Structural And Functional Study Of Mammalian Key Autophagy Protein Complex ATG2B-WDR45

Autophagy is a highly conserved subcellular degradation process among eukaryote cells.It can balance the physiological homeostasis as well as help the cell against adversity through degradation and recycling of cellular components.Autophagy is a highly regulated process,and PtdIns3P(Phosphatidylinositol 3-phosphate)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 autophagy.However,little is known about the biophysical properties and biological functions of the ATG2-ATG18/WIPI complex as a whole.In this study,we demonstrated that the mammalian Atg18 homolog WDR45/WIPI4 has a stronger binding capacity for mammalian ATG2 A or ATG2 B than the other three WIPIs.Furthermore,we performed cross-linking mass spectrometry and identified a set of highly cross-linked inter-molecular and intra-molecular lysine pairs.Based on the following mutagenesis analysis,we determined the conserved aromatic H/YF motif in the C-terminus of ATG2 A and ATG2 B that is crucial for complex formation.Next,we successfully purified the full-length Rattus norvegicus ATG2 B as well as the WDR45 protein and the ATG2B-WDR45 complex.We found that ATG2 B can bind liposomes independently of PtdIns3 P or WDR45.We went further to perform the three-dimensional reconstruction of ATG2 B protein and ATG2B-WDR45 complex by single-particle electron microscopy,which revealed a club-shaped heterodimer with an approximate length of 22 nm.The elongated shape and multivalent protein and lipid binding capacity make this complex quite similar to some known membrane tethering complexes like HOPS complex and TRAPPIII complex.Finally,we generated the Wipi2,Wdr45,Atg2 a,Atg2b single knock-out and Atg2 a & Atg2 b double knock-out cell line using Cas9 based method.Comparing the autophagy phenotypes among the cell lines mentioned above,we found significant similarity between Wipi2 and Atg2 b cell line,and between Wdr45 and Atg2 a cell line.Besides,the Atg2 a & Atg2 b double knock-out cell line shows accumulated small vesicles and autophagy marker proteins.Thus,we thought that mammalian ATG2 A and ATG2 B protein may play different but yet overlapped roles in autophagosome formation.Combining the biochemical and structual data above,we propose that ATG2B-WDR45 complex might contribute to autophagosome formation by directly mediating autophagy precursor membrane fusion.