A tethering complex dimer catalyzes trans-SNARE complex formation in intracellular membrane fusion

Neurons fire by releasing neurotransmitters via fusion of synaptic vesicles with the plasma membrane. Fusion can be evoked by an incoming signal from a preceding neuron or can occur spontaneously. Synaptic vesicle fusion requires the formation of trans complexes between SNAREs as well as Ca2+ ions. Wang et al. (2014. J. Cell Biol. http://dx.doi.org/jcb.201312109) now find that the Ca2+-binding protein Calmodulin promotes spontaneous release and SNARE complex formation via its interaction with the V0 sector of the V-ATPase.

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α-Synuclein kinetically regulates the nascent fusion pore dynamics

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2021742118 ◽

2021 ◽

Vol 118 (34) ◽

pp. e2021742118

Author(s):

Rohith K. Nellikka ◽

Bhavya R. Bhaskar ◽

Kinjal Sanghrajka ◽

Swapnali S. Patil ◽

Debasis Das

Keyword(s):

Complex Formation ◽

Membrane Fusion ◽

Snare Complex ◽

Fusion Pore ◽

Open Probability ◽

Intrinsically Disordered ◽

Pore Properties ◽

Snare Complex Formation ◽

Vesicular Secretion ◽

Pore Dynamics

α-Synuclein (α-synFL) is central to the pathogenesis of Parkinson’s disease (PD), in which its nonfunctional oligomers accumulate and result in abnormal neurotransmission. The normal physiological function of this intrinsically disordered protein is still unclear. Although several previous studies demonstrated α-synFL’s role in various membrane fusion steps, they produced conflicting outcomes regarding vesicular secretion. Here, we assess α-synFL’s role in directly regulating individual exocytotic release events. We studied the micromillisecond dynamics of single recombinant fusion pores, the crucial kinetic intermediate of membrane fusion that tightly regulates the vesicular secretion in different cell types. α-SynFL accessed v-SNARE within the trans-SNARE complex to form an inhibitory complex. This activity was dependent on negatively charged phospholipids and resulted in decreased open probability of individual pores. The number of trans-SNARE complexes influenced α-synFL’s inhibitory action. Regulatory factors that arrest SNARE complexes in different assembly states differentially modulate α-synFL’s ability to alter fusion pore dynamics. α-SynFL regulates pore properties in the presence of Munc13-1 and Munc18, which stimulate α-SNAP/NSF–resistant SNARE complex formation. In the presence of synaptotagmin1(syt1), α-synFL contributes with apo-syt1 to act as a membrane fusion clamp, whereas Ca2+•syt1 triggered α-synFL–resistant SNARE complex formation that rendered α-synFL inactive in modulating pore properties. This study reveals a key role of α-synFL in controlling vesicular secretion.

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Phosphatidylinositol 3,5-Bisphosphate Regulates Yeast Vacuole Fusion at the Transition between trans-SNARE Complex Formation and Hemifusion

10.1101/390062 ◽

2018 ◽

Author(s):

Gregory E. Miner ◽

Katherine D. Sullivan ◽

Annie Guo ◽

Brandon C. Jones ◽

Matthew L. Starr ◽

...

Keyword(s):

Complex Formation ◽

Membrane Fusion ◽

Molecular Switch ◽

Snare Complex ◽

Negative Regulator ◽

Cellular Functions ◽

Hyperosmotic Shock ◽

Yeast Vacuole ◽

Vacuole Fusion ◽

Snare Complex Formation

ABSTRACTPhosphoinositides (PIs) regulate myriad cellular functions including membrane fusion, as exemplified by the yeast vacuole, which uses various PIs at different stages of fusion. In light of this, the effect of phos-phatidylinositol 3,5-bisphosphate [PI(3,5)P2] on vacuole fusion remains unknown. PI(3,5)P2 is made by the PI3P 5-kinase Fab1/PIKfyve and has been characterized as a regulator of vacuole fission during hyperosmotic shock where it interacts with the TRP family Ca2+ channel Yvc1. Here we demonstrate that exogenously added dioctanoyl (C8) PI(3,5)P2 abolishes homotypic vacuole fusion. This effect was not linked to interactions with Yvc1, as fusion was equally affected using yvc1Δ vacuoles. Thus, the effects of C8-PI(3,5)P2 on fusion versus fission operate through distinct mechanisms. Further testing showed that C8-PI(3,5)P2 inhibited vacuole fusion after the formation of trans-SNARE pairs. Although SNARE complex formation was unaffected we found that C8-PI(3,5)P2 strongly inhibited hemifusion. Overproduction of endogenous PI(3,5)P2 by the fab1T2250A hyperactive kinase mutant also inhibited at the hemifusion stage, bolstering the model in which PI(3,5)P2 inhibits fusion when present elevated levels. Taken together, this work identifies a novel function for PI(3,5)P2 as a negative regulator of vacuolar fusion. Moreover, it suggests that this lipid acts as a molecular switch between fission and fusion.

