Studies On The Function And Interaction Between ATG1 And ATG5 In Spodoptera Litura

Autophagy exists in all eukaryotic cells widely,it's evolutionarily conserved.Now it's generally accepted that autophagy is a defense and regulatory mechanism under stress,it provides necessary raw and recycling material for the reconstruction,regeneration and repair of cells.In the process of autophagy,autophagy related genes regulate each phase precisely,and different ATG proteins play their own unique and key roles.Although the molecular mechanism during autophagy involving of a series of ATG genes has been clarified to some extent,but of which details still remains a mystery.ATG1 protein,a serine/threonine protein kinase,is a critical autophagy related protein in the process of autophagy.When autophagy initiates,ATG1-ATG13 compounds can recruit other ATG proteins and induce autophagy.In Lepidoptera insects,the role of ATG1 in the specific regulatory mechanism of cell autophagy is still not very clear,the research on its interacting proteins is not clear,neither.Spodoptera litura,a polyphagous insect,which belongs to Noctuidae,caused serious harm for many crops.Based on the Spodoptera litura,we study the molecular mechanism of autophagy involving of ATG1,the results are of potential value to agricultural pests prevention and control in our country.This study first demonstrated the interaction between ATG 1 and ATG5 in Spodoptera litura via GST pull down,Co-Immunoprecipitation and bimolecular fluorescence complementation.Gene and protein sequences of SlAtg1 were analyzed,which indicated that SI ATG 1 has three functional structure domains,which are kinase domain,LIR structure domain and ATG 13-binding region,respectively;According to the structure of the domain,the gene was truncated.The colocalization experiment,bimolecular fluorescence complementation and GST pull down technology were used to confirm that the interaction between ATG1 with ATG5 mainly relied on kinase domain;Then we gradually truncated 27,100,200,300 amino acids at N terminal of ATG1,and the specific interaction sites may be at 1-100AA of ATG1's N terminal,which was explored by bimolecular fluorescence complementation;And co-transfection of ATG 1-Flag and GFP-ATG5 in Sl-HP cells showed that ATG1 can promote the ATG5-ATG12 conjugating and the degradation of ATG5.All together,the results suggest that ATG5 may be a physiological substrates of ATG 1,ATG 1 had effects on the stability of ATG5 and ATG5-ATG12 coupling via some unknown mechanisms.