Lipid-anchored Synaptobrevin Provides Little or No Support for Exocytosis or Liposome Fusion

Abstract

SNARE proteins catalyze many forms of biological membrane fusion, including Ca2؉ -triggered exocytosis. Although fusion mediated by SNAREs generally involves proteins anchored to each fusing membrane by a transmembrane domain (TMD), the role of TMDs remains unclear, and previous studies diverge on whether SNAREs can drive fusion without a TMD. This issue is important because it relates to the question of the structure and composition of the initial fusion pore, as well as the question of whether SNAREs mediate fusion solely by creating close proximity between two membranes versus a more active role in transmitting force to the membrane to deform and reorganize lipid bilayer structure. To test the role of membrane attachment, we generated four variants of the synaptic v-SNARE synaptobrevin-2 (syb2) anchored to the membrane by lipid instead of protein. These constructs were tested for functional efficacy in three different systems as follows: Ca 2؉ -triggered dense core vesicle exocytosis, spontaneous synaptic vesicle exocytosis, and Ca 2؉ -synaptotagmin-enhanced SNARE-mediated liposome fusion. Lipid-anchoring motifs harboring one or two lipid acylation sites completely failed to support fusion in any of these assays. Only the lipid-anchoring motif from cysteine string protein-␣, which harbors many lipid acylation sites, provided support for fusion but at levels well below that achieved with wild type syb2. Thus, lipid-anchored syb2 provides little or no support for exocytosis, and anchoring syb2 to a membrane by a TMD greatly improves its function. The low activity seen with syb2-cysteine string protein-␣ may reflect a slower alternative mode of SNARE-mediated membrane fusion.Nearly all biological fusion mediated by SNARE proteins employs at least one t-SNARE and one v-SNARE anchored to the membrane by a transmembrane domain (TMD) 3 (1-3). Ca 2ϩ -triggered exocytosis of neurotransmitter and hormone from many cells requires the vesicle-SNARE synaptobrevin 2 (syb2) and plasma membrane-SNARE syntaxin, both of which have a TMD (4, 5). Mutations in the TMD alter flux through exocytotic fusion pores in a manner consistent with structural models of fusion pores formed by the TMDs of syntaxin (6, 7) and syb2 (8). Yeast t-and v-SNAREs anchored to membranes by geranylgeranyl moieties instead of a TMD support docking but not fusion (9). SNAREs anchored to membranes by lipid instead of a TMD support lipid mixing of liposomes only when the lipid moiety is long enough to span a lipid bilayer or multiple lipid moieties are present (10) or an accessory protein is present (11).Although early work supported a role of SNARE TMDs in membrane fusion, this issue has become controversial. Recent work on fusion of yeast vacuoles suggests that some SNAREs with lipid anchors can support lipid mixing, content mixing, or both, but other SNAREs cannot function when their TMD is replaced by a lipid anchor (12). Lipid-anchored forms of syntaxin and syb2 were reported to rescue neurotransmitter release at synapses as effectively as wild type ...

The Multifaceted Role of SNARE Proteins in Membrane Fusion

“…Moreover, the replacement of the SNARE transmembrane domain by a lipid anchor was shown to inhibit the fusion of vacuoles (Rohde et al, 2003) or of reconstituted proteoliposomes (Chang et al, 2016). In the latter study three different fusion constructs, namely Ca 2+ -triggered dense core vesicle exocytosis, spontaneous synaptic vesicle exocytosis, and Ca 2+ -synaptotagmin-enhanced SNARE-mediated liposome fusion, were tested.…”

Energetics, kinetics, and pathway of SNARE folding and assembly revealed by optical tweezers

“…The stepwise SNARE zippering continues to the LD and the transmembrane domain (TMD), contributing to additional SNARE zippering energy . Yet, the functions of LD and TMD zippering remain a matter of debate . Highly positively charged, LD interacts strongly with membranes and may constitute a membrane anchor with the TMD .…”

Molecular mechanism of fusion pore formation driven by the neuronal SNARE complex

“…A number of studies in which either SNARE transmembrane domains were replaced by lipid anchors (7)(8)(9) or a TMD was partially deleted (10), or in which the C terminus of the syb2 TMD was extended by polar residues (11), indicated that fusion was inhibited or even arrested, pointing to an important function of the TMD in fusion pore formation. The precise nanomechanical mechanism of fusion and the structure of the nascent pore remain unclear.…”

“…The precise nanomechanical mechanism of fusion and the structure of the nascent pore remain unclear. One model proposes a lipid-based fusion pore (12), while other studies have suggested a proteinaceous fusion pore with the TMDs of stx1 and syb2 lining the fusion pore, like an ion channel or gap junction pore (9). Recent experiments using varied-sized nanodiscs (NDs) (13) support the hypothesis that the fusion pore is a hybrid structure composed of both lipids and proteins (14); however, there is still no structural model of the fusion pore.…”