Abstract
Parkinson's disease is associated with α-synuclein (α-syn), a cytosolic protein enriched in presynaptic terminals. The biological function of α-syn remains elusive; however, increasing evidence suggests that the protein is involved in the regulation of synaptic vesicle fusion, signifying the importance of α-syn-lipid interactions. We show that α-syn preferentially binds to GM1-rich, liquid-ordered lipid domains on cytoplasmic membranes by using unroofed cells, which encapsulates lipid complexity and cellular topology. Moreover, proteins (Rab3a, syntaxin-1A, and VAMP2) involved in exocytosis also localize with α-syn, supporting its proposed functional role in exocytosis. To investigate how these lipid/protein interactions influence α-syn at the residue level, α-syn was derivatized with an environmentally sensitive fluorophore (7-nitrobenz-2-oxa-1,3-diazol-4-yl [NBD]) at different N- and C-terminal sites. Measurements of NBD fluorescence lifetime distributions reveal that α-syn adopts a multitude of membrane-bound conformations, which were not recapitulated in simple micelle or vesicle models, indicating an exquisite sensitivity of the protein to the complex lipid environment. Interestingly, these data also suggest the participation of the C terminus in membrane localization, which is generally overlooked and thus emphasize the need to use cellularly derived and biologically relevant membranes for biophysical characterization. Collectively, our results demonstrate that α-syn is more conformationally dynamic at the membrane interface than previously appreciated, which may be important for both its physiological and pathological functions.
Highlights
Parkinson’s disease is associated with α-synuclein (α-syn), a cytosolic protein enriched in presynaptic terminals
To investigate direct α-syn–lipid interactions on unroofed cells, we employed site-specific Cys variants (V26C, V40C, and Y136C), α-Synuclein (α-syn), a widely studied neuronal protein involved in the pathology of Parkinson’s disease, remains an enigma because its physiological functions are not well understood [1]. α-Syn is concentrated in presynaptic terminals, where it has been proposed to be involved in synaptic vesicle docking, fusion, clustering, and homeostasis [2, 3]
The lipophilic DiD exhibited a diffusive staining behavior, indicative of an intact phospholipid bilayer (Fig. 1 A, Left), whereas fluorescently labeled cholera toxin B (CT-B), a protein that binds to GM1, a ganglioside enriched in lipid rafts [41, 42], showed numerous puncta distributed across the unroofed cell with a low degree of labeling in the surrounding membrane (Fig. 1 A, Right)
Summary
Parkinson’s disease is associated with α-synuclein (α-syn), a cytosolic protein enriched in presynaptic terminals. Measurements of NBD fluorescence lifetime distributions reveal that α-syn adopts a multitude of membrane-bound conformations, which were not recapitulated in simple micelle or vesicle models, indicating an exquisite sensitivity of the protein to the complex lipid environment. Contrary to anionic lipids [29], the protein exhibits the unusual behavior of preferring ordered gel phases of zwitterionic phospholipids in vitro [30, 31], driven by its enhanced affinity for lipid-packing defects [30] This observation is pertinent due to the similarity in the lipid composition to those found in cellular liquid-ordered lipid domains [32], enriched in cholesterol and sphingolipids associated with exocytic sites. Membrane-bound α-synuclein is conformationally dynamic, exquisitely sensitive to lipid/protein composition, enabling the protein to carry out its function
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