Identification And Functional Analysis Of Interaction Proteins Of Sl-Atg5 Of Spodoptera Litura

Autophagy is a stress reaction degrading biomacromolecules by lysosomes in order to adapt to changes of the external environment and it is highly conserved process existing in eukaryotic cells.Autophagy is mainly divided into three types:microautophagy,macroautophagy and chaperone-mediated autophagy(CMA).Studying of autophagy is of great significance,because autophagy closely related to a variety of physiological and pathological processes of cells.Autophagy is coordinated and controlled by different signaling pathways and multiple autophagy-related genes.Atg5 plays an important role in autophagy of yeast and mammalian cells.In cells,Atg5 once after synthesized interacts with Atg 12 to form a complex.This complex interacts with Atgl6 to promote the lipidation of Atg8 and is essential for the extension of autophagy bubble membrane.However,there are less known about interaction proteins and functions of Lepidopteran insect Atg5.Spodoptera litura(Fabricius)is a species of the genus Spodoptera from the Lepidoptera noctuidae family,which is omnivorous and overeating,causing damage to cotton,soybeans,tobacco,and other crops.In this study,we first identified that the over expression of Atg5-GFP localized in nucleus and cytoplasm by fluorescence microscope.However,Atg5-GFP and Atg 12-FLAG were simultaneously overexpressed to form complexes and were localized only in the cytoplasm;The Western Blot proved that the Atg5-Atg12 complex was more stable than Atg5 in the cells;Then,the influence of Atg5-Atg12 complex on cell autophagy and Atg3 complex was analyzed by Western Blot.The results showed that only overexpression of Atg5-Atg12 complex did not significantly affect the level of autophagy and the formation of Atg3-Atgl2 complex;Then,using pull down technology,GST-SlAtg5 as bait and Sl-HP cell total protein as prey to get Sl-Atg5 interaction proteins,it is preliminarily analyzed that 26 proteins can interact with Atg5,which can be divided into 13 types.Finally,the role of SlAtg5 in the autophagy induced by Bt toxin in insect cells was studied by RNAi technique.It was suggested that after the knockdown of Atg5,the sensitivity of the cells to toxins decreased,the conversion of Atg8 and Atg8-PE was blocked,and the level of autophagy decreased significantly;In addition,Sl-Atg1 in the upstream of Sl-Atg5 can interact with each of Sl-Atg6,Sl-Atg101 and Sl-Atg8 respectively by using the bimolecular fluorescence complementation.The results of this study suggest that Atg5 plays an indispensable role in the regulation of autophagy.Exploring and studying the interaction proteins and functions of Atg5 are of great significance for further studying the molecular mechanism of cell autophagy.