AbstractWe characterized phenotypes in RBL-2H3 mast cells transfected with human alpha synuclein (a-syn) using stimulated exocytosis of recycling endosomes as a proxy for similar activities of synaptic vesicles in neurons. We found that low expression of a-syn inhibits stimulated exocytosis and that higher expression causes slight enhancement. NMR measurements of membrane interactions correlate with these functional effects: they are eliminated differentially by mutants that perturb helical structure in the helix 1 (A30P) or NAC/helix-2 (V70P) regions of membrane-bound a-syn, but not by other PD-associated mutants or C-terminal truncation. We further found that a-syn (but not A30P or V70P mutants) associates weakly with mitochondria, but this association increases markedly under conditions of cellular stress. These results highlight the importance of specific structural features of a-syn in regulating vesicle release, and point to a potential role for a-syn in perturbing mitochondrial function under pathological conditions.