Molecular Mechanism Of Golgi Protein GOLPH3 Regulating Autophagy Through MTOR Signaling Pathway

Autophagy is an evolutionary conserved catabolic process,which is regulated coordinately by a great number of proteins and lipids.GOLPH3,a peripheral membrane protein located at TGN,plays an important role in maintaining the morphology and structure of the Golgi apparatus and the vesicle transport.GOLPH3 also functions as a proto-oncogene to promote tumorigenesis and its development.It can also regulate the activity of m TORC1 and affect the sensitivity of cells to Rapamycin.Here,we found that GOLPH3 is involved in autophagy,and GOLPH3 knockdown promotes cell autophagy.Compared with control cells,GOLPH3 knockdown cells possess higher levels of ULK1,FIP200 and LC3.Furthermore,treatment with Bafilomycin A1(Baf A1)also results in the increased LC3-II levels,which indicats that GOLPH3 plays a role at the early stages of autophagosome formation.m TORC1 is composed of three core components: m TOR,Raptor,and m LST8.m TORC2 also contains m TOR and m LST8,and the core protein Rictor.It was found that Raptor as a “bridge” molecule in the m TORC1 complex,linking the m TORC1 kinase domain with the downstream substrate of m TORC1.While the 36 KDa m LST8 can specifically bind the m TORC1 catalytic domain and stabilize the m TORC1 complex.m LST8 is more important for m TORC2.Deletion of m LST8 affect m TOR association with the cofactors Rictor and m Sin1,therefore interfering the m TORC2 activity.We found that GOLPH3 colocalized with m LST8 and m TOR,a core component of m TORC1/2,and immunoprecipitation experiments indicated that GOLPH3 interacted with m TOR and m LST8.In addition,knockdown GOLPH3 resulted in the less recruitment of Raptor and m LST8 to m TOR,while the recruited Rictor was not affected.VPS74(the orthologue of human GOLPH3)was found to interact with PI4 P phosphatase SAC1 in yeast cells.VPS74 regulates the activity of SAC1.Knockdownof VPS74 or GOLPH3 increase the level of PI4 P on Golgi.Here,we show that GOLPH3 in mammalian cells also interacts with SAC1,and GOLPH3 knockdown results in the increase in the level of PI4 P on Golgi.Previous reports that the increase of PI4 P on Golgi will damage the lysosomal degradation.Based on this,we investigated the effect of GOLPH3 knockdown on lysosomal degradation.We found that GOLPH3 knockdown significantly decrease the level of lysosomal hydrolase CTSB and CTSD,which indicates lysosomal degradation is impaired.Together,we found that GOLPH3 is a key player in the recruitment and assembly of m TOR complex.Therefore,GOLPH3 regulates the early autophagy process through modulating the m TOR signaling.On the other hand,GOLPH3 interacts with SAC1,affecting the distribution of PI4 P in Golgi,which is vital for the function of lysosomal degradation.