The Function And Mechanism Of Tcb3 And SYT1 In The Interaction Between Endoplasmic Reticulum And Cell Membrane

The endoplasmic reticulum(ER)forms a dynamic inner membrane network structure and is distributed throughout the cell.It communicates with plasma membrane(PM)and other membranous organelles through membrane contact sites(MCS).ER-PM MCS is essential for calcium homeostasis,lipid metabolism and signal transductions,which are mediated by a variety of functional proteins.Lipid transfer protein(LTP)senses and transports lipids between membranes and plays a central role in ER-PM MCS.Most LTPs span and tether two opposing membranes when performing lipid exchange,highlighting the important role of LTPs in maintaining the MCS architecture.However,there are often multiple regulates play the same function at MCS,which increases the difficulty of identifying the lipid transfer function of LTPs.Extended-synaptotagmins(E-Syts)are proposed as a type of membrane tethering and lipid transfer protein in mammalian cells,which play important roles in maintaining ER-PM MCS.It has been reported that the tricalbins(Tcbs)and synaptotagmins(SYTs),the homologs of E-Syts in yeast and plants,are also located in ER-PM MCS.However,the function and mechanisms of these proteins in MCS are still unclear.In this study,we developed an in vitro-reconstituted lipid transfer system and systematically studied the function and mechanisms of yeast Tcb3 and Arabidopsis SYT1.We expressed and purified Tcb3 protein and their mutants.We found that Tcb3 can associate to liposomes,and this association is regulated by Ca²⁺.In lipid transfer assays,Tcb3 can even transfer lipids between different membrane structures without Ca²⁺.When Ca²⁺is added,the rate of lipid transfer is significantly enhanced,indicating that Tcb3 is a Ca²⁺regulated LTP.Tcb3 can modulate lipid transfers in Ca²⁺-independent and-dependent manners,which are both mediated through the synaptotagmin-like mitochondrial lipid-binding protein(SMP)domain.By liposome tethering assay,we found that Ca²⁺can significantly promote Tcb3-mediated liposome tethering,suggesting that Ca²⁺improves the efficiency of lipid transfer by strengthening the connections between the opposed membranes.Further mechanism studies showed that Tcb3 transfers lipids through the interaction between the SMP domain and the negatively charged phosphatidylserine(PS).There are four C2 domains in Tcb3,while only C2B domain does not bind to Ca²⁺.We found that the Ca²⁺-stimulated lipid transfers reduced by the mutations of the Ca²⁺binding sites on the C2C or C2D domain,indicating that the stimulatory activity of Ca²⁺requires intact Ca²⁺-binding sites on both the C2C and C2D domains of Tcb3.In addition to Tcb3,we also found that Tcb2 can also carry out lipid transfer function,indicating that regulation of lipid transfer is a conserved function of tricalbins.It is difficult to obtain Arabidopsis SYT1 protein from E.coli.We used insect cell sf9expression system to obtain active SYT1 protein.In the reconstitution system,we found that SYT1 binds to liposomes and can also mediate lipid transfer.Unlike Tcb3,SYT1mainly transfers lipids in the Ca²⁺-independent manner.The regulation of lipid transfer by Ca²⁺is weak.Through lipid transfer assays,we found that SYT1-mediated lipid transfer is also carried out through the SMP domain.Unexpectedly,the C2 domain of SYT1significantly stimulates lipid transfer even in the absence of Ca²⁺.Our results indicate that SYT1-mediated lipid transfer is driven by both the C2 domains and SMP domain.The C2domains play important regulatory roles in both Ca²⁺-independent and Ca²⁺-dependent lipid transfer processes.In summary,our experimental results prove that yeast Tcb3 and plant SYT1 are both LTPs that can mediate lipid transfer in ER-PM MCS,indicating that lipid transfer is a conserved and important function of the E-Syt protein family.Although both of the lipid transfer is mediated through the SMP domain,Tcb3 and SYT1 take divergent regulatory mechanisms in the MCS of yeast and Arabidopsis.