Synergistic actions of v-SNARE transmembrane domains and membrane-curvature modifying lipids in neurotransmitter release.

Madhurima Dhara,Mazen Makke,Maria Mantero Martinez,Dieter Bruns,Yvonne Schwarz,Ralf Mohrmann

doi:10.7554/elife.55152

Madhurima Dhara, Mazen Makke + Show 4 more

Open Access

https://doi.org/10.7554/elife.55152

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Journal: eLife	Publication Date: May 11, 2020
Citations: 19	License type: CC BY 4.0

Affiliation: Saarland University, Otto-von-Guericke University Magdeburg

Abstract

Vesicle fusion is mediated by assembly of SNARE proteins between opposing membranes. While previous work suggested an active role of SNARE transmembrane domains (TMDs) in promoting membrane merger (Dhara et al., 2016), the underlying mechanism remained elusive. Here, we show that naturally-occurring v-SNARE TMD variants differentially regulate fusion pore dynamics in mouse chromaffin cells, indicating TMD flexibility as a mechanistic determinant that facilitates transmitter release from differentially-sized vesicles. Membrane curvature-promoting phospholipids like lysophosphatidylcholine or oleic acid profoundly alter pore expansion and fully rescue the decelerated fusion kinetics of TMD-rigidifying VAMP2 mutants. Thus, v-SNARE TMDs and phospholipids cooperate in supporting membrane curvature at the fusion pore neck. Oppositely, slowing of pore kinetics by the SNARE-regulator complexin-2 withstands the curvature-driven speeding of fusion, indicating that pore evolution is tightly coupled to progressive SNARE complex formation. Collectively, TMD-mediated support of membrane curvature and SNARE force-generated membrane bending promote fusion pore formation and expansion.

Highlights

Membrane fusion is the key event in many cell biological processes like exocytosis, intracellular cargo trafficking and even fertilization
We show for the first time that naturally occurring transmembrane domains (TMDs) variants of different v-sensitive-factor attachment receptor proteins (SNAREs) isoforms have a decisive influence on the rate of fusion pore expansion
Using curvature-inducing agents together with the expression of defined VAMP2 mutants, we show that favorable curvature generation in either leaflet fully restores the functional deficits of rigidifying the v-SNARE TMD helix

Summary

Introduction

Membrane fusion is the key event in many cell biological processes like exocytosis, intracellular cargo trafficking and even fertilization. Sequence-specific back bone dynamics of isolated TMD model helices (probed by hydrogen/deuterium exchange) enhanced the fusogenicity of liposomes in in vitro assays (Stelzer et al, 2008; Quint et al, 2010), pointing to an active role of the v-SNARE TMD in the fusion mechanism Overall, these results showed that the function of v-SNARE TMDs clearly goes beyond simple membrane anchoring, but left the mechanisms of the underlying protein-lipid interplay unclear. Membrane-incorporated lipids like lysophosphatidylcholine (LPC) or oleic acid (OA) affected fusion induction and subsequent pore expansion in a membrane leaflet-specific fashion, correlating with their intrinsic curvature preference of the cytoplasmic and extracellular leaflet in the context of highly bent fusion intermediates These results indicate that membrane mechanics represent a rate-limiting energy barrier for Ca2+-triggered fusion of chromaffin granules, which proceeds via the formation of a membrane stalk intermediate into a lipidic fusion pore. Slowing of fusion pore expansion by a v-SNARE variant with a rigid TMD was fully rescued by either intracellular OA or extracellular LPC, indicating that v-SNARE transmembrane anchors and phospholipids cooperate in membrane remodeling by supporting membrane curvature at the fusion pore neck

Results

Discussion

Materials and methods

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