Vacuolar trafficking and vesicle fusion machineries at the Arabidopsis trans-Golgi network

Endomembrane trafficking is required for maintaining diverse cellular processes. Disruption of endomembrane trafficking affects cell viability, stress responses, development and other processes. The Arabidopsis trans-Golgi network (TGN) in the endomembrane system is an important organelle where several sorting events take place. The SYP41 complex, a protein complex at the Arabidopsis TGN, is involved in vacuolar trafficking. SYP41 interacts with AtVPS45, SYP61 and VTI12, which are proteins that are involved in vesicle transport to the vacuole. A previously uncharacterized protein was identified as a SYP41-interacting protein by co-immunoprecipitation, and was named TNO1 (TGN-localized SYP41-interacting protein). TNO1 localizes to the TGN and is involved in salt tolerance and vacuolar trafficking. TNO1 is important for the localization of SYP61, suggesting that TNO1 may assist recruitment of SYP61 to the SYP41 complex. Application of the trafficking inhibitor Brefeldin A indicated that TNO1 may be also involved in membrane fusion and in maintaining TGN structure. These results suggest that membrane fusion and stability of the TGN are important for salt tolerance and vacuolar trafficking in Arabidopsis.;Vesicle fusion is an essential process for maintaining the structure and function of the endomembrane system. The SYP41 complex consists of an SM protein, SNAREs, and TNO1. A role for the SYP41 complex in membrane fusion has been shown previously using in vitro fusion assays. In Arabidopsis, there are three SYP4 and four VTI1 family proteins. Here we used a proteoliposome fusion assay to demonstrate that other proteins in the SYP4 family and two members of the VTI1 family (VTI11 and VTI12) are also able to drive vesicle fusion. The results indicate that all members of the SYP4 family can also function with VTI12 in membrane fusion, and that VTI11 can substitute for VTI12 in this reaction.;In addition, we have demonstrated that AtVPS45 interacts with SYP4 protein complexes via direct binding to SYP4 family proteins in vitro. Addition of AtVPS45 reduces the efficiency of membrane fusion between vesicles containing SYP4 and VTI1 family proteins, suggesting that strong binding of AtVPS45 to SYP4 family proteins may inhibit membrane fusion between these vesicles. These results demonstrate that functional redundancy of VTI11 with VTI12 and regulation of vesicle fusion by VPS45 are mediated by interaction with SYP4 family SNAREs.