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. 2016 Apr 1;5:e11792. doi: 10.7554/eLife.11792

Figure 2. 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).

DOI: http://dx.doi.org/10.7554/eLife.11792.004

Figure 2.

Figure 2—figure supplement 1. Western blot of His-Ub pull-down mHtt103QP in RQC mutants.

Figure 2—figure supplement 1.

mHtt103QP-GFP ubiquitinated by His-tagged ubiquitin was pulled-down by Ni-beads and detected by GFP antibody.
DOI: http://dx.doi.org/10.7554/eLife.11792.005

Figure 2—figure supplement 2. FRAP assay of mHtt103QP aggregate in Wt and RQC mutants.

Figure 2—figure supplement 2.

(a) Representative images of mHtt103QP-GFP aggregate before and after laser bleach. (b) Relative fluorescence of the bleached region.
DOI: http://dx.doi.org/10.7554/eLife.11792.006

Figure 2—figure supplement 3. Ltn1-GFP co-localize with mHtt103QP-mRFP.

Figure 2—figure supplement 3.

DOI: http://dx.doi.org/10.7554/eLife.11792.007

Figure 2—figure supplement 4. mHtt levels chase after cycloheximide treatment.

Figure 2—figure supplement 4.

(a) Representative Western blots of soluble and aggregated mHtt103QP from Wt and ltn1Δ strains. (b) Quantitafication of three repeats.
DOI: http://dx.doi.org/10.7554/eLife.11792.008

Figure 2—figure supplement 5. mHtt103QP aggregate in ltn1Δtae2Δ is also co-localized with dense actin structures.

Figure 2—figure supplement 5.

DOI: http://dx.doi.org/10.7554/eLife.11792.009