BACKGROUND
the endomembrane system in plants, consisting of the endoplasmic reticulum, golgi apparatus, trans-golgi network , prevacuolar compartment , vacuole and endosomes, has important roles throughout development, in responses to stress conditions and in defense responses  <cit> .
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  <cit> .
soluble n-ethylmaleimide-sensitive factor attachment protein receptor  proteins have a central role in vesicle trafficking in the recognition and fusion between vesicle and target membranes  <cit> .
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  <dig> 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  <cit> .
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  <cit> .
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  <cit> .
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  <cit> .
the arabidopsis tgn contains the syp <dig> family of closely-related snares, which has  <dig> members, syp <dig>  syp <dig> and syp <dig>  <cit> .
syp <dig> and syp <dig> each interact with the t-snare syp <dig> and v-snare vti <dig> in addition to the sm  protein vps <dig>  a potential regulator of vesicle fusion  <cit> .
there have been conflicting reports regarding the possible functional redundancy of the syp <dig> family members.
syp <dig> and syp <dig> were originally reported to be essential proteins in arabidopsis, with syp <dig> and syp <dig> knockout single mutations being gametophyte lethal  <cit> , suggesting that each syp <dig> 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 syp41syp42syp <dig> triple mutant.
vacuolar and secretory trafficking was defective in a syp42syp <dig> double mutant, suggesting a function of the syp <dig> family in multiple trafficking pathways  <cit> .
the reason for the discrepancies between these two studies is not clear.
the vti <dig> snare family consists of four genes  in arabidopsis but only vti <dig> and vti <dig> are expressed at significant levels  <cit> .
vti <dig> and vti <dig> have high amino acid sequence identity , but they function in different trafficking pathways; vti <dig> is involved in trafficking to the lytic vacuole, while vti <dig> is involved in trafficking of storage proteins  <cit> .
mutant phenotypes of vti <dig> and vti <dig> are also distinct.
a vti <dig> mutant shows defects in shoot gravitropism, whereas a vti <dig> mutant is defective in the autophagy pathway and a vti11/vti <dig> double mutant is lethal  <cit> .
although vti <dig> and vti <dig> have different functions in vacuolar trafficking, overexpressed vti <dig> or a mutant of vti <dig> that changes its specificity can substitute for vti <dig> in the vti <dig> mutant  <cit> , and vti <dig> is also able to interact with syp <dig> and syp <dig> when vti <dig> is not available  <cit> .
these genetic studies suggest that vti <dig> may be able to functionally substitute for vti <dig> in the syp <dig> snare complex to drive vesicle fusion, although this has not been addressed directly.
previously, either syp <dig> or syp <dig>  together with vti <dig> and the multifunctional snare ykt61/ <dig>  were found to be sufficient to drive lipid mixing of liposomes in vitro <cit> .
in yeast and mammalian cells, ykt <dig> is involved in vacuolar trafficking and recycling from endosomes to the tgn by interacting with vti1p  <cit> .
in arabidopsis, there are two proteins related to yeast ykt <dig>  and both of them are able to drive membrane fusion with syp <dig> and vti <dig> in vitro, suggesting that they may be functionally redundant  <cit> .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 syp <dig> and/or syp <dig> substitute for syp <dig> in the in vitro lipid mixing assays, thus providing further evidence to distinguish between overlapping vs. distinct functions of these snares?
; and can vti <dig> also drive fusion in combination with syp <dig> snares and ykt61/ <dig>  providing biochemical evidence that vti <dig> may be able to functionally substitute for vti <dig> in the syp <dig> snare complex when vti <dig> is absent, thus supporting the genetic studies  <cit> ?
we show that syp <dig> or syp <dig> 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 syp <dig> family member, indicating that functional overlap between vti <dig> and vti <dig> is mediated by interaction and fusion activity with syp <dig> family proteins.
