The Role Of Rab11A Effector FIP5 In The FLCN-Rab11A Interaction

The BHD syndrome is a genetic disorder in animals.It is generally accepted that mutatons of FLCN is a major cause of the diease,but the precise relationships between FLCN and BHD are not clear yet.There is evidence that FLCN regulates the visicle trafficking processes via interating withseveral GTPases.Our recentstudy revealed that FLCN is an interacting protein of Rab11 A,a key regulator of recycling transport.FLCN binds to Rab11 A via its DENN domain,and inhibits the transport of the amino acid transporter PAT1 to the lysosome and promotes its localization on the plasma membrane.As a result,FLCN can maintain the lysosomal amino acid poll and positively regulates the mTORC1 signalling pathway.This finding revealed a new role of FLCN during the recycling transport and provides new clues to elucidate the FLCN functions.In the currentstudy,we took the human embryonic kidney cells HEK293 as a model and applied various biochemical approaches to investigated the FLCN-Rab11 A interaction.The following results were obtained.(1)In order to investigate if FLCN can directly bind to Rab11 A,we generated human FLCN proteins using the prokaryotic expression system,and generated human Rab11 A proteins by the eukaryotic expression system.We performed the in vitro pull down assay and found FLCN could not bind Rab11 A directly.This resultsuggests that FLCN may interact with Rab11 A through certainscaffold proteins.(2)FIP5 is a widley expressed Rab11 A effector.To answer if FIP5 is ascaffold protein to mediate the FLCN-Rab11 A interaction,we genraetdstable FIP5 knockdown cell lines.We found in these FIP5 knockdown cells,the mTORC1 activity was normal and the FLCN-Rab11 A interaction was not affected.To confirm the role of FIP5 during FLCN-Rab11 A interaction,we used the CRISPR/Cas9 system to generate FIP5 knockout cell clines.We found FLCN deletion did not affect the mTORC1 activity and the FLCN-Rab11 A interaction.Based on these results,we propose that FLCN interacts with Rab11 A via co-factors,and FIP5 does not play a major role during this process.