Autophagy is crucial for maintaining cellular homeostasis and is linked to various dis-eases.In Saccharomyces cerevisiae,the Polymyxin B Sensitivity 2(Pbs2)protein is a member of the mitogen-activated protein kinase(M...Autophagy is crucial for maintaining cellular homeostasis and is linked to various dis-eases.In Saccharomyces cerevisiae,the Polymyxin B Sensitivity 2(Pbs2)protein is a member of the mitogen-activated protein kinase(MAPK)family and plays a role in mitophagy.To explore the potential role of Pbs2 in macroautophagy,we engineered wild-type and PBS2-deficient cells using plasmid construction and yeast transforma-tion techniques,followed by a series of autophagy assays.First,after nitrogen star-vation,the levels of autophagic activity were evaluated with the classical GFP-Atg8 cleavage assay and the Pho8Δ60 activity assay at different time points.Deleting PBS2 significantly decreased both GFP-Atg8 protein cleavage and Pho8Δ60 activity,indicat-ing that Pbs2 is essential for macroautophagy.Furthermore,the influence of Pbs2 on macroautophagy was shown to be independent of Hog1,a well-known downstream factor of Pbs2.Second,the Atg8 lipidation assay demonstrated that Atg8 lipidation levels increased upon PBS2 deletion,suggesting that Pbs2 acts after Atg8 lipidation.Third,the proteinase K protection assay indicated that the loss of PBS2 led to a higher proportion of closed autophagosomes,implying that Pbs2 impacts the later stages of macroautophagy following autophagosome closure.In conclusion,Pbs2 regulates the late stages of macroautophagy induced by nitrogen starvation.展开更多
基金National Natural Science Foundation of China,Grant/Award Number:31970044 and 32370805。
文摘Autophagy is crucial for maintaining cellular homeostasis and is linked to various dis-eases.In Saccharomyces cerevisiae,the Polymyxin B Sensitivity 2(Pbs2)protein is a member of the mitogen-activated protein kinase(MAPK)family and plays a role in mitophagy.To explore the potential role of Pbs2 in macroautophagy,we engineered wild-type and PBS2-deficient cells using plasmid construction and yeast transforma-tion techniques,followed by a series of autophagy assays.First,after nitrogen star-vation,the levels of autophagic activity were evaluated with the classical GFP-Atg8 cleavage assay and the Pho8Δ60 activity assay at different time points.Deleting PBS2 significantly decreased both GFP-Atg8 protein cleavage and Pho8Δ60 activity,indicat-ing that Pbs2 is essential for macroautophagy.Furthermore,the influence of Pbs2 on macroautophagy was shown to be independent of Hog1,a well-known downstream factor of Pbs2.Second,the Atg8 lipidation assay demonstrated that Atg8 lipidation levels increased upon PBS2 deletion,suggesting that Pbs2 acts after Atg8 lipidation.Third,the proteinase K protection assay indicated that the loss of PBS2 led to a higher proportion of closed autophagosomes,implying that Pbs2 impacts the later stages of macroautophagy following autophagosome closure.In conclusion,Pbs2 regulates the late stages of macroautophagy induced by nitrogen starvation.
基金JohnE.Fogarty International Center-NIH-NIAID基金（TW005926）志谢：本实验所用质粒pDHt/SK和根瘤农杆菌EHA105系Kyung J.Kwon-Chung教授惠赠,质粒pALX223系Alex Andrianopou-los教授惠赠,在此特表示衷心的感谢.