RESULTS
expression of recombinant snares in escherichia coli
snares used in this study are shown schematically in figure 1a.
recombinant proteins were expressed in e. coli and purified via their n-terminal his <dig> tag using ni-nta resin.
purified proteins were separated by sds-page and stained with coomassie blue.
the purified proteins migrated at their expected mw with the exception of syp <dig> .
the predicted size of syp <dig> is approximately  <dig> kda, but recombinant syp <dig> migrated at around  <dig> kda, presumably due to structural hindrance during migration in the gel .
this size difference was also observed in a previous study of epitope-tagged syp <dig> in transgenic arabidopsis plants  <cit> .
syp <dig> and syp <dig> can also drive liposome fusion
previously, proteoliposome fusion assays using syp <dig>  vti <dig> and ykt <dig> demonstrated that these proteins were sufficient to drive full lipid mixing between liposomes, as a proxy for vesicle fusion  <cit> .
conflicting reports in the literature have suggested that the syp <dig> family snares have either distinct  <cit>  or redundant  <cit>  functions.
to further address this issue, we tested whether other syp <dig> family proteins can substitute for syp <dig> in driving lipid mixing in combination with vti <dig> and ykt <dig>  recombinant syp <dig>  syp <dig> or syp <dig> was reconstituted into phospholipid vesicles mimicking plant membrane composition  containing  <dig> mol%  <dig> -dioleoyl-sn-glycero-3-phosphocholine ,  <dig> mol% 1-palmitoyl-2-oleoyl-sn-glycero-3-)  and  <dig> mol% 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-ethanolamine  using a bio-beads/dialysis method  <cit> .
vti <dig> was reconstituted into separate vesicles  of the same lipid composition, except for the addition of  <dig>  mol% each of n-  and rhodamine-labeled fluorescent lipids  replacing an equal amount of dopc.
the efficiency of reconstitution of proteins into liposomes was analyzed by sds-page and staining with coomassie blue, and the reconstitution efficiency as determined by densitometry was around 50-60% for each protein .
the basis for the in vitro liposome lipid mixing assay is loss of fluorescence resonance energy transfer  due to dilution of the fluorescent lipids.
donor vesicles contain nbd and rhodamine-labeled fluorescent lipids, whereas acceptor vesicles are unlabeled.
nbd fluorescence in donor vesicles is quenched by rhodamine resulting in very low initial fluorescence.
when donor and acceptor vesicle membranes fuse, dilution of the fluorescent lipids leads to a decrease in nbd fluorescence quenching, causing an increased nbd fluorescence which can be monitored by fluorescence spectrophotometry.
acceptor vesicles containing syp <dig>  syp <dig> or syp <dig> and donor vesicles containing vti <dig> were mixed together with the soluble form of ykt <dig>  which is normally present as a lipid-modified form for membrane association in yeast and lacks a transmembrane domain .
this led to a rapid increase in nbd fluorescence due to dilution of the fluorescent lipids caused by membrane fusion .
these data indicate that syp <dig>  syp <dig> and syp <dig> can all drive lipid mixing of liposomes, and that syp <dig> and syp <dig> can function with vti <dig> and ykt <dig> in snare complexes in vitro, as previously suggested by their co-immunoprecipitation  <cit> .
no fusion was observed when ykt <dig> was omitted from each fusion reaction, suggesting ykt <dig> is also required for membrane fusion with syp <dig> and vti <dig> family members and that lipid mixing is dependent on the presence of the snares .
this result provides support for the idea of functional overlap between the syp <dig> snares in vesicle fusion.
vti <dig> can drive membrane lipid mixing in combination with syp <dig> family snares
in arabidopsis, four genes  comprise the vti <dig> family, with vti <dig> only expressed in arabidopsis suspension cells  <cit> .
vti <dig> and vti <dig> are v-snares localized to the pvc and tgn, respectively  <cit> .
vti <dig> and vti <dig> were previously shown to compensate for each other in each single knockout mutant, as a vti11vti <dig> double mutant is embryo lethal, suggesting they are at least partially functionally redundant  <cit> .
niihama et al.
