The Localization Of Bridge Lipid Transfer Protein SHIP164 And Its Mechanism Of Regulating Lipid Transport Between Golgi And Endosome

Background:Intracellular lipid transporters transfer lipids across opposing membranes at MCSs through two modes of action – shuttling or bridging.Bridge transporters feature an extended channel,most likely lined with hydrophobic residues that bind tens of lipids at once.These bridge lipid transporters can be recruited to MCSs via two adaptors resident on two opposing membranes of organelles,for instance,mammallian Vps13 proteins,show much higher lipid extracting activities than shuttle transporters,called bridge lipid transporters proteins.Studies have pointed out that SHIP164 interacts with the SNARE(Soluble NSF attachment receptor)-related protein Syntaxin-6,which may play a role in the cargo transport from early/recycling endosomes to the Golgi apparatus.A recent study showed that the Nterminus of SHIP164 contains a conserved Vps13/Chorein domain,simillar to VPS13 protein,and SHIP164 is also a bridge-like lipid transfer protein.Clinical studies have shown that SHIP164 is an important risk factor for Parkinson’s disease,and its mutation is directly related to this severe neurodegenerative disease.At present,the cellular localization,precise functional scenario and molecular mechanism of SHIP164 are still unclear,leading to the lack of understanding of the pathological mechanism of Parkinson’s disease caused by SHIP164 mutations.Objective:To explore cellular localization,precise functional scenario and molecular basis of SHIP164.Methods:1)Screen adaptors of SHIP164 through high-throughput live-cell imaging using the small GTPase/ATPase library or by mass spectrometry analysis.2)Determine the nature of interactions between SHIP164 and its adapter proteins.3)Observe SHIP164-mediated early endosome-Golgi interaction.4)Functionally,SHIP164 was knocked down,the morphological changes of endosomes and the Golgi apparatus or the condition of cell growth were observed.5)Analyze the lipid species bound by SHIP164 in cells through non-targeted lipidomics;detect the changes in endosomal membrane lipid species or ratio after SHIP164 deletion.Results:1)Identify the Golgi-associated ATPase RhoBTB3 and the early endosomal protein Vps26B(retromer complex component)as the adaptors’ protein of SHIP164.2)SHIP164,Vps26 B,and RhoBTB3 forms a tethering complex at Golgi-endosome contacts sites.3)In RPE1 cells,deletion of SHIP164 specifically reduced the size and number of early endosome budding structures(EE buds)marked by Vps26 B and actin,led to decreased number but increased size of EEs.4)SHIP164 bound to glycerophospholipids,and some sphingolipids.SHIP164 depletion specifically reduced the level of LPC/PC in the Rab14-labeled EE but the levels of Cer,PE,PG was not significantly affected.5)The loss of SHIP164 led to a significant decrease in the level of sphingomyelin(SM)in the plasma membrane(PM).6)There are frequent and dynamic contacts between Golgi vesicles and EEs.Conclusion:The bridge-like lipid transporter SHIP164 is a soluble protein in the cytosol.The Golgiassociated ATPase RhoBTB3 cooperates with the retromer component Vps26 B enriched at EE buds to recruit SHIP164 to Golgi-early endosomal membrane contacts.The SHIP164-RhoBTB3 interaction were mediated by eight conserved hydrophobic residues in SHIP164-RBID.The RhoBTB3 D532 E mutation significantly attenuated the recruitment of SHIP164.Functionally,the lipid transfer activity of SHIP164 was required for EE bud biogenesis,and inhibition of its activity significantly affected early/circulating endosome morphology,size,and number,as well as a reduction of SM trafficking to PM,and impaired cell growth.The RhoBTB3-positive Golgi vesicles underwent frequent and dynamic contacts(kiss and run)with EEs.Based on the above findings,we propose a novel model,in which SHIP164 promotes EE bud biogenesis by mediating lipid transfer from the Golgi vesicles to EE buds.