α-Synuclein is an abundant presynaptic protein and a primary component of Lewy 
bodies in Parkinson disease. Although its pathogenic role remains unclear, in 
healthy nerve terminals α-synuclein undergoes a cycle of membrane binding and 
dissociation. An α-synuclein binding assay was used to screen for vesicle 
proteins involved in α-synuclein membrane interactions and showed that 
antibodies directed to the Ras-related GTPase Rab3a and its chaperone RabGDI 
abrogated α-synuclein membrane binding. Biochemical analyses, including density 
gradient sedimentation and co-immunoprecipitation, suggested that α-synuclein 
interacts with membrane-associated GTP-bound Rab3a but not to cytosolic 
GDP-Rab3a. Accumulation of membrane-bound α-synuclein was induced by the 
expression of a GTPase-deficient Rab3a mutant, by a dominant-negative GDP 
dissociation inhibitor mutant unable to recycle Rab3a off membranes, and by 
Hsp90 inhibitors, radicicol and geldanamycin, which are known to inhibit Rab3a 
dissociation from membranes. Thus, all treatments that inhibited Rab3a recycling 
also increased α-synuclein sequestration on intracellular membranes. Our results 
suggest that membrane-bound GTP-Rab3a stabilizes α-synuclein on synaptic 
vesicles and that the GDP dissociation inhibitor·Hsp90 complex that controls 
Rab3a membrane dissociation also regulates α-synuclein dissociation during 
synaptic activity.