<cit>  found that expression of vti <dig> was increased in a vti <dig> mutant, and a single amino acid substitution in vti <dig> which changes the cellular localization of vti <dig> to the pvc suppressed the vti <dig> mutant phenotype.
we hypothesize that vti <dig>  in addition to vti <dig>  is also able to mediate vesicle fusion in combination with members of the syp <dig> family and that the specificity of snare complex formation in vivo is due at least in part to the distinct localization of vti <dig> and vti <dig>
to test this hypothesis, vti <dig> was reconstituted into donor vesicles  containing fluorescent lipids, and proteoliposome lipid mixing assays were performed with acceptor vesicles containing each syp <dig> family member and donor vesicles containing vti <dig>  all in the presence of ykt <dig>  as shown in figure  <dig>  all syp <dig> family proteins were able to drive rapid lipid mixing with donor vesicles containing vti <dig>  suggesting that the syp <dig> family is also able to form functional snare complexes with vti <dig>  when the maximum percentage of lipid mixing  containing one syp <dig> family member with vesicles containing either vti <dig> or vti <dig> was compared, syp <dig> and syp <dig> showed similar maximum percentages.
however, syp <dig> showed more lipid mixing with vti <dig> than vti <dig> by 5-10%.
this difference was found to be statistically significant , suggesting that vti <dig> functions with syp <dig> better than vti <dig> in vitro , although whether this has biological significance in vivo is unknown.
we have shown that both vti <dig> and vti <dig> can drive lipid mixing in combination with syp <dig> family members .
to test whether the lipid mixing is driven by the specific interaction between individual syp <dig> and vti <dig> family members rather than a general requirement that any snare can fulfill, memb <dig>  a v-snare involved in endoplasmic reticulum to golgi anterograde trafficking and fusion at the arabidopsis cis-golgi, was synthesized and incorporated into donor vesicles  <cit> .
little lipid mixing was seen between vesicles containing memb <dig> and vesicles containing syp <dig> family snares in the presence of ykt <dig>  indicating that the reactions are specific for the tested snares .
as a further confirmation that the observed lipid mixing is specifically dependent on the vti <dig> family, soluble fragments of vti <dig> and vti <dig>  were synthesized lacking the transmembrane domain.
these soluble vti <dig> or vti <dig> fragments were mixed with ykt <dig> and acceptor vesicles containing one syp <dig> family member, followed by incubation for  <dig> min before adding reconstituted donor vesicles and measuring lipid mixing .
the soluble vti <dig> and vti <dig> proteins inhibited the lipid mixing of vesicles containing their full length version.
when cross inhibition  was examined, the same inhibition was observed, presumably due to occupation of the entire available syp <dig> family member by the soluble version of vti <dig> or vti <dig> proteins, thus preventing interaction with the full length version .
although syp <dig> preferentially works with vti <dig>  vti12s can still block vti <dig> fusion.
the disassembly of snare complexes requires nsf and snap  <cit> .
thus, the preformed syp <dig> snare complex containing vti12s and ykt <dig> cannot interact with vti <dig>  because the syp <dig> snare complex is locked.
these data indicate that the full-length proteins cannot displace the soluble fragments and confirm that lipid mixing is mediated by the interaction between snares on the acceptor and donor vesicles.
these results demonstrate that individual syp <dig> family members can interact with vti <dig> during lipid mixing in addition to their interaction with vti <dig>  inhibition by soluble vti <dig> and vti <dig> suggests that vti <dig> and vti <dig> may have similar abilities to drive membrane fusion.
this implies that vti <dig> can substitute for vti <dig> in catalyzing membrane fusion via interaction with the same syp <dig> family complexes to which vti <dig> binds in vivo and provides a molecular explanation for the ability of vti <dig> and vti <dig> to partially compensate for each other in the respective mutants.
discussion
the arabidopsis genome encodes three proteins that are members of the syp <dig> family and four members of the vti <dig> family.
