Adding hydrophobicity or positive charges to the cytosolic half of the α-synuclein 3-11 helix increases membrane association and S129 phosphorylation.

The neuronal protein α-synuclein is centrally involved in the neurodegeneration occurring in Parkinson's disease and related synucleinopathies. α-Synuclein's membrane-induced 3-11 helix conformation has a hydrophobic membrane-embedded half and a hydrophilic cytosolic half. Here, we studied the significance of (a) the surprising hydrophobicity of amino-acids at cytosol-exposed helix position 8; (b) the absence of positively charged lysine/arginine from all cytosol-exposed positions (1-5-8-9). We found that (a) further increasing hydrophobicity or adding lysine, but not glutamate, at position 8 augments both membrane interaction and S129 phosphorylation; (b) adding lysines at cytosol-exposed positions 1, 5, 8, or 9 has similar effects. Variants abundantly present in membranes by biochemical fractionation markedly colocalized with transferrin-receptor (an endosomal marker) in immunofluorescence-microscopy, indicating accumulation at vesicle membranes. Thus, we observed a striking correlation between membrane attraction and S129 phosphorylation, relevant for understanding α-synuclein biology in health and disease.