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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    • "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.
    Molecular biology of the cell 04/2015; 26(12). DOI:10.1091/mbc.E15-01-0040 · 4.47 Impact Factor
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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.
    Genes to Cells 11/2013; 19(1). DOI:10.1111/gtc.12106 · 2.81 Impact Factor
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    ABSTRACT: In the translation process, translating ribosomes usually move on an mRNA until they reach the stop codon. However, when ribosomes translate an aberrant mRNA, they stall. Then, ribosomes are rescued from the aberrant mRNA, and the aberrant mRNA is subsequently degraded. In eukaryotes, Pelota (Dom34 in yeast) and Hbs1 are responsible for solving general problems of ribosomal stall in translation. In archaea, aPelota and aEF1α, homologous to Pelota and Hbs1, respectively, are considered to be involved in that process. In recent years, great progress has been made in determining structures of Dom34/aPelota and Hbs1/aEF1α. In this review, we focus on the functional roles of Dom34/aPelota and Hbs1/aEF1α in ribosome rescue, based on recent structural studies of them. We will also present questions to be answered by future work.
    BIOPHYSICS 01/2013; 9:131-140. DOI:10.2142/biophysics.9.131
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