Htt requires HAP1 interacting domain to facilitate the transport of Golgi-derived vesicles. A) HEK cells were transfected with pARIS-mCherry-httQ23 or a deletion mutant for the minimal HAP1 interaction domain (denoted as pARIS-mCherry-httQ23-ΔHAP1) in the absence or presence of HAP1-GFP. Exogenous htt was immunoprecipitated (IP) from cell lysates using a HA antibody and immunocomplexes were analyzed for the presence of HAP1-GFP. Immunoprecipitations with mouse IgGs were used as a specificity control. (B) Golgi reformation assays were done in HeLa cells stably expressing GFP-mannosidase II as described previously. Representative image of a pARIS-mCherry-httQ23-ΔHAP1 expressing cell failing to reconstitute the GA after NZ washout. Scale bar 10 μm. (C) Quantification of the Golgi dispersion as the mean volume per Golgi particle (μm3) before and after NZ washout for the different treatments. Results were obtained from 3 independent experiments in which 190 cells were scored. One way ANOVA followed by Fisher's Post Hoc test: ***p < 0.0001. NS, non significant. All comparisons t = 0 vs t = 120; siRNA-htt 0.073 ± 0.008 vs 0.158 ± 0.06; siRNA-htt + pARIS-mCherry-httQ23: 0.222 ± 0.035 vs 3.763 ± 0.712; siRNA-htt + pARIS-mCherry-httQ23-ΔHAP1: 0.073 ± 0.080 vs 0.159 ± 0.060.
Pardo et al. Molecular Brain 2010 3:17 doi:10.1186/1756-6606-3-17