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SNARE Complex Formation Is Triggered by Ca 2+ and Drives Membrane Fusion

Cell ◽

10.1016/s0092-8674(00)80727-8 ◽

1999 ◽

Vol 97 (2) ◽

pp. 165-174 ◽

Cited By ~ 298

Author(s):

Yu A Chen ◽

Suzie J Scales ◽

Sejal M Patel ◽

Yee-Cheen Doung ◽

Richard H Scheller

Keyword(s):

Complex Formation ◽

Membrane Fusion ◽

Snare Complex ◽

Snare Complex Formation

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Faculty Opinions recommendation of CAPS drives trans-SNARE complex formation and membrane fusion through syntaxin interactions.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1166306.627256 ◽

2009 ◽

Author(s):

Nils Brose

Keyword(s):

Complex Formation ◽

Membrane Fusion ◽

Snare Complex ◽

Snare Complex Formation

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Phosphatidylinositol 3,5-bisphosphate regulates the transition between trans-SNARE complex formation and vacuole membrane fusion

Molecular Biology of the Cell ◽

10.1091/mbc.e18-08-0505 ◽

2019 ◽

Vol 30 (2) ◽

pp. 201-208 ◽

Cited By ~ 8

Author(s):

Gregory E. Miner ◽

Katherine D. Sullivan ◽

Annie Guo ◽

Brandon C. Jones ◽

Logan R. Hurst ◽

...

Keyword(s):

Complex Formation ◽

Membrane Fusion ◽

Molecular Switch ◽

Snare Complex ◽

Cellular Functions ◽

Vacuole Membrane ◽

Hyperosmotic Shock ◽

Outer Leaflet ◽

Vacuole Fusion ◽

Snare Complex Formation

Phosphoinositides (PIs) regulate a myriad of cellular functions including membrane fusion, as exemplified by the yeast vacuole, which uses various PIs at different stages of fusion. In light of this, the effect of phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) on vacuole fusion remains unknown. PI(3,5)P2 is made by the PI3P 5-kinase Fab1 and has been characterized as a regulator of vacuole fission during hyperosmotic shock, where it interacts with the TRP Ca2+ channel Yvc1. Here we demonstrate that exogenously added dioctanoyl (C8) PI(3,5)P2 abolishes homotypic vacuole fusion. This effect was not linked to Yvc1, as fusion was equally affected using yvc1Δ vacuoles. Thus, the effects of C8-PI(3,5)P2 on fusion and fission operate through distinct mechanisms. Further testing showed that C8-PI(3,5)P2 inhibited vacuole fusion after trans-SNARE pairing. Although SNARE complex formation was unaffected, we found that C8-PI(3,5)P2 blocked outer leaflet lipid mixing. Overproduction of endogenous PI(3,5)P2 by the fab1T2250A hyperactive kinase mutant also inhibited the lipid mixing stage, bolstering the model in which PI(3,5)P2 inhibits fusion when present at elevated levels. Taken together, this work identifies a novel function for PI(3,5)P2 as a regulator of vacuolar fusion. Moreover, it suggests that this lipid acts as a molecular switch between fission and fusion.

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Phosphatidylinositol and phosphatidylinositol-3-phosphate activate HOPS to catalyze SNARE assembly, allowing small headgroup lipids to support the terminal steps of membrane fusion

Molecular Biology of the Cell ◽

10.1091/mbc.e21-07-0373 ◽

2021 ◽

pp. mbc.E21-04-0191

Author(s):

Thomas Torng ◽

William Wickner

Keyword(s):

Phosphatidic Acid ◽

Membrane Fusion ◽

Snare Complex ◽

Rab Gtpases ◽

Intracellular Membrane ◽

Complex Assembly ◽

Membrane Tethering ◽

Intracellular Membrane Fusion ◽

Phosphatidylinositol 3 ◽

Hops Complex

Intracellular membrane fusion requires Rab GTPases, tethers, SNAREs of the R, Qa, Qb, and Qc families, and SNARE chaperones of the Sec17 (SNAP), Sec18 (NSF), and SM (Sec1/Munc18) families. The vacuolar HOPS complex combines the functions of membrane tethering and SM catalysis of SNARE assembly. HOPS is activated for this catalysis by binding to the vacuolar lipids and Rab. Of the 8 major vacuolar lipids, we now report that phosphatidylinositol and phosphatidylinositol-3-phosphate are required to activate HOPS for SNARE complex assembly. These lipids plus ergosterol also allow full trans-SNARE complex assembly, yet do not support fusion, which is reliant on either phosphatidylethanolamine (PE) or on phosphatidic acid (PA), phosphatidylserine (PS), and diacylglycerol (DAG). Fusion with a synthetic tether and without HOPS, or even without SNAREs, still relies on either PE or on PS, PA, and DAG. These lipids are thus required for the terminal bilayer rearrangement step of fusion, distinct from the lipid requirements for the earlier step of activating HOPS for trans-SNARE assembly.

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