Misfolded α-synuclein (A-syn) is widely recognized as the primal cause of neurodegenerative diseases including Parkinson’s disease and dementia with Lewy bodies. The normal cellular function of A-syn has, however, been elusive. There is evidence that A-syn plays multiple roles in the exocytotic pathway in the neuron, but the underlying molecular mechanisms are unclear. A-syn has been known to interact with negatively charged phospholipids and with vesicle SNARE protein VAMP2. Using single-vesicle docking/fusion assays, we find that A-syn promotes SNARE-dependent vesicles docking significantly at 2.5 μM. When phosphatidylserine (PS) is removed from t-SNARE-bearing vesicles, the docking enhancement by A-syn disappears and A-syn instead acts as an inhibitor for docking. In contrast, subtraction of PS from the v-SNARE-carrying vesicles enhances vesicle docking even further. Moreover, when we truncate the C-terminal 45 residues of A-syn that participates in interacting with VAMP2, the promotion of vesicle docking is abrogated. Thus, the results suggest that the A-syn’s interaction with v-SNARE through its C-terminal tail and its concurrent interaction with PS in trans through its amphipathic N-terminal domain facilitate SNARE complex formation, whereby A-syn aids SNARE-dependent vesicle docking.
Recently, Parkinson’s disease-associated α-synuclein (αS) has emerged as an important regulator for SNARE-dependent vesicle fusion. However, it is controversial if excessive accumulation of αS, even in the absence of aggregation, impairs neurotransmission. Here we use a single vesicle fusion assay with ms time resolution capable of dissecting the impact of αS on each step of membrane fusion. Unlike the previous results from various
in vitro
, cellular, and
in vivo
studies, we find that non-aggregated αS promotes vesicle merger even at exorbitant concentrations. The enhancement has been seen as much as 13 fold. Delving into the kinetics of the intermediate states for vesicle fusion reveals that αS stimulates vesicle docking without altering the dynamics of bilayer merger (lipid mixing). However, minute amounts of soluble aggregated species abolish SNARE-dependent bilayer merger completely. Thus, the results show that excessive accumulation of non-aggregated αS may not be toxic for neurotransmitter release.
SNARE-dependent membrane fusion is essential for neurotransmitter release at the synapse. Recently, α-synuclein has emerged as an important regulator for membrane fusion. Misfolded α-synuclein oligomers are potent fusion inhibitors. However, the function of normal α-synuclein has been elusive. Here, we use the single vesicle-to-supported bilayer fusion assay to dissect the role of α-synuclein in membrane fusion. The assay employs 10 kD Rhodamine B-dextran as the content probe that can detect fusion pores larger than ∼6 nm. We find that the SNARE complex alone is inefficient at dilating fusion pores. However, α-synuclein dramatically increases the probability as well as the duration of large pores. When the SNARE-interacting C-terminal region of α-synuclein was truncated, the mutant behaves the same as the wild-type. However, the double proline mutants compromising membrane-binding show significantly reduced effects on fusion pore expansion. Thus, our results suggest that α-synuclein stimulates fusion pore expansion specifically through its membrane binding.
SNARE complex formation, which is believed to drive intracellular membrane fusion, transits through multiple conformational states along the membrane fusion pathway. The SNARE intermediates are biologically important because they serve as targets for fusion regulators and clostridial neurotoxins. Spin labeling EPR has contributed significantly to the understanding of the structures and the dynamics of SNARE intermediates. In particular, the EPR lineshape analysis, which is highly sensitive to protein conformational changes such as the local coil-to-helix transition, has revealed the sequential compacting steps leading to formation of the highly stable four helix bundle.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.