The Mechanisms Of Lysosomal Localization Of PAT1 And Their Influences On MTORC1

mTORC1（mechanistic/mammalian target of rapamycin complex1）is an evolutionarily conserved protein kinase complex,which is a master regulator of cellular growth in response to a variety of environmental cues.Dysregulation of mTORC1 has been associated with multiple diseases.According to a current model,the amino acids stored within the lysosome are a direct signal to activate mTORC1.However,the mechanisms of how the lysosomal amino acids are maintained remain largely a mystery.PAT1（proton-coupled amino acid transporter 1）is an unbiquitoulsy expressed amino acid transporter that contains 11transmembrane domains.Within the cells,PAT1 is often localized on the lysosomal surface and was believed to negatively regulate mTORC1 through decreasing the luminal amino acid level.PAT1 is also expressed on several other endosomes,including the nuclei.So far,the mechanisms controlling the subcellular localization of PAT1 have not been carefully studied.Our previous study has revealed that the localization of PAT1 on lysosome was dynamic and affected by the amino acid levels.In order to better understand the mechanisms of PAT1 localization and its influence on mTORC1,we took the human renal epithelial cell HEK293 as the main research model,and carefully investiagated the subcellular localizations of tagged PAT1.The following results were obtained:1.Using EGFP-PAT1 as a target,we found it could be cleaved around the ⁴⁷TTWF⁵⁰motif.This resulted in the loss of the N-terminal cytosolic region of PAT1.2.We identificated in cleaved fragment a consensus tyrosine-based lysosomal targeting signal,³⁸YQRF⁴¹（YxxΦ）.Replacing the critical 38^th tyrosine with an alanine decreased the localization of PAT1 on the lysosome but increased its localization on the plasma membrane,and could not inhibit mTORC1 activity.This result reveals that the ³⁸YQRF⁴¹ motif assists the transport of EGFP-PAT1 to the lysosome.3.Amino acid starvation induced PAT1 cleavage,which was inhibited by amino acid stimulation,or individually glutamine.These results suggest that amino acids may promote the lysosomal amino acid level through inducing PAT1 cleavage.4.Using the immunofluencent method,we found the second cytoplasmic region of PAT1 contains important lysosomal targeting signals.5.The ¹¹³FVDY¹¹⁶ motif in the second cytoplasmic region of PAT1 guides the transport of PAT1 from the endoplasmic reticulum to the Golgi apparatus.Missing of this motif caused the retention of PAT1 in the endoplasmic reticulum and overexpression of the mutant could not inhibit mTORC1.6.Ectopic expression of PAT1 on mitochondria could not inhibit mTORC1.This result provides further evidence that PAT1 inhibits mTORC1 on the lysosome.In summary,two major findings were obtained in this study.First,amino acids can induce PAT1 cleavage,resulting in the loss of a lysosomal localization signal,³⁸YQRF⁴¹,thereby inhibiting the transport of PAT1 to the lysosomes and promoting mTORC1 activation.Second,the second cytoplasmic region of PAT1 contains important lysosomal localization signals.And the ¹¹³FVDY¹¹⁶ motif directs the newly synthesized PAT1 transport from endoplasmic reticulum to Golgi apparatus.Mislocalization of PAT1 on the endoplasmic reticulum or mitochondria could not inhibit mTORC1.The above results indicate that PAT1can inhibite mTORC1 on the lysosome.