The Mechanism Of Amino Acid Transporter PAT1 In MTORC1 Regulation

The mechanistic target of rapamycin 1(mTORC1)is a key regulator of cell growth,metabolism,aging and so on.The available evidence suggests that lysosome is a key site for mTORC1 activation.When there are high levels of amino acids(particularly Leucine,Arginine and Glutamine)in the lysosome,mTORC1 is recruited to the lysosome,on which it can be activated by Rhed,which is a lysosome-associated small GTPase.According to this model,the lysosomal amino acids are a critical signal to activate mTORC1.However,how the level of lysosomal amino acids is regulated has not been carefully investigated.PAT1(Proton-coupled Amino acid Transporter 1)is a widely expressed membrane protein.Inside the cells,PAT1 is mainly localized on the lysosome,transports both proton and amino acids into the cytosol.Although it is known that PAT1 can regulate m TORC1,but the underling mechanism is not clear yet.In the current study,I used HEK293 cells as a model to investigate the significance of the localization and transport function of PAT1 during mTORC1 regulation,and obtained the following discoveries:(1)The stable HEK293 cells expressing myc-PAT1 were generated.Under the steady state condition,the mTORC1 activity did not show significant differences between the control HEK293 and the myc-PAT1 cells.When the cells were synchronized by starvation,with a short period time of amino acids replenishment(10 minutes),the mTORC1 activities were decreased in the myc-PAT1 overexpressing cells;with a longer amino acids replenishment,the mTORC1 activities were similar bwteen the control HEK 293 and the myc-PAT1 cells.(2)The disulfide fond formed between C180 and C329 of the PAT1 protein is critical for amino acids loading.I generated stable cell lines expressing two mutated PAT1,including C180A(sM)and C180/329/A(dM).Both mutations did not affect the lysosomal localization of PAT1.However,overexpression of the mutant proteins could not inhibit mTORC1.(3)PAT1 is a proton-coupled amino acids transporter.This binding with proton is critical for the transport function of PAT1.I generated HEK 293 cells stably expressing the proton-binding mutant,H55 A.The mutant protein could be localized on the lysosome.However,overexpression of the H55 A mutant could not inhibit mTORC1.(4)Tryptophan and its derivative,5-hydroxytryptamine(5-HTP),are competitive inhibitors of the PAT1's transport function.I found Try and 5-HT could antagonize the PAT1's activity to inhibit mTORC1.(5)The PAT1 mutant cell lines(PAT1-/-)were generated using the CRISPR/Cas9 system.Comapred with the wild-type control cells(HEK 293T),the mTORC1 activity was not apparently changed in the PAT1-/-cells.In the absence of amino acids,the mTORC1 activity was decreased in both wild-type(HEK 293)and the PAT1-/-cells,but the decrease was slower in the PAT1-/-cells.On the other hand,the mTORC1 activity was decreased faster in the myc-PAT1-overexprsing cells.These results suggest the expression level of PAT1 is a factor affecting the mTROC1 activity.Base on these findings,I conclude that PAT1 controls mTORC1 in an amino acids-sensitive manner that requires its amino acids transport function of PAT1.These data support a model that amino acids inhibit the expression of PAT1 on the lysosome.This allows a relatively high level of lysosmal amino acids,which can activate mTORC1.