The Role Of COP? Coat Protein SEC24 In Mammalian Macroautophagy

Autophagy is an evolutionarily conserved pathway in which cytoplasmic components are sequestered within double-membrane vesicles called autophagosomes and then transported into lysosomes or vacuoles for degradation.COP? vesicles derived from the endoplasmic reticulum are important membrane sources of autophagosomes.And their well-known biological function is the transport of cargos from the endoplasmic reticulum to the Golgi apparatus.When starvation induces autophagy,COP? vesicles can be transferred to autophagy pathway to provides a membrane for autophagosomes.SEC24 is the coat protein of COP? vesicles.There are four subunits of SEC24 in mammalian cells: SEC24 A,SEC24B,SEC24 C and SEC24 D.And they are divided into two groups according to homology: SEC24A/SEC24 B and SEC24C/SEC24 D.However,the role of different subunits in mammalian autophagy is not yet clear.To solve this problem,we start with single knockdown of SEC24 A,SEC24B,SEC24 C,SEC24D subtypes and double knockdown of SEC24A/SEC24 B,SEC24C/SEC24 D subunits to examine the effects of different SEC24 on mammalian autophagy,identify the SEC24 subunit and confirm its action site that regulates autophagy.In this thesiss,the four subunits of SEC24,SEC24 A,SEC24B,SEC24 C,and SEC24 D were first knocked down using si RNA interference technology.In addition,SEC24A/SEC24 B and SEC24C/SEC24 D were double knocked down,then we verified the knockdown effect by Western Blot and QPCR.Starvation of the knocked down cell line induces autophagy.Western Blot and immunofluorescence were used to detect the levels of autophagy substrate protein p62 and autophagy marker protein LC3-II.The results showed that the LC3-II level was reduced compared with negative control under starvation in SEC24A-depleted cells or in SEC24D-depleted cells.And the accumulation of autophagy substrate p62 suggests that the starvation-induced autophagy pathway is blocked.After treatment with Bafilomycin A1,the level of LC3-II increased and was still lower than negative control.It suggested that autophagy defects occurred in the early stage of autophagosome formation.However,starvationinduced autophagy activity had no significant effect in SEC24B-depleted cells.In SEC24C-depleted cells,LC3-II levels were higher than negative control under starvation conditions,and the autophagy substrate p62 decreased,suggesting that starvation-induced autophagy activity increased.Instead,it has the opposite effect of knocking down SEC24 D.Therefore,the effect of SEC24A/SEC24 B double knockdown to inhibit autophagy is mainly achieved by knocking down SEC24 A.The double knockdown of SEC24C/SEC24 D does not affect autophagy.The main reason is that the knockdown of SEC24 C and the knockdown of SEC24 D show the opposite effect of autophagy.The results indicate that SEC24 A and SEC24 D are required for starvation-induced autophagy.Subsequently,we analyze the sites where SEC24 A and SEC24 D regulate the starvation-induced autophagy.We transfected RFP-GFP-LC3 tandem fluorescent plasmid to detect the number of autophagosomes and autolysosomes.The results showed that the number of autophagosomes and autolysosomes were reduced in SEC24 A or SEC24D-depleted cells.It indicates that the autophagy defect occurred in the early autophagosome formation stage which is consistent with the Western Blot results of LC3-II.We then used proteinase K sensitivity experiments to identify whether autophagy defects occurred before or after autophagosome closure.By comparing the sensitivities of GFP-LC3 and p62 to proteinase K with and without surfactants in SEC24 A or SEC24D-depleted cells,increased sensitivity of GFP-LC3 and p62 to proteinase K indicates that they are not protected by autophagosomes,and autophagy defects occur before autophagosomes are closed.The results showed that under starvation-induced autophagy in SEC24 A or SEC24D-depleted cells,the number of ATG16L1 and ATG14 L puncta structures decreased,while the number of ATG9 A puncta structures did not change significantly.The association of LC3 with ATG9 A,ATG14L and ATG16L1 were all increased in SEC24 A or SEC24D-depleted cells.These data suggest that SEC24 A and SEC24 D act upstream of ATG16L1 and ATG14 L,and lead to the accumulation of ATG protein in early autophagy membrane structure.In summary,we show the important role of SEC24 in mammalian cells in starvation-induced autophagy,and identify SEC24 A and SEC24 D are required for autophagy in mammalian cells.Then we found that SEC24 A and SEC24 D acted in the early stage of autophagy before autophagosome closure and further found that knockdown of SEC24 A or SEC24 D affected the formation of puncta structures of ATG16L1 and ATG14 L,and caused the accumulation of ATG protein in the early autophagy membrane structure.Our data reveal a key role of SEC24 subunits in mammalian macroautophagy.It advances the understanding of the mechanism of autophagosome completion and also provides information for the important role of COP? vesicles and their coat proteins in macroautophagy.