SNAREs fuse membranes in several steps. Trans‐SNARE complexes juxtapose membranes, induce hemifused stalk structures, and open the fusion pore. A recent penetration model of fusion proposed that SNAREs force the hydrophilic C‐termini of their transmembrane domains through the hydrophobic core of the membrane(s). In contrast, the indentation model suggests that the C‐termini open the pore by locally compressing and deforming the stalk. Here we test these models in the context of yeast vacuole fusion. Addition of small hydrophilic tags renders bilayer penetration by the C‐termini energetically unlikely. It preserves fusion activity, however, arguing against the penetration model. Addition of large protein tags to the C‐termini permits SNARE activation, trans‐SNARE pairing, and hemifusion but abolishes pore opening. Fusion proceeds if the tags are detached from the membrane by a hydrophilic spacer or if only one side of the trans‐SNARE complex carries a protein tag. Thus, both sides of a trans‐SNARE complex can drive pore opening. Our results are consistent with an indentation model in which multiple SNARE C‐termini cooperate in opening the fusion pore by locally deforming the inner leaflets.
The attachment of bulky protein domains at the luminal C‐termini of SNARE proteins arrests membrane fusion in a hemifused state. Since bulky tags interfere with clustering of several C‐termini in a small region, this suggests that several SNARE C‐termini collectively act on a small patch to deform the inner membrane leaflet and open a fusion pore, providing support for the indentation model.
SNARE‐mediated membrane fusion proceeds through a stalk‐like hemifusion intermediate.
Opening the fusion pore in this intermediate requires SNARE transmembrane domains.
The luminal C‐termini of these transmembrane domains must approach each other in a small membrane region to open the pore.
EMBO Reports (2016) 17: 1590–1608
- Received February 15, 2016.
- Revision received July 27, 2016.
- Accepted August 12, 2016.
- © 2016 The Authors