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Spatial sequestration and detoxification of Huntingtin by the ribosome quality control complex.

ABSTRACT: Huntington disease (HD) is a neurological disorder caused by polyglutamine expansions in mutated Huntingtin (mHtt) proteins, rendering them prone to form inclusion bodies (IB). We report that in yeast, such IB formation is a factor-dependent process subjected to age-related decline. A genome-wide, high-content imaging approach, identified the E3 ubiquitin ligase, Ltn1 of the ribosome quality control complex (RQC) as a key factor required for IB formation, ubiquitination, and detoxification of model mHtt. The failure of ltn1? cells to manage mHtt was traced to another RQC component, Tae2, and inappropriate control of heat shock transcription factor, Hsf1, activity. Moreover, super-resolution microscopy revealed that mHtt toxicity in RQC-deficient cells was accompanied by multiple mHtt aggregates altering actin cytoskeletal structures and retarding endocytosis. The data demonstrates that spatial sequestration of mHtt into IBs is policed by the RQC-Hsf1 regulatory system and that such compartmentalization, rather than ubiquitination, is key to mHtt detoxification.

SUBMITTER: Yang J

PROVIDER: S-EPMC4868537 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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Spatial sequestration and detoxification of Huntingtin by the ribosome quality control complex.

Yang Junsheng J Hao Xinxin X Cao Xiuling X Liu Beidong B Nyström Thomas T

eLife 20160401

Huntington disease (HD) is a neurological disorder caused by polyglutamine expansions in mutated Huntingtin (mHtt) proteins, rendering them prone to form inclusion bodies (IB). We report that in yeast, such IB formation is a factor-dependent process subjected to age-related decline. A genome-wide, high-content imaging approach, identified the E3 ubiquitin ligase, Ltn1 of the ribosome quality control complex (RQC) as a key factor required for IB formation, ubiquitination, and detoxification of mo ...[more]

PMID: 27033550

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Project description:Protein conformational maladies such as Huntington Disease are characterized by accumulation of intracellular and extracellular protein inclusions containing amyloid-like proteins. There is an inverse correlation between proteotoxicity and aggregation, so facilitated protein aggregation appears cytoprotective. To define mechanisms for protective protein aggregation, a screen for suppressors of nuclear huntingtin (Htt103Q) toxicity was conducted. Nuclear Htt103Q is highly toxic and less aggregation prone than its cytosolic form, so we identified suppressors of cytotoxicity caused by Htt103Q tagged with a nuclear localization signal (NLS). High copy suppressors of Htt103Q-NLS toxicity include the polyQ-domain containing proteins Nab3, Pop2, and Cbk1, and each suppresses Htt toxicity via a different mechanism. Htt103Q-NLS appears to inactivate the essential functions of Nab3 in RNA processing in the nucleus. Function of Pop2 and Cbk1 is not impaired by nuclear Htt103Q, as their respective polyQ-rich domains are sufficient to suppress Htt103Q toxicity. Pop2 is a subunit of an RNA processing complex and is localized throughout the cytoplasm. Expression of just the Pop2 polyQ domain and an adjacent proline-rich stretch is sufficient to suppress Htt103Q toxicity. The proline-rich domain in Pop2 resembles an aggresome targeting signal, so Pop2 may act in trans to positively impact spatial quality control of Htt103Q. Cbk1 accumulates in discrete perinuclear foci and overexpression of the Cbk1 polyQ domain concentrates diffuse Htt103Q into these foci, which correlates with suppression of Htt toxicity. Protective action of Pop2 and Cbk1 in spatial quality control is dependent upon the Hsp70 co-chaperone Sti1, which packages amyloid-like proteins into benign foci. Protein:protein interactions between Htt103Q and its intracellular neighbors lead to toxic and protective outcomes. A subset of polyQ-rich proteins buffer amyloid toxicity by funneling toxic aggregation intermediates to the Hsp70/Sti1 system for spatial organization into benign species.

Project description:Translation elongation rates are regulated to ensure proper conformation and biological function of proteins. Translation of either non-stop mRNA or transcripts coding for poly-basic sequences induces ribosome stalling, and the arrest product is degraded by the ribosome-mediated quality control system (RQC). During this process, the stalled ribosome is dissociated into subunits, and the polypeptide is ubiquitinated by the E3 ubiquitin ligase Listerin on the 60S large ribosomal subunit (LSU) leading to subsequent proteasomal degradation. However, it is largely unknown how stalled ribosomes are recognized and dissociated into subunits. Here we report that ubiquitination of the ribosomal protein uS10 by the E3 ubiquitin ligase Hel2 is required for the production of the RQC substrate. RQC-trigger (RQT) factors, a RNA helicase-family protein Slh1/Rqt2, ubiquitin binding protein Cue3/Rqt3 and yKR023W/Rqt4, were also required for the primary steps of RQC, and associated with Hel2-ribosome complexes. Rqt2-4 factors were dispensable for the ubiquitination of uS10 by Hel2/Rqt1 and associated with ribosomes independent of the ubiquitination of uS10. However, the ubiquitin-binding activity of Rqt3 were crucial to trigger RQC. Cryo-electron microscopy (cryo-EM) analysis revealed that Hel2 bound ribosomes are in an rotated state containing hybrid state AP- and PE-tRNAs. Furthermore, ribosome profiling revealed that short footprints, hallmarks of hybrid state ribosomes18, were accumulated at tandem CGA rare codons at the beginning of the poly arginine stalling sequence and long footprints at subsequent codons, respectively. Short footprints at CGA codons were decreased in rqt1 mutant but drastically increased in uS10 mutants defective in the ubiquitination or rqt2 mutant. Collectively, our results demonstrate that Hel2 stabilizes ratcheted ribosomes leading to ubiquitination of uS10. Subsequently, Rqt2-4 factors target these hybrid state ribosomes specifically, allowing subsequent RQC reactions.

Dataset Information

Spatial sequestration and detoxification of Huntingtin by the ribosome quality control complex.

Publications

Spatial sequestration and detoxification of Huntingtin by the ribosome quality control complex.

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