The Study Of How PI4P Phosphatase And PI4Ks Regulate Autophagy And Homeostasis

Inter-organelle contacts enable the direct exchange of substances between organelles to coordinate various biological events in cells.The endoplasmic reticulum,as a large membranous tubular system distributed throughout the cytoplasm,can establish extensive contacts with other membranous organelles in cells.Anchored proteins located on the endoplasmic reticulum mediate contacts between the endoplasmic reticulum and other organelles through interactions with proteins located on the membranes of other organelles.These contacts are important for maintaining cellular physiological functions,but how they are established,regulated,and what functions they perform remain unclear.In normal cultured cells,phosphatidylinositol-4-phosphate(PI4P)enriched on the surface of Golgi recruits PI4P-binding proteins such as Oxysterol binding protein(Osbp).The latter binds to VAMP-associated proteins(VAPs)located on the endoplasmic reticulum,mediating contact between the endoplasmic reticulum and Golgi apparatus as well as lipid transport between these two organelles.It has been found that PI4 P is enriched on autolysosomes during autophagy,but its role remains unclear.In order to study the function and regulation of PI4 P on autolysosomes,we systematically investigated the role of PI4P-related kinases,phosphatase and PI4 P effectors during autophagy in Drosophila fat bodies.In this study,we found that knocking down PI4 P phosphatase Sac1 results in degenerated phenotypes in the fly eyes and abnormal accumulation of various autophagy markers in the fat body tissues.Further experimental results showed that the reduction of Sac1 promotes the initiation of autophagy and does not affect the fusion of autophagosome and lysosome,but leads to the weakening of acidity of autolysosomes and the decrease of autophagy flux.PI4 P reporter GFP-P4M-Sid M signals were increased in Sac1 RNAi tissues,suggesting that the levels of PI4 P on autolysosomes were elevated.We then overexpressed or knockdown three phosphatidylinositol 4-kinases(PI4Ks)in the fat body tissues to observe their localization,changes in PI4 P levels,and distribution patterns of autophagy markers.The results showed that the three PI4 Ks regulate PI4 P levels in the PI4 P pool in different parts of the cell,and they are all required for autophagy.Pi4KIIα is a key enzyme that produces PI4 P on autolysosomes,which can be transferred to the autolysosomes during starvation.Subsequently,we investigated whether PI4 P binding proteins were involved in autophagy and found that PI4 P enriched on autolysosomes recruits Osbp and ceramide transfer protein(Cert).When Osbp or Cert was knocked down,the levels of PI4 P on the autolysosomes were increased and the autophagy defect phenotypes were similar to but less severe than that when Sac1 was reduced.In addition,neurodegeneration was also present in the fly eyes when reduced Osbp or Cert expression.Further studies showed that PI4 P levels on autolysosomes increased when the endoplasmic reticulum VAP protein Vap33 was knocked down.Vap33 is an important protein mediating contact between the endoplasmic reticulum and other organelles.In normal cultured cells,Vap33 can bind to proteins such as Osbp/Cert to mediate lipid transport between the Golgi apparatus and the endoplasmic reticulum.We found the endoplasmic reticulum-autolysosome contacts were increased in tissues when knocked down Sac1 or overexpressed Osbp.Overexpression of a mutant form of Osbp that cannot transfer cholesterol but can bind to Vap33 further increased the endoplasmic reticulumautolysosome contacts.Based on these results,we propose a model: Similar to the endoplasmic reticulumGolgi contact and lipid transport mediated by PI4P-Osbp/Cert-VAP-Sac1 in normal cultured cells,during starvation,the autolysosomes recruit Pi4KIIα on their surface to generate PI4 P,and establish the endoplasmic reticulum-autolysosome contacts through the PI4P-binding proteins Osbp and Cert.The contacts promote PI4 P to be transported to the endoplasmic reticulum and dephosphorylated by Sac1 to drive the transport of cholesterol and other lipids between the endoplasmic reticulum and autolysosomes,maintaining the normal functions of autolysosomes and neuronal homeostasis.By translocating PI4 Ks in response to stimulation,phospholipid signaling molecules are located on different organelles,and then the same downstream effector molecules are recruited to mediate contact between endoplasmic reticulum and different organelles and materials exchange,reflecting the exquisite regulation of organelle contacts in cells.In conclusion,this study reveals a new regulation mechanism of intracellular organelle contacts,and provides rich reference information for understanding the physiological functions of PI4P-related kinases,phosphatase and downstream effector proteins.