Spatial sequestration and detoxification of Huntingtin by the ribosome quality control complex
- University of Gothenburg, Sweden
Figures
Figure 1
Screen approach and mHtt IB-forming mutants.
(a) Aggregation of different mHtt reporters as indicated. (b)Morphology of mHtt103QP aggregates (red) in young and old (1.6 and 12.6 bud scars (white), respectively) cells. Scale=2 μm. Bar graph shows percentages of Class3 cells in young and old cells. Mean ± s.d. (c) Schematic description of the HCM-based screen. (d) Htt103QP aggregation 0, 60, 120 and 180 min after HTT103QP induction. (e)Representative pictures of Class 0, 1, 2 and 3 cells. (f) Mutants displaying increased% of Class 3 cells, grouped according to functions. Y-axis shows fold increase relative to wild type. (g) Physical (red) and genetic (green) interaction between Class 3 genes/proteins and their quantitative interaction (thickness of grey lines) with mHtt103QP as indicated.
Figure 2 with 5 supplements
Role of RQC in mHtt103QP IB formation ubiquitination and toxicity.
(a, d)Htt103QP aggregate numbers (% Class 1,2&3 cells; see Figure 1) in mutants as indicated. W1542E encodes a ubiquitin-ligase-defect Ltn1 protein. HSF1-R206S encodes a hyper-active Hsf1. The hsf1-848 is a conditional ts mutant while HSF1ΔCAD lacks the c-terminal trans-activating domain. Scale=2 μm. Bar graphs show % of Class 1, 2 and 3 cells in each strain. Mean ± s.d. (b)Ubiquitination of mHtt103QP in strains from ‘a’. (c) Htt103QP stability in WT and ltn1Δ cells after a block in protein synthesis. Mean ± s.d. e-g. Fitness (see Materials and methods) of strains carrying pYES2-mHtt103QP-GFP compared to pYES2-GFP. Results from Galactose (mHtt induced) and Glucose (mHtt repressed) are shown. Ratios were calculated from the mean of three repeats (error bars are 95% confidence intervals) for WT, RQC, and rnq1∆ mutants (e) HSF1-R206S (f) and hsf1-848 (g).
Figure 2—figure supplement 1
Western blot of His-Ub pull-down mHtt103QP in RQC mutants.
mHtt103QP-GFP ubiquitinated by His-tagged ubiquitin was pulled-down by Ni-beads and detected by GFP antibody.
Figure 2—figure supplement 2
FRAP assay of mHtt103QP aggregate in Wt and RQC mutants.
(a) Representative images of mHtt103QP-GFP aggregate before and after laser bleach. (b) Relative fluorescence of the bleached region.
Figure 2—figure supplement 3
Ltn1-GFP co-localize with mHtt103QP-mRFP.
https://doi.org/10.7554/eLife.11792.007
Figure 2—figure supplement 4
mHtt levels chase after cycloheximide treatment.
(a) Representative Western blots of soluble and aggregated mHtt103QP from Wt and ltn1Δ strains. (b) Quantitafication of three repeats.
Figure 2—figure supplement 5
mHtt103QP aggregate in ltn1Δtae2Δ is also co-localized with dense actin structures.
https://doi.org/10.7554/eLife.11792.009
Figure 3 with 2 supplements
Role of actin in Htt103QP detoxification.
(a)Co-localization of mHtt103QP IBs and UBC9ts IPODs. (b, c) Actin structures (Red; phalloidin) and mHtt103QP (Green; GFP) aggregates in WT and ltn1Δ analyzed by 3D-SIM. Scale=1 μm. (d)Essential ts-alleles increasing toxicity of mHtt103QP, grouped according to biological processes. (e) Functional enrichment analysis of mHtt103QP-sensitive ts mutants. (f) Number of actin-associated aggregates and aggregate-free actin structures in WT and ltn1Δ cells. Mean ± s.d. g. Endocytotic activity in WT and ltn1Δ cells analyzed by FM4-64FX uptake to vacuoles. Mean ± s.d. (h)A model of the regulation of mHtt103QP IB formation and toxicity by RQC components and Hsf1.
Figure 3—figure supplement 1
FM4-64FX stained cells.Images of FM4-64FX stained cells corresponding to Figure 3g.
Representative cells carrying pYES2-GFP ('Vector') or pYES2-mHtt103QP-GFP ('103QP') were shown.
ltn1Δ.
Figure 3—figure supplement 2
Actin integrity of Wt and a. Actin staining of Wt and ltn1Δ cells, b. Quantification of actin depolarization of Wt and ltn1Δ cells, see Materials and methods for details.
https://doi.org/10.7554/eLife.11792.012Videos
Video 1
3D structures of mHtt103QP aggregate and actin in WT.
mHtt103QP aggregates (green) and actin (red) structures of a WT cell shown in Figure 3b.
Video 2
3D structures of mHtt103QP aggregate and actin in ltn1Δ.
mHtt103QP aggregates (green) and actin (red) structures of a ltn1Δ cell shown in Figure 3c.
Additional files
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Supplementary file 1
List of confirmed mutants from the HCM-based screen that have increased Class 3 cells.
- https://doi.org/10.7554/eLife.11792.015
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Supplementary file 2
List of ts alleles that increased mHtt103QP toxicity in SGA screen.
- https://doi.org/10.7554/eLife.11792.016
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Supplementary file 3
List of S. cerevisiae strains and plasmids.
A. List of S. cerevisiae strains. B. List of plasmids
- https://doi.org/10.7554/eLife.11792.017
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Spatial sequestration and detoxification of Huntingtin by the ribosome quality control complex
eLife 5:e11792.
https://doi.org/10.7554/eLife.11792
