The BEACH and MFS domains are sufficient for WDR81 recruitment onto Htt97Q aggregates.

(A) Schematic representations of full-length and truncations of human WDR81 protein analyzed in present study. Indicated mutations of WDR81 are shown in the BEACH and MFS domains. (B and C) Recruitment of indicated truncations of EGFP-WDR81 onto Htt97Q-RFP aggregates (left panel). Plasmids of Htt97Q-RFP and EGFP-WDR81 were co-transfected into HeLa cells at the same time. 48 h later, recruitment of indicated proteins was observed by fluorescent microscopy. Quantification of the percentage of polyQ foci positive for indicated EGFP-WDR81 truncations is shown in the right panel (C). 200 cells were analyzed for the recruitment in each group. (D and E) Recruitment of mCh-WDR81 without BEACH domain (651–1940) or mCh-WDR81 without MFS domain onto Htt97Q-EGFP aggregates (upper panel). Plasmids of Htt97Q-EGFP and mCh-WDR81 were co-transfected into HeLa cells at the same time. 48 h later, recruitment of indicated proteins was observed by fluorescent microscopy. Quantification of the percentage of polyQ foci positive for indicated mCh-WDR81 truncations is shown in the bottom panel (E). 215 cells were analyzed for the recruitment in each group. (F and G) Colocalization of Htt97Q-EGFP foci with wild-type (WT) or mutant mCherry-WDR81 (mCh-WDR81). Quantification of the percentage of polyQ foci positive for indicated WT or mutant mCh-WDR81 is shown in the bottom panel (G). 210 cells were analyzed for the recruitment in each group. (H-L) Immunoblotting analysis of Htt97Q-EGFP and autophagic proteins without or with overexpression of WT or mutant mCh-WDR81 in WDR81 KO HeLa cells. Fold changes of Htt97Q-EGFP (I) and autophagic proteins (J-L) were normalized to control (vector). Scale bars represent 10 μm in all panels. For quantifications, means±SEM were derived from three independent experiments and analyzed using t -tests or ANOVA. ** P <0.01. ***P <0.001.