v-SNARE transmembrane domains function as catalysts for vesicle fusion

Abstract

Vesicle fusion is mediated by an assembly of SNARE proteins between opposing membranes, but it is unknown whether transmembrane domains (TMDs) of SNARE proteins serve mechanistic functions that go beyond passive anchoring of the force-generating SNAREpin to the fusing membranes. Here, we show that conformational flexibility of synaptobrevin-2 TMD is essential for efficient Ca ²⁺-triggered exocytosis and actively promotes membrane fusion as well as fusion pore expansion. Specifically, the introduction of helix-stabilizing leucine residues within the TMD region spanning the vesicle’s outer leaflet strongly impairs exocytosis and decelerates fusion pore dilation. In contrast, increasing the number of helix-destabilizing, ß-branched valine or isoleucine residues within the TMD restores normal secretion but accelerates fusion pore expansion beyond the rate found for the wildtype protein. These observations provide evidence that the synaptobrevin-2 TMD catalyzes the fusion process by its structural flexibility, actively setting the pace of fusion pore expansion.

DOI: http://dx.doi.org/10.7554/eLife.17571.001

eLife digest

Neurons signal to other cells by releasing chemicals known as neurotransmitters. The neurotransmitters are stored in the neuron in small membrane-bound compartments called vesicles. When a neuron receives an electrical impulse, this ultimately triggers the vesicles to fuse with the cell membrane and release their contents into the gap between the neurons. This process is known as exocytosis. Other cells called neuroendocrine cells, which can receive signals from neurons, also undergo exocytosis to release chemicals into the bloodstream.

A group of membrane-bound proteins called SNAREs help a vesicle to fuse with the cell membrane. SNARE proteins are embedded in both the vesicle and cell membrane, and force them into close proximity. When the two membranes make contact, a small channel called the fusion pore forms and expands to release the vesicle’s contents out of the cell.

Synaptobrevin-2 is a SNARE protein found in the vesicle membrane. The part of the protein that sits in the membrane is called the transmembrane domain; however, it is not clear whether this domain plays any role in membrane fusion.

The transmembrane domain of synaptobrevin-2 is rich in certain amino acids that are thought to make it flexible, thereby allowing it to bend and tilt in the membrane. Dhara, Yarzagaray et al. altered these amino acids in such a way that made this domain either more or less flexible than in the normal protein. The results show that in both neurons and a type of neuroendocrine cell called chromaffin cells, exocytosis is significantly reduced and the fusion pores open more slowly when the transmembrane domain is less flexible. By contrast, exocytosis occurs normally when the transmembrane domain is more flexible; however, the fusion pore expands more rapidly than normal.

These results suggest that the flexibility of the transmembrane domain of synaptobrevin-2 promotes membrane fusion and sets the pace at which the fusion pore expands. It is likely that the transmembrane domain disturbs the surrounding membrane in a way that enables these events to happen. Further work is needed to address whether this is the case.

DOI: http://dx.doi.org/10.7554/eLife.17571.002