Roles of Dom34:Hbs1 in Nonstop Protein Clearance from Translocators for Normal Organelle Protein Influx

Department of Chemistry, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan.
Cell Reports (Impact Factor: 8.36). 09/2012; 2(3):447-53. DOI: 10.1016/j.celrep.2012.08.010
Source: PubMed


Because messenger RNAs without a stop codon (nonstop mRNAs) generate stalled ribosomes, cells have developed a mechanism allowing degradation of nonstop mRNAs and their translation products (nonstop proteins) in the cytosol. Here, we observe the fate of nonstop proteins destined for organelles such as the endoplasmic reticulum (ER) and mitochondria. Nonstop mRNAs for secretory-pathway proteins in yeast generate nonstop proteins that become stuck in the translocator, the Sec61 complex, in the ER membrane. These stuck nonstop secretory proteins avoid proteasomal degradation in the cytosol, but are instead released into the ER lumen through stalled ribosome and translocator channels by Dom34:Hbs1. We also found that nonstop mitochondrial proteins are cleared from the mitochondrial translocator, the TOM40 complex, by Dom34:Hbs1. Clearance of stuck nonstop proteins from organellar translocator channels is crucial for normal protein influx into organelles and for normal cell growth, especially when nonstop mRNA decay does not function efficiently.

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Available from: Shuh-Ichi Nishikawa, Feb 12, 2014
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    • "The situations under which stalled nascent secretory proteins can become cytosolically exposed for possible ubiquitination are not clear. In yeast, enforced stalling at an ER translocon was targeted by the ribosome rescue factors Dom34 and Hbs1 (Izawa et al., 2012). In the absence of this pathway, the stall could not be efficiently resolved, resulting in impaired translocation due to limited translocon availability . "
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    ABSTRACT: Cytosolic ribosomes that stall during translation are split into subunits, and nascent polypeptides trapped in the 60S subunit are ubiquitinated by the ribosome quality control (RQC) pathway. Whether the RQC pathway can also target stalls during cotranslational translocation into the ER is not known. Here, we report that Listerin and NEMF, core RQC components, are bound to translocon-engaged 60S subunits on native ER membranes. RQC recruitment to the ER in cultured cells is stimulated by translation stalling. Biochemical analyses demonstrated that translocon-targeted nascent polypeptides that subsequently stall are polyubiquitinated in 60S complexes. Ubiquitination at the translocon requires cytosolic exposure of the polypeptide at the ribosome-Sec61 junction. This exposure can result from either failed insertion into the Sec61 channel or partial backsliding of translocating nascent chains. Only Sec61-engaged nascent chains early in their biogenesis were relatively refractory to ubiquitination. Modelling based on recent 60S-RQC and 80S-Sec61 structures suggests that the E3 ligase Listerin accesses nascent polypeptides via a gap in the ribosome-translocon junction near the Sec61 lateral gate. Thus, the RQC pathway can target stalled translocation intermediates for degradation from the Sec61 channel. © 2015 by The American Society for Cell Biology.
    Full-text · Article · Apr 2015 · Molecular biology of the cell
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    • "These observations , together with cofractionation of Listerin with 60S-RNCs, argue that Listerin accesses stalled RNCs only after 40S subunit removal. This model is consistent with studies in yeast showing Ltn1 copurification with 60S (Bengtson and Joazeiro, 2010;Brandman et al., 2012;Defenouillè re et al., 2013) and stabilization of nascent chains in strains lacking the Pelota homolog Dom34 (Izawa et al., 2012;Verma et al., 2013).In addition to Ltn1 and ribosome splitting factors, genetic studies in yeast have identified additional components in the ribosome-associated quality control (RQC) pathway. The ribosome-associated proteins Asc1 (Brandman et al., 2012;Kuroha et al., 2010) and Hel2 (Brandman et al., 2012) facilitate stalling at poly-basic residues, such as lysines encoded by poly(A) tails. "
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    ABSTRACT: During ribosome-associated quality control, stalled ribosomes are split into subunits and the 60S-housed nascent polypeptides are poly-ubiquitinated by Listerin. How this low-abundance ubiquitin ligase targets rare stall-generated 60S among numerous empty 60S is unknown. Here, we show that Listerin specificity for nascent chain-60S complexes depends on nuclear export mediator factor (NEMF). The 3.6 Å cryo-EM structure of a nascent chain-containing 60S-Listerin-NEMF complex revealed that NEMF makes multiple simultaneous contacts with 60S and peptidyl-tRNA to sense nascent chain occupancy. Structural and mutational analyses showed that ribosome-bound NEMF recruits and stabilizes Listerin's N-terminal domain, while Listerin's C-terminal RWD domain directly contacts the ribosome to position the adjacent ligase domain near the nascent polypeptide exit tunnel. Thus, highly specific nascent chain targeting by Listerin is imparted by the avidity gained from a multivalent network of context-specific individually weak interactions, highlighting a new principle of client recognition during protein quality control. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Full-text · Article · Jan 2015 · Molecular Cell
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    • "The Dom34:Hbs1 complex stimulates the endonucleolytic cleavage of mRNA induced by translation arrest in vivo (Doma & Parker 2006; Chen et al. 2010; van den Elzen et al. 2010; Kobayashi et al. 2010) and dissociates the subunits of stalled ribosomes in vitro (Shoemaker et al. 2010; Pisareva et al. 2011; Shoemaker & Green 2011). In addition, Dom34:Hbs1 dissociates stalled ribosomes at the 3′ end of nonstop mRNA and stimulates its degradation by exosomes in vivo (Kobayashi et al. 2010; Izawa et al. 2012). However, the relationship between Dom34:Hbs1-dependent subunit dissociation of stalled ribosomes and Ltn1-dependent rapid degradation of nonstop protein products remains to be resolved. "
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    ABSTRACT: Quality control systems eliminate aberrant proteins derived from aberrant mRNAs. Two E3 ubiquitin ligases, Ltn1 and Not4, are involved in proteasomal protein degradation coupled to translation arrest. Here, we evaluated nonstop and translation arrest products degraded in a poly(A) tail-independent manner. Ltn1 was found to degrade aberrant nonstop polypeptides derived from nonstop mRNA lacking a termination codon, but not peptidyl-tRNA, even in the absence of the ribosome dissociation complex Dom34:Hbs1. The receptor for activated C kinase (RACK1/ASC1) was identified as a factor required for nascent peptide-dependent translation arrest as well as Ltn1-dependent protein degradation. Both Not4 and Ltn1 were involved in the degradation of various arrest products in a poly(A) tail-independent manner. Furthermore, carboxyl terminus-truncated degradation intermediates of arrest products were stabilized in a cdc48-3 mutant defective in unfolding or the disassembly related to proteasomal degradation. Thus, we propose that stalled ribosomes may be dissociated into subunits and that peptidyl-tRNA on the 60S subunit is ubiquitinated by Ltn1 and Cdc48 is required for the degradation following release from tRNA.
    Preview · Article · Nov 2013 · Genes to Cells
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