The Screening Of Sec24-interacting Proteins For Protein Kinases Involved In Macroautophagy In Saccharomyces Cerevisiae.

Autophagy is a catabolic process in which unwanted proteins and organelles are transported to vacuoles or lysosomes for degradation.Macroautophagy,a type of autophagy,is crucial for maintaining cell homeostasis and survival,and the changes in the activity of macroautophagy are related to various diseases.The membrane structures of many different components are involved in this process.COPII vesicles commonly transport substances between the endoplasmic reticulum and Golgi apparatus,but when macroautophagy is initiated,COPII vesicles start playing their role in the macroautophagy pathway.It is observed that the Sec24 subunit of the COPII coat complex facilitates the entry of COPII vesicles into the macroautophagy pathway.The phosphorylation of Sec24 by Hrr25 is a critical step that enables COPII vesicles to participate in the macroautophagy process in saccharomyces cerevisiae.Hrr25 belongs to the CK1 family,and it is known to target proteins that contain phosphorylated serine or threonine residues.This finding shows that CK1 family member can phosphorylates proteins that have already been phosphorylated by the upstream protein kinases.Therefore,it is speculated that some other protein kinases may be involved in macroautophagy which phosphorylates the Sec24 subunit of proteins upstream of Hrr25.Given this scientific assumption,we carried out the screening of proteins interacting with Sec24 to identify the possible Sec24 interacting macroautophagy associated protein kinases.Firstly,protein kinases interacting with Sec24 were screened by mass spectrometry.The integrated expression function of plasmid p RS305 was used to modify the genome of wild-type yeast cells to obtain Sec24-13 Myc protein-expressing yeast strains.Subsequently,Immunoprecipitation of Sec24-13 Myc was performed to identify the proteins interacting with Sec24 using mass spectrometry.The results showed that there was a total of 1279 trusted Sec24 interacting proteins.Out of 1279 Sec24 interacting proteins,28 identified as protein kinases.It was found that 22 out of 28 protein kinases interacted with Sec24 only after macroautophagy induction.After an extensive literature review,a total of 34 candidate protein kinases interacting with Sec24 were selected.Furthermore,candidate Sec24 interacting protein kinases were evaluated for their connection to macroautophagy,and protein kinases showing no connection were excluded.Among 34 protein kinases,13 were found to have possible involvement in macroautophagy.GFP-Atg8 cleavage and alkaline phosphatase activity assay were used to verify the macroautophagy activity level after the knockout of candidate proteins.The macroautophagy activity levels decreased after the knockout of seven protein kinases,Bub1,Cmk1,Cmk2,Ire1,Pbs2,Rim15,and Snf1,which indicated their involvement in macroautophagy.The remaining six protein kinases,Cka2,Chk1,Hog1,Kkq8,Yck1,and Yck2,were failed to show a role in macroautophagy hence excluded.Then it was verified whether the candidate protein kinases interacting with Sec24 were involved in macroautophagy.After obtaining 34 protein kinases interacting with Sec24,the protein kinases that had been verified to be unrelated to macroautophagy were excluded,other 13 protein kinases with the possibility of involvement in macroautophagy were screened out as candidate protein kinases.GFP-ATg8 cleavage and alkaline phosphatase activity assay were used to verify the level of macroautophagy after the knockout of candidate proteins.It was concluded that the levels of macroautophagy decreased after the knockout of seven protein kinases,Bub1,Cmk1,Cmk2,Ire1,Pbs2,Rim15 and Snf1,indicating that these protein kinases were involved in macroautophagy.And six protein kinases can be ruled out,they are Cka2,Chk1,Hog1,Kkq8,Yck1 and Yck2.After identifying seven Sec24 interacting macroautophagy associated protein kinases,these were evaluated for their degree of involvement in macroautophagy.Among these,three protein kinases,Ire1,Rim15,Snf1 were showed a solid connection to macroautophagy,compared to the remaining four protein kinases,Bub1,Cmk1,Cmk2,Pbs2.In this study,we screened out the Sec24 interacting macroautophagy-related protein kinases,which tend to modify the macroautophagy activity.The findings of our study have provided concrete evidence of the involvement of Sec24 interacting protein kinases in macroautophagy,which could help in a more comprehensive understanding of the macroautophagy process in the future.