Are there too many or too few SNAREs in proteoliposomes?

Phospholipid vesicles referred to as li-posomesallowreconstitutionofproteinfunction in the absence of other pro-teins.Liposomeshavebeenwidelyusedin exploring viral fusion proteins and,more recently, proteins that mediateintracellular fusion. An evolutionarilyconserved family of proteins calledSNAREs (soluble N-ethylmaleimide-sensitive factor attachment proteinreceptors) is critically important inmembrane and protein trafﬁcking ineukaryotic cells including docking andfusion of synaptic vesicle carryingv-SNARE VAMP with plasma mem-brane carrying t-SNAREs SNAP-25and syntaxin (1). The speciﬁc role ofSNAREs in intracellular fusion hasbeen explored by many groups and inmany experimental systems. Howeverthe important question whether pairingof v- and t-SNAREs directly mediatesfusion or acts upstream of it remainsunder debate (1–5). One of the mostimportant arguments that SNAREsmight act as fusogens came fromreports on fusion between SNARE-bearing proteoliposomes (6). Two arti-cles in this and previous issues providenew insights into fusogenic activity ofreconstituted SNAREs (7,8). Do SNAREs represent minimal fu-sion machinery? According to one ofthe conventional deﬁnitions, a fusionprotein is a protein that is critical forfusioninabiologicallyrelevantcontextand is sufﬁcient to promote merger ofboth outer and inner leaﬂets of themembranes in model systems. SNAREssatisfy this deﬁnition, as evidenced byfusion reported for SNAREs reconsti-tuted in lipid bilayers (6,9–11) andexpressed at cell surface (12). A stron-gerdeﬁnitionwouldadditionallyrequirethis reconstituted fusion to be com-parable with the biologically relevantone in rates of both lipid and contentmixing under conditions that mimicphysiological conditions; in particular,in dependency onthe lipid andmediumcompositions; and in the numbers ofthe proteins required for fusion. Thesurface density of fusion proteins de-terminesnotonlytheratesoffusionbutalso the observed fusion phenotypes,with fewer proteins required for themerger of outer leaﬂets (hemifusion)than for complete fusion (10,13). In many earlier articles, SNARE–mediated liposome fusion was studiedatveryhighsurfacedensitiesofSNAREswith protein/lipid ratios of v-SNAREVAMP as high as 1:20–50 (6,10) cor-responding to 300–750 proteins pervesicle. This exceeds v-SNARE densi-ties characteristic for synaptic vesicles,widely quoted as 30–100 v-SNAREsper vesicle (3) but might be as low as10–15v-SNAREs/vesicle(protein/lipid;1:1000) as estimated in Dennisonet al. (8). Two new articles explore thefusogenic activity of proteoliposomeswith SNARE (synaptobrevin) surfacedensityintherangefrom1:950to1:120.Both groups formed proteoliposomesbyadirectreconstitutionmethod,wheredetergent-solubilized SNAREs wereadded to preformed liposomes ratherthan by a comicellization techniquebased on mixing of proteins and lipidsindetergent.Whereasmostoftheearlierwork on SNARE proteoliposomes wasdone on comicellization proteolipo-somes (6,11) but see Lu et al. (10),proteoliposomes prepared by direct re-constitution are much more homoge-neousinsizeandproteincontent(7)andthus are better suited for analysis of theeffects of the surface density of recon-stituted proteins. In contrast to studiesusing proteoliposomes with high sur-face densities of SNAREs (6,10), bothChen et al. and Dennison et al. reportthat at lower protein/lipid ratios of upto 1:120–250, fusion is very slow andinefﬁcient (7,8), and does not exceed4% even after 75 min (8). At theseproteindensities,fusionisnotobservedeven when efﬁcient docking of proteo-liposomes is induced by subfusogenicconcentrations of polyethylenglycol(PEG) (8), indicating that in artiﬁcialsystems, a high density of SNAREs isneeded not only to increase dockingprobability but for the actual fusionreaction as well. Interestingly, for thesame average protein density, proteoli-posomes formed by comicellization aremorefusogenic.Thismightbeexplainedby the heterogeneity of these liposomescontaining a fraction of smaller andapparently more fusogenic vesicles thatare enriched in SNAREs (7). At high enough density, SNAREsmediate robust lipid mixing betweenproteoliposomes (7,10), and, even atlower densities, reconstituted SNAREspromote fusion driven by PEG (8). Inbothcases,SNARE-promotedfusionbe-tween liposomes proceeds by the samefusion-through-hemifusion pathway asdiverse biological fusion reactions in-cluding intracellular fusion (13). Basedon the analysis of the effect ofSNARE proteins on the kinetics ofPEG-induced fusion, Dennison et al.(8) conclude that SNAREs promoteformation of a local hemifusion con-nection, referred to as a stalk interme-diate (13). Finding that similar to manyother fusion reactions (13), includingSNARE-dependent fusion betweenyeast vacuoles (5), SNARE-dependentliposome fusion observed at high pro-tein densities is inhibited by lysophos-phatidylcholine (7) further strengthensthis conclusion.Although work on SNARE proteoli-posomes, including the new studies,has already brought important insightsinto the mechanisms of the interactions