Mechanistic Studies Of The Interactions Of Arl8b With The RUN Domains Of PLEKHM1 And SKIP

Autophagy is one of the main mechanisms that cells deal with various internal and external cell stresses.It can transport unnecessary or harmful substances to lysosomes for degradation and recycling.During autophagy,the degradation of various substrates depends on the membrane fusion of autophagosome and lysosome,while the spatial encounter of autophagosome and lysosome depends on the directional transport of lysosome.At present,many key proteins involved in the membrane fusion of autophagosome and lysosome as well as the directional transport of lysosome have been found,such as small G protein Arl8b,tethering factor PLEKHM1,HOPS complex and SKIP,but their related working mechanisms are still unknown.The research work in this thesis mainly focuses on elucidating the molecular mechanism of small G protein Arl8b in mediating the directional transport of lysosome and the membrane fusion of autophagosome and lysosome in the autophagy pathway.In chapter 3,we studied the interaction mechanism between the small G protein Arl8b,which mediates the membrane fusion of autophagosome and lysosome,and the tethering factor PLEKHM1.Firstly,we qualitatively analyzed the interaction between Arl8b and PLEKHM1 using analytical gel filtration chromatography method,and found that there is a direct interaction between the GTP-bound active Arl8b and the Nterminal RUN domain of PLEKHM1.Then,we solved the crystal structure of the active Arl8b and PLEKHM1 RUN complex by X-ray crystallography method,and elucidated the detailed molecular mechanism underlying the interaction between Arl8b and PLEKHM1.In addition,we also detected the interactions of the relevant subunits of HOPS complex with Arl8b and PLEKHM1,and found that Arl8b directly interacts with the N-terminal region of VPS41,while PLEKHM1 interacts weakly with the C-terminal region of VPS39.In chapter 4,we have conducted an in-depth research study on the interaction mechanism between small G protein Arl8b and SKIP protein,which mediates the directional transport of lysosomes in cells.Firstly,we used analytical gel filtration chromatography and isothermal titration calorimetry to qualitatively and quantitatively analyze the interaction between Arl8b and SKIP,and found that,similar to PLEKHM1,the active Arl8b can directly interact with the N-terminal RUN domain of SKIP.Subsequently,we determined the crystal structure of the Arl8b/SKIP complex and revealed the molecular mechanism of the interaction between Arl8b and SKIP in detail.After analyzing and comparing the structures of the Arl8b/PLEKHM1 and Arl8b/SKIP complexes,we found that although their overall structures are highly similar,there are significant differences in the binding interface residues,indicating that the two complexes play different roles in the autophagy pathway.In conclusion,in this thesis,through systematic biochemical and structural studies,we determined,for the first time,the crystal structures of small G protein Arl8b in complex with the RUN domains of PLEKHM1 and SKIP,respectively,and uncovered the detailed molecular mechanism of Arl8b for binding to PLEKHM1 and SKIP,and revealed a novel binding mode of the RUN domain for interacting with other proteins.Our work provides a good basis for further understanding the molecular mechanism of membrane fusion of autophagosome and lysosome as well as the directional transport of lysosome in autophagy pathway.