Trans-complex formation by proteolipid channels in the terminal phase of 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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Capture and release of partially zipped trans-SNARE complexes on intact organelles

The Journal of Cell Biology ◽

10.1083/jcb.200811082 ◽

2009 ◽

Vol 185 (3) ◽

pp. 535-549 ◽

Cited By ~ 77

Author(s):

Matthew L. Schwartz ◽

Alexey J. Merz

Keyword(s):

Fusion Protein ◽

Membrane Fusion ◽

Attachment Protein ◽

Binding Partner ◽

Straightforward Method ◽

Protein Receptors ◽

Trans Complex ◽

In Trans ◽

Capture And Release ◽

Protein Attachment

Soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptors (SNAREs) are hypothesized to trigger membrane fusion by complexing in trans through their membrane-distal N termini and zippering toward their membrane-embedded C termini, which in turn drives the two membranes together. In this study, we use a set of truncated SNAREs to trap kinetically stable, partially zipped trans-SNARE complexes on intact organelles in the absence of hemifusion and content mixing. We show that the C-terminal zippering of SNARE cytoplasmic domains controls the onset of lipid mixing but not the subsequent transition from hemifusion to full fusion. Moreover, we find that a partially zipped nonfusogenic trans-complex is rescued by Sec17, a universal SNARE cochaperone. Rescue occurs independently of the Sec17-binding partner Sec18, and it exhibits steep cooperativity, indicating that Sec17 engages multiple stalled trans-complexes to drive fusion. These experiments delineate distinct functions within the trans-complex, provide a straightforward method to trap and study prefusion complexes on native membranes, and reveal that Sec17 can rescue a stalled, partially zipped trans-complex.

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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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