previously, syp <dig>  vti <dig> and ykt61/ <dig> were found to be important components of the membrane fusion machinery at the arabidopsis tgn  <cit> .
here, we demonstrate that other members of the syp <dig> and vti <dig> families are also able to drive rapid lipid mixing in vitro, and provide further evidence that family members have functional overlap.
we use a lipid mixing assay based on dequenching of lipid fluorescence as a proxy for membrane fusion  <cit> .
we have previously shown that syp41-driven lipid mixing results from full fusion between the lipid bilayers, and not simply from hemi-fusion of the outer leaflets  <cit> .
the other combinations of snares used here show similar lipid mixing efficiencies, suggesting that they also result in full membrane fusion.
it has been shown that lipid mixing precedes and is more efficient than content mixing during in vitro liposome fusion  <cit> ; determination of whether the snares used in this work can also lead to content mixing will require a specific assay for this process  <cit> .
the degree of functional overlap between syp <dig> family members has been controversial in the literature.
one report indicated that each family member is essential for viability, suggesting that each syp <dig> protein has a unique and essential function  <cit> .
a second report, however, indicated substantial overlap in function, with no phenotype observed for single mutants and combinations of mutations required to observe trafficking defects  <cit> ; a triple syp41syp42syp <dig> mutation was lethal.
the reason for these discrepancies is unknown, but our in vitro results indicate that all three syp <dig> family members can drive lipid mixing with the same combinations of other snares, providing support for functional overlap between family members.
it has been shown that syp <dig> and syp <dig> interact with only one vti <dig> family protein, vti <dig>  in vivo, suggesting syp <dig> and syp <dig> are involved in membrane fusion with vesicles containing vti <dig> as a v-snare  <cit> .
this was confirmed by an in vitro lipid mixing assay using reconstituted syp <dig> or syp <dig> and vti <dig> .
however, syp <dig> showed an unexpected preference for vti <dig> in the in vitro lipid mixing assay .
it is possible that the difference between immunoprecipitation results using arabidopsis cell extract and lipid mixing assays using recombinant proteins could be caused by the distinct localization of vti <dig> and vti <dig> in vivo.
vti <dig> is hypothesized to function as a v-snare that targets vesicles containing the vacuolar sorting receptor vsr <dig> and ssvsd-containing vacuolar cargo from the tgn to the pvc, and vti <dig> is thought to be involved in trafficking to storage vacuoles via recycling of vesicle trafficking components from the pvc to the tgn, although functional overlap between the two proteins is evident  <cit> .
vti <dig> localizes to the pvc and interacts with syp <dig> and syp <dig>  while vti <dig> localizes to the tgn  <cit> .
the different localization of vti <dig> versus vti <dig> precludes the possibility of interaction of vti <dig> with syp <dig> family members at the tgn under normal conditions.
although vti <dig> and vti <dig> seem to be involved in distinct trafficking pathways, they can compensate for loss of the other protein in vti <dig> and vti <dig> single mutants  <cit> .
in addition, more vti <dig> is expressed in a vti <dig> mutant, presumably to make up for loss of vti <dig>  and a single amino acid substitution in vti <dig> can partially suppress the phenotype of vti <dig> mutants  <cit> .
these results suggest that vti <dig> family proteins can bind to non-cognate interaction partners when the other vti <dig> family member is not available.
this possibility was tested using in vitro lipid mixing assays, showing that vti <dig> was able to mediate vesicle fusion by interacting with syp <dig> family members.
this result indicates that compensation by vti <dig> in vti <dig> mutants in vivo is likely to be via the ability to form a functional snare complex with the vti <dig> binding partners.
the inverse may also be true, that vti <dig> may be able to interact with the normal vti <dig> binding partners under the appropriate circumstances.
in vitro fusion assays using recombinant syp <dig> and syp <dig> could provide further information about the redundant functions of vti <dig> and vti <dig>  our data support the hypothesis that the distinct subcellular localization of vti <dig> and vti <dig> in vivo is the primary determinant of fusion specificity of this family, rather than the innate biochemical properties of the proteins themselves.
