Architecture of the ATG2B-WDR45 complex and an aromatic Y/HF motif crucial for complex formation |
| |
Authors: | Jing-Xiang Zheng Yan Li Yue-He Ding Jun-Jie Liu Mei-Jun Zhang Meng-Qiu Dong |
| |
Institution: | 1. State Key Laboratory of Membrane Biology, Tsinghua University-Peking University Joint Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, China;2. Joint Graduate Program of Peking-Tsinghua-National Institute of Biological Science, Tsinghua University, Beijing, China;3. Ministry of Education Key Laboratory of Protein Sciences, Tsinghua-Peking Joint Center for Life Sciences, Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing, China;4. National Institute of Biological Sciences, Beijing, China;5. Joint Graduate Program of Peking-Tsinghua-National Institute of Biological Science, Tsinghua University, Beijing, China;6. Ministry of Education Key Laboratory of Protein Sciences, Tsinghua-Peking Joint Center for Life Sciences, Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing, China |
| |
Abstract: | PtdIns3P signaling is critical for dynamic membrane remodeling during autophagosome formation. Proteins in the Atg18/WIPI family are PtdIns3P-binding effectors which can form complexes with proteins in the Atg2 family, and both families are essential for macroautophagy/autophagy. However, little is known about the biophysical properties and biological functions of the Atg2-Atg18/WIPI complex as a whole. Here, we demonstrate that an ortholog of yeast Atg18, mammalian WDR45/WIPI4 has a stronger binding capacity for mammalian ATG2A or ATG2B than the other 3 WIPIs. We purified the full-length Rattus norvegicus ATG2B and found that it could bind to liposomes independently of PtdIns3P or WDR45. We also purified the ATG2B-WDR45 complex and then performed 3-dimensional reconstruction of the complex by single-particle electron microscopy, which revealed a club-shaped heterodimer with an approximate length of 22 nm. Furthermore, we performed cross-linking mass spectrometry and identified a set of highly cross-linked intermolecular and intramolecular lysine pairs. Finally, based on the cross-linking data followed by bioinformatics and mutagenesis analysis, we determined the conserved aromatic H/YF motif in the C terminus of ATG2A and ATG2B that is crucial for complex formation. |
| |
Keywords: | aromatic amino acids ATG2 autophagy cross-linking mass spectrometry PROPPIN single-particle electron microscopy WDR45 WIPI-family proteins |
|
|