Mechanism Of Intracellular Localization Of Amino Acid Transporter PAT1 Regulated By BHD Syndrome Factor FLCN

Mutations of the FLCN(folliculin)gene have been linked with the BHD(Birt-Hogg-Dubé)syndrome.The available evidence suggests that FLCN is involved in multiple biological processes,including signal transduction,cell-cell adhesion,biogenesis of lysosomes and mitochondria,energy and nutritional homeostasis,and so on,but the precise reasons of why loss of FLCN can casue BHD remain elusive.A previous structure study has revealed a DENN-like domain on the C-terminus of the FLCN protein.This provides a hint that FLCN may regulate vesicular trafficking processes by interacting with the Rab GTPases.However,because the cargos transported by FLCN are largely unknown,the putative functions of FLCN during vesicular trafficking lack experimental evidence.The proton-assisted amino acid transporter 1(PAT1,SLC36A1)is a widely expressed transmembrane protein in mammals.In many cell types,PAT1 is mainly localized on the lysosome,but it was also detected on several other locations,including the plasma membrane and podosomes,the synaptic vesicles and axons of neurons,and even the nuclei of the rat smooth muscle cells.These results indicate that the intracellular localizations of PAT1 are regulated by diverse mechanisms,and the functions of PAT1 in different cell types are related to its specific localizations.Using the human embryonic kidney 293(HEK293)cells as a model,we have discovered before that FLCN inhibited the lysosomal localization of PAT1.When FLCN was suppressed,PAT1 became accumulated on the lysosome.This tends to accelerate the efflux of the luminal amino acids into the cytosol.Because the lysosomal amino acids are a critical signal to stimulate mTORC1,which is a key regulator of cellullar nutrient homeostasis,the mTORC1 activity was decreased in response to the reduced lysosomal amino acids.In eukaryotic cells,the membrane proteins are transported mainly through the vesicular trafficking pathways.We therefore suspect that PAT1 is probably a cargo transported by FLCN.In order to explore the mechanisms of how FLCN regulates PAT1 localization and uncover the potential roles of FLCN duing vesicular trafficking,in this study,I took HEK293 cells as a model,and applied various cell biology and biochemical techniques to investigate the PAT1 transport pathways and the underling mechanisms regulated by FLCN.The following results were obtained.1.PAT1 was detected on various membrane organelles.The immunofluorescent staining data revealed that PAT1 was co-localized with the markers of diverse organelles,including LAMP1(lysosome),Rab5A(eraly endosome),EEA1(early endosome),TGN38(trans-Golgi network),Chmp5(multivesicular body),Rab7(late endosome),M6PR(late and recycling endosomes),and Rab11A(recycling endosome).PAT1 was mainly localized on the lysosomes,but it was also localized on other endosomes.We think PAT1 can be transported through both endocytic and recycling pathways.2.The internalization assay reveraled that PAT1 could move from the plasma membrane into the cell,and eventually target on the lysosome.This indicates PAT1 could be transported to the lysosome through the indirect transport pathway.3.FLCN inhibits the localization of PAT1 on the lysosome and promotes its targeting on the plasma membrane,thereby positively regulating mTORC1.Suppression of FLCN by RNAi or FLCN deletion increased PAT1 on the lysosome and decreased it on the plasma membrane.As a result,the mTORC1 activity was decreased.When amino acids were missing,PAT1 became accumulated on the lysosome.This result could be reversed by FLCN overexpression.As a result,the mTORC1 activity was maintained even under the condition of amino acids deficiency.4.FLCN interacts with Rab11 A,which is a key regulator of the recycling transport pathway.The co-immunoprecipitation(co-IP)assay revealed that FLCN weakly bond with Rab5 A,and it preferentially bond with Rab7 A and Rab11 A.In addition,we found FLCN bond with Rab11 A through its DENN domain.5.Rab11 A inhibits the localization of PAT1 on the lysosome and promotes its localization on the plasma membrane.Suppression of Rab11 A by RNAi,or overexpression of a dominant negative mutant Rab11A(S25N),caused the increase of PAT1 on the lysosome and decrease of PAT1 on the plasma membrane,resulting in mTORC1 down-regulation.Conversely,overexpressing the active Rab11A(Q70L)inhibited the lysosomal localization of PAT1 and reversed the inhibitory effect of PAT1 on mTORC1.6.The in vitro GEF activity assay revealed that FLCN did not modulate the Rab11 A activity directly.This result is consistent with a previous report.Instead,we found FLCN promoted the binding of PAT1 with Rab11 A.Without FLCN,the interaction between PAT1 with Rab5 A or Rab7 A was not affected,but the PAT1-Rab11 A interaction was clearly decreased.Based on these results,I attempted to make two conclusions.First,FLCN is a new effector of Rab11A;FLCN interacts with Rab11 A through its C-terminal DENN domain.Second,FLCN promotes the binding of PAT1 to Rab11 A,thereby accelerating the recycling of PAT1.This study identifies the relationship between FLCN and Rab11 A and a new role of PAT1 during recycling transport.These findings can provide new clues to understand the BHD symptoms.