Unroofing site-specific α-synuclein-lipid interactions at the plasma membrane

Parkinson's disease is associated with alpha-synuclein (alpha-syn), a cytosolic protein enriched in presynaptic terminals. The biological function of alpha-syn remains elusive; however, increasing evidence suggests that the protein is involved in the regulation of synaptic vesicle fusion, signifying the importance of alpha-syn-lipid interactions. We show that alpha-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 alpha-syn, supporting its proposed functional role in exocytosis. To investigate how these lipid/protein interactions influence alpha-syn at the residue level, alpha-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 alpha-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 alpha-syn is more conformationally dynamic at the membrane interface than previously appreciated, which may be important for both its physiological and pathological functions.