CONCLUSIONS
in conclusion, we have demonstrated that each syp <dig> family member at the tgn, together with either vti <dig> or vti <dig>  can drive proteoliposome lipid mixing in vitro.
these data provide support for functional overlap between syp <dig> family members  <cit>  rather than distinct essential functions  <cit> , an issue which has been controversial in the literature.
they also indicate that vti <dig> can substitute for vti <dig> in driving lipid mixing with syp <dig>  suggesting that they have similar biochemical properties and that their specificity in different trafficking pathways in vivo is mainly due to their different subcellular localizations.

 the endomembrane system in plants, consisting of the endoplasmic reticulum, golgi apparatus, trans-golgi network, prevacuolar compartment, vacuole and endosomes, has important roles throughout development, in responses to stress conditions and in defense responses. 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. soluble n-ethylmaleimide-sensitive factor attachment protein receptor proteins have a central role in vesicle trafficking in the recognition and fusion between vesicle and target membranes. 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 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. the arabidopsis tgn contains the syp family of closely-related snares, which has members, syp syp and syp. syp and syp each interact with the t-snare syp and v-snare vti in addition to the sm protein vps a potential regulator of vesicle fusion. there have been conflicting reports regarding the possible functional redundancy of the syp family members. syp and syp were originally reported to be essential proteins in arabidopsis, with syp and syp knockout single mutations being gametophyte lethal, suggesting that each syp 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 syp41syp42syp triple mutant. vacuolar and secretory trafficking was defective in a syp42syp double mutant, suggesting a function of the syp family in multiple trafficking pathways. the reason for the discrepancies between these two studies is not clear. the vti snare family consists of four genes in arabidopsis but only vti and vti are expressed at significant levels. vti and vti have high amino acid sequence identity, but they function in different trafficking pathways; vti is involved in trafficking to the lytic vacuole, while vti is involved in trafficking of storage proteins. mutant phenotypes of vti and vti are also distinct. a vti mutant shows defects in shoot gravitropism, whereas a vti mutant is defective in the autophagy pathway and a vti11/vti double mutant is lethal. although vti and vti have different functions in vacuolar trafficking, overexpressed vti or a mutant of vti that changes its specificity can substitute for vti in the vti mutant, and vti is also able to interact with syp and syp when vti is not available. these genetic studies suggest that vti may be able to functionally substitute for vti in the syp snare complex to drive vesicle fusion, although this has not been addressed directly. previously, either syp or syp together with vti and the multifunctional snare ykt61/ were found to be sufficient to drive lipid mixing of liposomes in vitro. in yeast and mammalian cells, ykt is involved in vacuolar trafficking and recycling from endosomes to the tgn by interacting with vti1p. in arabidopsis, there are two proteins related to yeast ykt and both of them are able to drive membrane fusion with syp and vti 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 syp and/or syp substitute for syp in the in vitro lipid mixing assays, thus providing further evidence to distinguish between overlapping vs. distinct functions of these snares?; and can vti also drive fusion in combination with syp snares and ykt61/ providing biochemical evidence that vti may be able to functionally substitute for vti in the syp snare complex when vti is absent, thus supporting the genetic studies? we show that syp or syp 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 syp family member, indicating that functional overlap between vti and vti is mediated by interaction and fusion activity with syp family proteins.  