Presynaptic nerve terminals release neurotransmitters repeatedly, often at high frequency, and in relative isolation from neuronal cell bodies. Repeated release requires cycles of SNARE-complex assembly and disassembly, with continuous generation of reactive SNARE-protein intermediates. Although many forms of neurodegeneration initiate presynaptically, only few pathogenic mechanisms are known, and the functions of presynaptic proteins linked to neurodegeneration, such as α-synuclein, remain unclear. Here, we find that maintenance of continuous presynaptic SNARE-complex assembly required a non-classical chaperone activity mediated by synucleins. Specifically, α-synuclein directly bound to the SNARE-protein synaptobrevin-2/VAMP2, and promoted SNARE-complex assembly. Moreover, triple knockout mice lacking synucleins developed age-dependent neurological impairments, exhibited decreased SNARE-complex assembly, and perished prematurely. Thus, synucleins may function to sustain normal SNAREcomplex assembly in a presynaptic terminal during aging.In presynaptic terminals, neurotransmitter release requires a tightly coordinated membrane fusion machinery whose central components are soluble NSF attachment protein receptor (SNARE) and Sec1/Munc18-like proteins (1-3). Terminals release neurotransmitters thousands of times per minute; during each release reaction, SNARE-complex assembly and disassembly generates highly reactive unfolded SNARE protein intermediates, rendering the terminals potentially vulnerable to activity-dependent degeneration. Indeed, much evidence points to presynaptic terminals as an initiation site for neurodegeneration (4-6), and knockout (KO) of at least one presynaptic chaperone protein, cysteine string protein-α (CSPα), causes fulminant neurodegeneration in mice (7). Synucleins are abundant presynaptic proteins that are expressed from three genes (α-, β-and γ-synuclein; 8). α-Synuclein is involved in neurodegeneration (9-11), and γ-synuclein may contribute to progression of many types of cancer (12). Synucleins may modify neurotransmitter release (13,14), but their physiological functions remain unknown. Strikingly, transgenic expression of α-synuclein abolishes the lethal neurodegeneration induced by KO of CSPα, whereas deletion of endogenous synucleins accelerates this neurodegeneration (15). CSPα KO mice exhibit decreased levels of the SNARE protein SNAP-25 and impaired SNARE-complex assembly, suggesting a link between SNARE-complex assembly and neurodegeneration. α-* To whom correspondence should be addressed: tcs1@stanford.edu. HHMI Author Manuscript HHMI Author Manuscript HHMI Author ManuscriptSynuclein rescues SNARE-complex assembly but not SNAP-25 levels (15). This result indicates that α-synuclein may enhance SNARE-protein function, and thereby compensate for the CSPα deletion. To address this hypothesis, we here examine the role of α-synuclein in SNARE-complex assembly and in the maintenance of continuous SNARE-cycling in presynaptic terminals over the lifetime of an animal.We imm...

Physiologically, α-synuclein chaperones soluble NSF attachment protein receptor (SNARE) complex assembly and may also perform other functions; pathologically, in contrast, α-synuclein misfolds into neurotoxic aggregates that mediate neurodegeneration and propagate between neurons. In neurons, α-synuclein exists in an equilibrium between cytosolic and membrane-bound states. Cytosolic α-synuclein appears to be natively unfolded, whereas membrane-bound α-synuclein adopts an α-helical conformation. Although the majority of studies showed that cytosolic α-synuclein is monomeric, it is unknown whether membrane-bound α-synuclein is also monomeric, and whether chaperoning of SNARE complex assembly by α-synuclein involves its cytosolic or membrane-bound state. Here, we show using chemical cross-linking and fluorescence resonance energy transfer (FRET) that α-synuclein multimerizes into large homomeric complexes upon membrane binding. The FRET experiments indicated that the multimers of membrane-bound α-synuclein exhibit defined intermolecular contacts, suggesting an ordered array. Moreover, we demonstrate that α-synuclein promotes SNARE complex assembly at the presynaptic plasma membrane in its multimeric membrane-bound state, but not in its monomeric cytosolic state. Our data delineate a folding pathway for α-synuclein that ranges from a monomeric, natively unfolded form in cytosol to a physiologically functional, multimeric form upon membrane binding, and show that only the latter but not the former acts as a SNARE complex chaperone at the presynaptic terminal, and may protect against neurodegeneration.Parkinson's disease | synapse | SNARE proteins | membrane fusion α -Synuclein is an abundant presynaptic protein that physiologically acts to promote soluble NSF attachment protein receptor (SNARE) complex assembly in vitro and in vivo (1-3). Point mutations in α-synuclein (A30P, E46K, H50Q, G51D, and A53T) as well as α-synuclein gene duplications and triplications produce early-onset Parkinson's disease (PD) (4-10). Moreover, α-synuclein is a major component of intracellular protein aggregates called Lewy bodies, which are pathological hallmarks of neurodegenerative disorders such as PD, Lewy body dementia, and multiple system atrophy (11-14). Strikingly, neurotoxic α-synuclein aggregates propagate between neurons during neurodegeneration, suggesting that such α-synuclein aggregates are not only intrinsically neurotoxic but also nucleate additional fibrillization (15-18).α-Synuclein is highly concentrated in presynaptic terminals where α-synuclein exists in an equilibrium between a soluble and a membrane-bound state, and is associated with synaptic vesicles (19)(20)(21)(22). The labile association of α-synuclein with membranes (23, 24) suggests that binding of α-synuclein to synaptic vesicles, and its dissociation from these vesicles, may regulate its physiological function. Membrane-bound α-synuclein assumes an α-helical conformation (25-32), whereas cytosolic α-synuclein is natively unfolded and monomeric (refs. 25, 2...

Misfolding accounts for the endoplasmic reticulum-associated degradation of mutant cystic fibrosis transmembrane conductance regulators (CFTRs), including deletion of Phe508 (DeltaF508) in the nucleotide-binding domain 1 (NBD1). To study the role of Phe508, the de novo folding and stability of NBD1, NBD2 and CFTR were compared in conjunction with mutagenesis of Phe508. DeltaF508 and amino acid replacements that prevented CFTR folding disrupted the NBD2 fold and its native interaction with NBD1. DeltaF508 caused limited alteration in NBD1 conformation. Whereas nonpolar and some aliphatic residues were permissive, charged residues and glycine compromised the post-translational folding and stability of NBD2 and CFTR. The results suggest that hydrophobic side chain interactions of Phe508 are required for vectorial folding of NBD2 and the domain-domain assembly of CFTR, representing a combined co- and post-translational folding mechanism that may be used by other multidomain membrane proteins.