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. Author manuscript; available in PMC: 2010 Sep 16.
Published in final edited form as: Cell. 2009 Apr 3;137(1):60–72. doi: 10.1016/j.cell.2009.03.018

Figure 5. Acetylation of Mutant Htt Enhances Its Clearance by Autophagy.

Figure 5

(A) Acetylation of mutant Htt at lysine 444 leads to increased recruitment of LC3 to autophagic vacuoles. RFP-Htt480-68Q and RFP-Htt480-68Q-KR were transfected into COS-7 cells together with GFP-LC3 and CFP-CBP-HAT. Live cells were sequentially scanned to detect distribution of Htt (red), LC3 (green), and CBP-HAT (blue). Scale bar, 10 μm. (a) Coexpression of RFP-Htt480-68Q with LC3 and CBP-HAT led to accumulation of LC3 puncta. (b) Cells transfected with RFP-Htt480-68Q-KR, LC3, and CBP-HAT displayed a marked decrease in the LC3 puncta. (c) LC3-G120A mutant does not form punctate structure. (d) The percentage of cells containing >5 puncta was scored in 150 cells per experiment. Values are expressed as means + SEM of three independent experiments.

(B) HeLa cell line, stably expressing YFP-LC3, was transfected with Htt590-97Q or Htt590-97Q-KR plus CFP-CBP-HAT or inactive CBP-HAT-DY. ANOVA analysis of the percentage of transfected cells with Htt puncta that colocalized with LC3 puncta revealed a significant effect of the presence of CBP-HAT (p < 0.001) and the requirement of K444 (p < 0.001).

(C) Accumulation of mutant acetyl-Htt after treatment with lysosomal inhibitor leupeptin (200 μM). β-tubulin was used as loading control; p < 0.001.

(D) Clearance of acetylated mutant Htt is impeded by inhibition of lysosomal enzymes. Levels of Htt590-97Q, monitored by radiolabeled pulse-chase, were determined after 1, 8, 16, and 24 hr by immunoprecipitation for Htt590-97Q using an N-terminal FLAG antibody. Coexpression of CBP-HAT significantly enhanced clearance of Htt590-97Q (filled square) compared to inactive CBP-HAT-DY (open square) (p < 0.05). This clearance required K444 (open diamond) (p < 0.005) and was impeded by 200 μM leupeptin (filled diamond) (p < 0.05). Significant degradation was achieved after a chase period of 16 hr and 24 hr (*p < 0.001). Densitometry measurements are expressed as % of signal after a 1 hr chase. Representative radiograms are shown. Three independent experiments were performed and are represented as mean + SEM. ANOVA reveals a significant difference across chase time (F(3,23) = 61.779, p < 0.0001), across group (F(3,23) = 19.656, p < 0.0001), and across an interaction between chase time and group (F(9,23) = 5.479, p = 0.005).

(E) Acetyl-Htt is preferentially localized to the cytoplasm. Levels of cytoplasmic and nuclear acetyl-Htt and CBP-HAT were determined by subcellular fractionation and western blotting. HDAC1 and β-tubulin were used as nuclear and cytoplasmic markers, respectively. Results are representative of three independent experiments.