The Role Of Spodoptera Litura ATG1 Complex In Autophagy

Autophagy is a process in which cells degrade their substances,which helps cells to maintain internal environment stable and balanced.A large number of experimental data have shown that the process of autophagy is a way of cell self-defense under the strict regulation of multiple autophagy-related genes(Atg).At the initial stage of autophagy,Atgl can bind many kinds of autophagy related proteins to form Atgl complex.The complex receives signals from the cell's nutritional status,recruits downstream Atg proteins to the assembly site of the pre-autophagosomal structure,and controls the formation of autophagosomes.It is known that the Atgl complex is mainly composed of Atg1-Atg13-Atg17-Atg29-Atg31 protein in Saccharomyces cerevisiae.The homolog of the mammalian Atgl complex is the ULK1 complex,this complex is mainly composed of ULK1-Atgl3-FIP200-Atg101 protein.The different components of Atgl complex in different species may lead to different regulatory mechanisms that control autophagy initiation.At present,there are relatively few studies on interaction proteins in the Atgl complex of lepidoptera insects,and the regulation mechanism of each subunit protein in the Atgl complex on autophagy is still unclear.In this experiment,we mainly studied the interaction proteins in the Spodoptera litura Atgl complex and explored the function of some autophagy-related proteins.In this experiment,the open reading frame of Atg13,Atg9 and FIP200 was cloned from the Spodoptera litura.Next,the insect cell expression vectors Atg13-Flag,Atg13-GFP,Atg101-GFP,and FIP200-GFP were constructed.Atg13 was co-expressed with Atgl,Atg101 and FIP200 proteins in S1-HP cells,and observing whether there was co-localization between the two proteins.Then,the insect cell bimolecular fluorescent expression vectors YFPN-Atgl3,Atg9-YFPC,and FIP200-YFPC were constructed.And it was investigated whether Atg 13 was the interaction protein of Atgl,Atg101 and FIP200 through the bimolecular fluorescence complementation technology,furthermore,whether there was interaction between Atgl and Atg9,FIP200 proteins.Finally,the effects of Atg13,AMPK,and Atg9 RNAi on autophagy were examined by interference experiments.The ORF total length of Atg13,FIP200 and Atg9 were 1281bp,3915bp and 2289bp respectively,the predicted amino acids were 426,1304 and 762 separately,the isoelectric points were 5.46,5.20 and 7.63 respectively.The results of immunofluorescence showed that Atg13 protein was co-localized with Atgl,Atg101 and FIP200 proteins,and co-localized in the cytoplasm of Sl-HP cells.The bimolecular fluorescence complementation experiments showed that Atg13 and Atgl had strong interaction signals,and small particles were distributed evenly in the cytoplasm.The interaction signal of Atg13 with Atg101,FIP200 proteins was relatively weak,and large particles were distributed unevenly in the cytoplasm.No obvious interaction signal was found between Atgl and Atg9,FIP200 proteins.The interference of Atg13 and AMPK prevented the induction of autophagy by toxin,and reduced the susceptibility of cells to toxins.This indicated that the interference of Atg13 and AMPK reduced the level of autophagy,however Atg9 RNAi was no significant effect on cell autophagy.These experimental results provide an important basis for revealing the role of SlAtg1 complex in autophagy.