Differential regulation of wild-type and mutant alpha-synuclein binding to synaptic membranes by cytosolic factors

In the present study, we analysed the ability of cytosolic factors to regulate α-syn binding to synaptic membranes. We show that co-incubation with brain cytosol significantly increases the membrane binding of normal and PD-linked mutant α-syn. To characterize cytosolic factor(s) that modulate α-syn binding properties, we investigated the ability of proteins, lipids, ATP and calcium to modulate α-syn membrane interactions. We report that lipids and ATP are two of the principal cytosolic components that modulate Wt and A53T α-syn binding to the synaptic membrane. We further show that 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (C16:0 PAF) is one of the principal lipids found in complex with cytosolic proteins and is required to enhance α-syn interaction with synaptic membrane. In addition, the impaired membrane binding observed for A30P α-syn was significantly mitigated by the presence of protease-sensitive factors in brain cytosol.These findings suggest that endogenous brain cytosolic factors regulate Wt and mutant α-syn membrane binding, and could represent potential targets to influence α-syn solubility in brain.The synuclein family of intrinsically unfolded proteins is composed of three homologous and evolutionarily-conserved members with poorly defined physiological roles [1]. Of these, α-synuclein (α-syn) has gained particular prominence due to its abundance in nerve terminals and its association with multiple neurodegenerative disorders including Parkinson disease (PD) [2]. α-Syn behaves as a peripherally associated membrane protein and can stably interact with synthetic phospholipid vesicles containing negatively charged head groups [3] via its amino-terminal domain, an amphipathic region comprising almost two-thirds of the protein and containing seven copies of an 11-residue repeat sequence [4]. Whereas the freely diffusible form of α-syn is natively unfolded, the N-terminal repeat region adopts an α-helical conformation upon binding to artificial ves