expression of recombinant snares in escherichia coli snares used in this study are shown schematically in figure 1a. recombinant proteins were expressed in e. coli and purified via their n-terminal his tag using ni-nta resin. purified proteins were separated by sds-page and stained with coomassie blue. the purified proteins migrated at their expected mw with the exception of syp. the predicted size of syp is approximately kda, but recombinant syp migrated at around kda, presumably due to structural hindrance during migration in the gel. this size difference was also observed in a previous study of epitope-tagged syp in transgenic arabidopsis plants. syp and syp can also drive liposome fusion previously, proteoliposome fusion assays using syp vti and ykt demonstrated that these proteins were sufficient to drive full lipid mixing between liposomes, as a proxy for vesicle fusion. conflicting reports in the literature have suggested that the syp family snares have either distinct or redundant functions. to further address this issue, we tested whether other syp family proteins can substitute for syp in driving lipid mixing in combination with vti and ykt recombinant syp syp or syp was reconstituted into phospholipid vesicles mimicking plant membrane composition containing mol% -dioleoyl-sn-glycero-3-phosphocholine, mol% 1-palmitoyl-2-oleoyl-sn-glycero-3-) and mol% 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-ethanolamine using a bio-beads/dialysis method. vti was reconstituted into separate vesicles of the same lipid composition, except for the addition of mol% each of n- and rhodamine-labeled fluorescent lipids replacing an equal amount of dopc. the efficiency of reconstitution of proteins into liposomes was analyzed by sds-page and staining with coomassie blue, and the reconstitution efficiency as determined by densitometry was around 50-60% for each protein. the basis for the in vitro liposome lipid mixing assay is loss of fluorescence resonance energy transfer due to dilution of the fluorescent lipids. donor vesicles contain nbd and rhodamine-labeled fluorescent lipids, whereas acceptor vesicles are unlabeled. nbd fluorescence in donor vesicles is quenched by rhodamine resulting in very low initial fluorescence. when donor and acceptor vesicle membranes fuse, dilution of the fluorescent lipids leads to a decrease in nbd fluorescence quenching, causing an increased nbd fluorescence which can be monitored by fluorescence spectrophotometry. acceptor vesicles containing syp syp or syp and donor vesicles containing vti were mixed together with the soluble form of ykt which is normally present as a lipid-modified form for membrane association in yeast and lacks a transmembrane domain. this led to a rapid increase in nbd fluorescence due to dilution of the fluorescent lipids caused by membrane fusion. these data indicate that syp syp and syp can all drive lipid mixing of liposomes, and that syp and syp can function with vti and ykt in snare complexes in vitro, as previously suggested by their co-immunoprecipitation. no fusion was observed when ykt was omitted from each fusion reaction, suggesting ykt is also required for membrane fusion with syp and vti family members and that lipid mixing is dependent on the presence of the snares. this result provides support for the idea of functional overlap between the syp snares in vesicle fusion. vti can drive membrane lipid mixing in combination with syp family snares in arabidopsis, four genes comprise the vti family, with vti only expressed in arabidopsis suspension cells. vti and vti are v-snares localized to the pvc and tgn, respectively. vti and vti were previously shown to compensate for each other in each single knockout mutant, as a vti11vti double mutant is embryo lethal, suggesting they are at least partially functionally redundant. niihama et al. found that expression of vti was increased in a vti mutant, and a single amino acid substitution in vti which changes the cellular localization of vti to the pvc suppressed the vti mutant phenotype. we hypothesize that vti in addition to vti is also able to mediate vesicle fusion in combination with members of the syp family and that the specificity of snare complex formation in vivo is due at least in part to the distinct localization of vti and vti to test this hypothesis, vti was reconstituted into donor vesicles containing fluorescent lipids, and proteoliposome lipid mixing assays were performed with acceptor vesicles containing each syp family member and donor vesicles containing vti all in the presence of ykt as shown in figure  all syp family proteins were able to drive rapid lipid mixing with donor vesicles containing vti suggesting that the syp family is also able to form functional snare complexes with vti when the maximum percentage of lipid mixing containing one syp family member with vesicles containing either vti or vti was compared, syp and syp showed similar maximum percentages. however, syp showed more lipid mixing with vti than vti by 5-10%. this difference was found to be statistically significant, suggesting that vti functions with syp better than vti in vitro, although whether this has biological significance in vivo is unknown. we have shown that both vti and vti can drive lipid mixing in combination with syp family members. to test whether the lipid mixing is driven by the specific interaction between individual syp and vti family members rather than a general requirement that any snare can fulfill, memb a v-snare involved in endoplasmic reticulum to golgi anterograde