecipitation followed by anti-Ub and Flag blotting. 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 protein degradation. VHL protein immunoblot in WT or ltn1Δ strains at steady state (zero time; 22°C) or 90min after cycloheximide addition and switch to 30°C or 37°C.

  2. Ltn1 targets newly synthesized non-stop proteins.
    Figure 2: Ltn1 targets newly synthesized non-stop proteins.

    a, NS expression during labelling for 1 min with [35S]methionine 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 labelling efficiency of total cellular proteins (c.p.m. per μg of whole-cell extract) in WT and ltn1Δ strains expressing NS protein. Bottom, corresponding mRNA levels (from Supplementary Fig. 3b). b, Degradation of newly synthesized NS protein is Ltn1 dependent. Cells labelled with a 1-min pulse (a) were chased with unlabelled methionine and cycloheximide before lysis and Flag immunoprecipitation. Squares, K0; diamonds, NS; black, WT strain; white, ltn1Δ. Error bars are shown below the data points for the WT and above for the ltn1Δ strain. Each curve was normalized to chase time = 0. The data in both panels are an average of two experiments (n = 2) each performed in duplicate, and are representative of four independent experiments. Error bars indicate s.d.

  3. Nascent polylysine peptides stall in ribosomes, cause translational arrest and trigger Ltn1-mediated ubiquitylation.
    Figure 3: Nascent polylysine peptides stall in ribosomes, cause translational arrest and trigger Ltn1-mediated ubiquitylation.

    a, K12 levels are regulated in an Ltn1 RING-dependent manner. Legend as for Fig. 1b. b, Ltn1 and K12 specifically co-immunoprecipitate. These results are part of the set in Fig. 1f. c, Proteasomal degradation of K12 is Ltn1 dependent. Immunoblot of whole-cell extracts. d, K12 ubiquitylation and degradation is Ltn1 dependent. K12 expressed in WT or ltn1Δ strains was Flag immunoprecipitated and used for immunoblots, as in Fig. 1g. Ub, ubiquitin. e, A nascent Lys12 tract located at various distances from the C terminus mediates translational arrest and Ltn1-dependent degradation. Expression of K0 and K12 constructs with zero to four 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 analysed by anti-Flag immunoblotting. Exposures were adjusted to facilitate comparison of the distribution of the proteins. The sedimentation of ribosomal particles was inferred from the A254 profile (see, for example, Fig. 4c and Supplementary Fig. 8c) and confirmed by reprobing blots for the 60S component, Rpl3. Bottom four panels: NS-expressing ltn1Δ/ski7Δ cell lysate was treated or not with EDTA before centrifugation. EDTA dissociates 80S ribosomes and promotes the loss of certain ribosomal components, slowing sedimentation of 40S and 60S subunits (indicated by S′). Rpl3 fractionates mostly with 80S ribosomes in the absence of EDTA, and with 60S′ subunits in its presence (see also Supplementary Fig. 8c). g, NS and K12, but not K0, co-immunoprecipitated with the 60S protein Rpl3. Lysates of ltn1Δ strains expressing the reporters were Flag immunoprecipitated, followed by anti-Rpl3 blotting.

  4. Ltn1 is predominantly associated with ribosomes.
    Figure 4: Ltn1 is predominantly associated with ribosomes.

    Strains expressing C-terminally Flag-tagged endogenous Ltn1 (ac) or Ltn1 ΔRING (a) were used in this figure. a, Ltn1 specifically co-immunoprecipitates with Rpl3. The indicated lysates were Flag immunoprecipitated, followed by anti-Rpl3 blotting. K0 and the untagged WT strain were negative controls. Arrowheads indicate Ltn1 and Ltn1 ΔRING; arrows indicate K0. b, Ltn1 is predominantly cytoplasmic. Pellet (P) and supernatant (S) samples were taken after centrifugation of lysate at 300g, 13,000g (13K) and 100,000g (100K). Blots were probed for Flag, for histone H3 dimethylated on Lys4, for Pgk1 and for Rpl3. c, Ltn1 is predominantly 60S-bound at steady state. Distribution of Ltn1 in sucrose-gradient fractions analysed by immunoblotting. Line tracing, A254 profile.

  5. Ltn1 confers resistance to stress caused by non-stop protein production.
    Figure 5: Ltn1 confers resistance to stress caused by non-stop protein production.

    Cultures of the indicated strains normalized to equal cell density were spotted in fivefold serial dilutions onto plates with rich medium (YPD), containing or lacking the indicated drugs.

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Author information

Affiliations

  1. Department of Cell Biology, The Scripps Research Institute, CB168, 10550 North Torrey Pines Road, La Jolla, California 92037, USA

    • Mario H. Bengtson &
    • Claudio A. P. Joazeiro

Contributions

C.A.P.J. and M.H.B. designed the studies, interpreted the data and wrote the manuscript. M.H.B. conducted the experiments.

Competing financial interests

The authors declare no competing financial interests.

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Supplementary information

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  1. Supplementary Information (3.1M)

    This file contains Supplementary Methods, a List of Genotypes, Supplementary Figures 1-9 with legends, Supplementary Table 1, and additional references.

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