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Journal: Autophagy
Article Title: Architecture of the ATG2B-WDR45 complex and an aromatic Y/HF motif crucial for complex formation
doi: 10.1080/15548627.2017.1359381
Figure Lengend Snippet: Liposome sedimentation analysis of WDR45 alone, ATG2B alone and the ATG2B-WDR45 complex. (A) TSF-tagged ATG2B alone or TSF-tagged WDR45 and 6xHis-tagged ATG2B were coexpressed in HEK293T cells, then subjected to large-scale affinity purification on a Strep-Trap affinity column and analyzed by size-exclusion chromatography using a Superose 6 10/300 column. The UV280 absorption curves are shown (the red dashed curve indicates ATG2B alone; the blue solid curve indicates the ATG2B-WDR45 complex). Both curves contain heterodimer peaks at 13.5 ml (peak 2, vertical black line). (B) Purified WDR45 alone, ATG2B alone and ATG2B-WDR45 complex analyzed by Coomassie staining. (C and D) Binding of the purified WDR45, ATG2B and ATG2B-WDR45 complex to PtdInsP-containing liposomes determined by liposome sedimentation assays. The purified protein or protein complex was incubated with (C) liposomes containing the indicated concentrations of PtdIns, PtdIns3P (0% to 15%) or (D) 15% PtdIns3P containing liposomes with different curvatures (100 nm, 400 nm, 1 μm) and then ultracentrifugated. After ultracentrifugation, proteins remaining in the supernatant (S) or those associated with liposomes in the pellet (P) were revealed by SDS-PAGE and Coomassie staining. The relative band density was calculated by normalizing either the supernatant or the pellet band density to their combined band density. The results are shown in the bar chart panel (n = 3) under corresponding Coomassie gel. “w.o. PtdIns or PtdIns3P” in subfigure (C) indicates liposomes without PtdIns or PtdIns3P. The 1 µm lane for ATG2B-WDR45 group in subfigure (D) is slightly shifted up due to a break in the gel.
Article Snippet: Detailed compositions (mole percentage) of the unilamellar liposomes used in this study were as follow: (1) 71% phosphatidylcholine (PC; Avanti, 850457C), 28.5% phosphatidylethanolamine (PE; Avanti, 850725C), 0.5% Lissamine Rhodamine B-labeled PE (Rhodamine-PE; Avanti, 810150C); (2) 15% PtdIns (Avanti, 840042C), 60% PC, 24.5% PE, 0.5% Rhodamine-PE; (3) 5%
Techniques: Sedimentation, Affinity Purification, Affinity Column, Size-exclusion Chromatography, Purification, Staining, Binding Assay, Incubation, SDS Page