trafficking and fusion at the arabidopsis cis-golgi, was synthesized and incorporated into donor vesicles. little lipid mixing was seen between vesicles containing memb and vesicles containing syp family snares in the presence of ykt indicating that the reactions are specific for the tested snares. as a further confirmation that the observed lipid mixing is specifically dependent on the vti family, soluble fragments of vti and vti were synthesized lacking the transmembrane domain. these soluble vti or vti fragments were mixed with ykt and acceptor vesicles containing one syp family member, followed by incubation for min before adding reconstituted donor vesicles and measuring lipid mixing. the soluble vti and vti proteins inhibited the lipid mixing of vesicles containing their full length version. when cross inhibition was examined, the same inhibition was observed, presumably due to occupation of the entire available syp family member by the soluble version of vti or vti proteins, thus preventing interaction with the full length version. although syp preferentially works with vti vti12s can still block vti fusion. the disassembly of snare complexes requires nsf and snap. thus, the preformed syp snare complex containing vti12s and ykt cannot interact with vti because the syp snare complex is locked. these data indicate that the full-length proteins cannot displace the soluble fragments and confirm that lipid mixing is mediated by the interaction between snares on the acceptor and donor vesicles. these results demonstrate that individual syp family members can interact with vti during lipid mixing in addition to their interaction with vti inhibition by soluble vti and vti suggests that vti and vti may have similar abilities to drive membrane fusion. this implies that vti can substitute for vti in catalyzing membrane fusion via interaction with the same syp family complexes to which vti binds in vivo and provides a molecular explanation for the ability of vti and vti to partially compensate for each other in the respective mutants. discussion the arabidopsis genome encodes three proteins that are members of the syp family and four members of the vti family. previously, syp vti and ykt61/ were found to be important components of the membrane fusion machinery at the arabidopsis tgn. here, we demonstrate that other members of the syp and vti families are also able to drive rapid lipid mixing in vitro, and provide further evidence that family members have functional overlap. we use a lipid mixing assay based on dequenching of lipid fluorescence as a proxy for membrane fusion. we have previously shown that syp41-driven lipid mixing results from full fusion between the lipid bilayers, and not simply from hemi-fusion of the outer leaflets. the other combinations of snares used here show similar lipid mixing efficiencies, suggesting that they also result in full membrane fusion. it has been shown that lipid mixing precedes and is more efficient than content mixing during in vitro liposome fusion; determination of whether the snares used in this work can also lead to content mixing will require a specific assay for this process. the degree of functional overlap between syp family members has been controversial in the literature. one report indicated that each family member is essential for viability, suggesting that each syp protein has a unique and essential function. a second report, however, indicated substantial overlap in function, with no phenotype observed for single mutants and combinations of mutations required to observe trafficking defects; a triple syp41syp42syp mutation was lethal. the reason for these discrepancies is unknown, but our in vitro results indicate that all three syp family members can drive lipid mixing with the same combinations of other snares, providing support for functional overlap between family members. it has been shown that syp and syp interact with only one vti family protein, vti in vivo, suggesting syp and syp are involved in membrane fusion with vesicles containing vti as a v-snare. this was confirmed by an in vitro lipid mixing assay using reconstituted syp or syp and vti. however, syp showed an unexpected preference for vti in the in vitro lipid mixing assay. it is possible that the difference between immunoprecipitation results using arabidopsis cell extract and lipid mixing assays using recombinant proteins could be caused by the distinct localization of vti and vti in vivo. vti is hypothesized to function as a v-snare that targets vesicles containing the vacuolar sorting receptor vsr and ssvsd-containing vacuolar cargo from the tgn to the pvc, and vti is thought to be involved in trafficking to storage vacuoles via recycling of vesicle trafficking components from the pvc to the tgn, although functional overlap between the two proteins is evident. vti localizes to the pvc and interacts with syp