The Regulatory Role Of Lysosomal Ion Channel Transient Receptor Potential Mucolipin-1 On Mechanistic Target Of Rapamycin Complex 1 During Starvation

Nutrient sensing is essential for cells and organisms to grow and survive in response to environmental changes.Macroautophagy?hereafter referred to as autophagy?is an evolutionary conserved cellular process in which cells degrade their own organelles to generate energy and nutrients to maintain cellular homoeostasis and support cell survival,especially under starvation.Autophagic lysosome reformation?ALR?is critical for maintaining lysosome homeostasis and cell survival wich is essential for contineuous autophagy during prolonged starvation.The mammalian target of rapamycin complex 1?mTORC1?is a ubiquitous protein kinase acting as a master regulator of autophagy.It is also a key nutrient sensor which coordinates cell growth and division in response to the availability of nutrients.mTORC1 is normally associated with the lysosome where it is stimulated by nutrients such as amino acids using an inside-out mechanism in which amino acids must accumulate in the lysosomal lumen to initiate signaling.Starvation causes mTORC1 falling off from the lysosome,causing mTORC1 inhibition and subsequent autophagy induction.Given that mTORC1 is critical for protein synthesis and cellular homeostasis,a feedback regulatory mechanism must exist to restore mTORC1 during starvation.However,the molecular mechanism underlying this feedback regulation is unclear.Intracellular Ca²⁺signaling has recently been recognized as an important player in the regulation of mTORC1 and autophagy,although its exact role remains a matter of debate.Recently,lysosomes have been found to be important intracellular Ca²⁺stores.Given that mTORC1 must be recruited to the surface of lysosomes to be activated,it is interesting to test whether lysosomal Ca²⁺regulates mTORC1 activity.Transient receptor potential mucolipin-1?TRPML1/MCOLN1?is an important lysosomal Ca²⁺release channel and we have systematically examined the role of TRPML1 on mTORC1 under nutrient limitation.To study the effect of TRPML1 on mTORC1,we upregulate TRPML1 by either TRPML1overexpression or ML-SA1 and find that both of them increase mTORC1 lysosomal localization and mTORC1 activity by releasing lysosomal Ca²⁺.Inversely,downregulation of TRPML1 using TRPML1-shRNA and ML-SI1 leads to a decrease in mTORC1 lysosomal localization and mTORC1 activity.These data suggest that TRPML1 is both sufficient and necessary for mTORC1 activation.Furthermore,we found that TRPML1 regulating lysosomal localization and mTORC1 activity is dependent on lysosomal Ca²⁺and calmodulin?CALM?.Lysosomal Ca²⁺released by TRPML1 binds to CALM and then activates it,thereby regulating lysosomal localization and activity of mTORC1.Interestingly,TRPML1 regulates mTORC1lysosomal localization and activity only under starvation but not normal fed condition.To further elucidate the molecular mechanism by which TRPML1 regulates mTORC1,we demonstrate that TRPML1 is activated by starvation and mTORC1's inhibition.Moreover,TRPML1 is normally inhibited due to phosphorylation at Ser571 and Ser576 by mTORC1 when mTORC1 is associated with lysosomes under fed conditions.Starvation results in mTORC1falling off from lysosomes and mTORC1 inhibition,subsequently leading to TRPML1activation by relieving mTORC1's inhibition on the channel.Although mTORC1 signaling is inhibited during initiation of autophagy,it is reactivated by degradation products such as amino acids during prolonged starvation and then triggers ALR.We find that both TRPML1 and CALM are required for mTORC1 reactivation during long-term starvation.More importantly,TRPML1 is required for cell survival during prolonged starvation.Our data suggest that lysosomal Ca²⁺signaling is an essential component of the canonical mTORC1-dependent autophagy pathway and TRPML1 provides a negative feedback regulation of mTORC1 to prevent mTORC1 from excess reduction during starvation.Therefore,we have suggested a negative feedback regulation of mTORC1 by the lysosomal channel TRPML1 using a CALM-dependent mechanism.TRPML1 is normally inhibited due to phosphorylation at Ser571 and Ser576 by mTORC1 when mTORC1 is associated with lysosomes under fed conditions.Starvation causes mTORC1 falling off from lysosomes and a subsequent decrease in mTORC1 activity.This further leads to TRPML1-CALM activation,promoting lysosomal mTORC1 recruitment and reactivation.The feedback regulation may be important for maintaining cellular homeostasis during starvation,as well as many other stress or disease conditions in which mTORC1 activity is defective.