The endomembrane system in plants, consisting of the endoplasmic reticulum, Golgi apparatus, trans-Golgi network (TGN), prevacuolar compartment (PVC), vacuole and endosomes, has important roles throughout development, in responses to stress conditions and in defense responses [1–3]. Transport between organelles of the endomembrane system is mediated by transport vesicles delivering appropriate proteins, lipids and polysaccharides. The correct trafficking of vesicles requires a number of proteins that function in processes from vesicle budding to vesicle fusion [4–6].
Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins have a central role in vesicle trafficking in the recognition and fusion between vesicle and target membranes [7, 8]. SNAREs have a coiled-coil domain which interacts with other SNAREs and typically have a C-terminal integral membrane domain for anchoring into the membrane. There are two functional types of SNAREs; v-SNAREs are inserted into the vesicular membrane, while t-SNAREs are located on the target membrane. SNAREs can be divided into 4 classes, Qa, Qb, Qc and R, depending on the presence of a conserved Q or R residue in their coiled-coil domain. Q-type SNAREs are usually located on the target membrane and R-type SNAREs are typically on the vesicular membrane . In general, two or three t-SNARE polypeptides form a cis-SNARE complex on the target membrane, which interacts with a v-SNARE on an incoming vesicle via their coiled-coil domains, forming a four-helix trans-SNARE complex . This trans-SNARE complex allows the vesicle to fuse with its target membrane and release its cargo. The trans-SNARE complex is typically formed only by the correct combination of v-and t-SNAREs, which is one mechanism for providing fusion specificity . The requirements for vesicle fusion have been studied extensively, and it has been demonstrated that SNARE complex formation is sufficient to drive membrane fusion in an in vitro proteoliposome fusion assay, suggesting that the SNAREs themselves form the core of the membrane fusion machinery [12–17].
The Arabidopsis TGN contains the SYP4 family of closely-related SNAREs, which has 3 members, SYP41, SYP42 and SYP43 [18, 19]. SYP41 and SYP42 each interact with the t-SNARE SYP61 and v-SNARE VTI12 in addition to the SM (Sec1/Munc18) protein VPS45, a potential regulator of vesicle fusion [18, 20, 21]. There have been conflicting reports regarding the possible functional redundancy of the SYP4 family members. SYP41 and SYP42 were originally reported to be essential proteins in Arabidopsis, with syp41 and syp42 knockout single mutations being gametophyte lethal , suggesting that each SYP4 family member had a distinct function. However, a more recent study found that members of this family had redundant or overlapping functions, with no or only subtle phenotypes for the single mutants but lethality for the syp41syp42syp43 triple mutant. Vacuolar and secretory trafficking was defective in a syp42syp43 double mutant, suggesting a function of the SYP4 family in multiple trafficking pathways . The reason for the discrepancies between these two studies is not clear.
The VTI1 SNARE family consists of four genes (VTI11, VTI12, VTI13 and VTI14) in Arabidopsis but only VTI11 and VTI12 are expressed at significant levels [19, 24–26]. VTI11 and VTI12 have high amino acid sequence identity (60%), but they function in different trafficking pathways; VTI11 is involved in trafficking to the lytic vacuole, while VTI12 is involved in trafficking of storage proteins . Mutant phenotypes of VTI11 and VTI12 are also distinct. A vti11 mutant shows defects in shoot gravitropism, whereas a vti12 mutant is defective in the autophagy pathway and a vti11/vti12 double mutant is lethal [26, 28]. Although VTI11 and VTI12 have different functions in vacuolar trafficking, overexpressed VTI12 or a mutant of VTI12 that changes its specificity can substitute for VTI11 in the vti11 mutant , and VTI11 is also able to interact with SYP41 and SYP42 when VTI12 is not available . These genetic studies suggest that VTI11 may be able to functionally substitute for VTI12 in the SYP4 SNARE complex to drive vesicle fusion, although this has not been addressed directly.
Previously, either SYP41 or SYP61, together with VTI12 and the multifunctional SNARE YKT61/62, were found to be sufficient to drive lipid mixing of liposomes in vitro. In yeast and mammalian cells, Ykt6 is involved in vacuolar trafficking and recycling from endosomes to the TGN by interacting with Vti1p [30–33]. In Arabidopsis, there are two proteins related to yeast Ykt6, and both of them are able to drive membrane fusion with SYP41 and VTI12 in vitro, suggesting that they may be functionally redundant .The requirement for only three SNARE proteins in the fusion reaction suggests that either a three-helix bundle may be formed at the Arabidopsis TGN or, more likely, two molecules of one component may be required to make a four-helix bundle.
Here, we use in vitro liposome lipid mixing assays to address two questions relating to TGN SNARE redundancy: can SYP42 and/or SYP43 substitute for SYP41 in the in vitro lipid mixing assays, thus providing further evidence to distinguish between overlapping vs. distinct functions of these SNAREs?; and can VTI11 also drive fusion in combination with SYP4 SNAREs and YKT61/62, providing biochemical evidence that VTI11 may be able to functionally substitute for VTI12 in the SYP4 SNARE complex when VTI12 is absent, thus supporting the genetic studies ? We show that SYP42 or SYP43 reconstituted into vesicles are also able to drive lipid mixing with VTI12-containing vesicles, supporting the idea of functional redundancy between these SNAREs. In addition, VTI11-containing vesicles are able to fuse with vesicles containing a SYP4 family member, indicating that functional overlap between VTI11 and VTI12 is mediated by interaction and fusion activity with SYP4 family proteins.