and syp while vti localizes to the tgn. the different localization of vti versus vti precludes the possibility of interaction of vti with syp family members at the tgn under normal conditions. although vti and vti seem to be involved in distinct trafficking pathways, they can compensate for loss of the other protein in vti and vti single mutants. in addition, more vti is expressed in a vti mutant, presumably to make up for loss of vti and a single amino acid substitution in vti can partially suppress the phenotype of vti mutants. these results suggest that vti family proteins can bind to non-cognate interaction partners when the other vti family member is not available. this possibility was tested using in vitro lipid mixing assays, showing that vti was able to mediate vesicle fusion by interacting with syp family members. this result indicates that compensation by vti in vti mutants in vivo is likely to be via the ability to form a functional snare complex with the vti binding partners. the inverse may also be true, that vti may be able to interact with the normal vti binding partners under the appropriate circumstances. in vitro fusion assays using recombinant syp and syp could provide further information about the redundant functions of vti and vti our data support the hypothesis that the distinct subcellular localization of vti and vti in vivo is the primary determinant of fusion specificity of this family, rather than the innate biochemical properties of the proteins themselves.  in conclusion, we have demonstrated that each syp family member at the tgn, together with either vti or vti can drive proteoliposome lipid mixing in vitro. these data provide support for functional overlap between syp family members rather than distinct essential functions, an issue which has been controversial in the literature. they also indicate that vti can substitute for vti in driving lipid mixing with syp suggesting that they have similar biochemical properties and that their specificity in different trafficking pathways in vivo is mainly due to their different subcellular localizations. 
the arabidopsis thaliana trans-golgi network resident snares syp and vti along with ykt61/ have been shown to function in vesicle fusion in vitro, consistent with immunoprecipitation results showing their interaction in arabidopsis cell extracts. our data provide biochemical evidence for functional overlap in membrane fusion between members of the syp or vti snare groups, supporting previous genetic data suggesting redundancy. fusion is mediated by t-snare fusion proteins on the target membrane and v-snares on the vesicle membrane; v-and t-snares interact with each other, driving vesicle fusion with the target membrane. here we used a proteoliposome fusion assay to demonstrate that syp and syp can substitute for syp in driving lipid mixing, providing support for functional overlap between family members. conflicting published results have indicated that syp family members are either functionally redundant or have distinct and essential functions; the reason for this discrepancy is unclear. we show that vti can substitute for vti in in vitro lipid mixing reactions, providing molecular support for the genetic evidence for partial functional redundancy in vivo. vesicle fusion is an essential process for maintaining the structure and function of the endomembrane system.
 vesicle fusion is an essential process for maintaining the structure and function of the endomembrane system. fusion is mediated by t-snare fusion proteins on the target membrane and v-snares on the vesicle membrane; v-and t-snares interact with each other, driving vesicle fusion with the target membrane. the arabidopsis thaliana trans-golgi network resident snares syp and vti along with ykt61/ have been shown to function in vesicle fusion in vitro, consistent with immunoprecipitation results showing their interaction in arabidopsis cell extracts. conflicting published results have indicated that syp family members are either functionally redundant or have distinct and essential functions; the reason for this discrepancy is unclear.  here we used a proteoliposome fusion assay to demonstrate that syp and syp can substitute for syp in driving lipid mixing, providing support for functional overlap between family members. previous reports have also suggested that vti and vti snares show partial overlap in function, despite having mostly distinct localizations and binding partners. we show that vti can substitute for vti in in vitro lipid mixing reactions, providing molecular support for the genetic evidence for partial functional redundancy in vivo.  our data provide biochemical evidence for functional overlap in membrane fusion between members of the syp or vti snare groups, supporting previous genetic data suggesting redundancy. membrane fusionsnaretrans-golgi networkvesicle trafficking 