Alpha-synuclein (a-syn) is a major component of Lewy bodies, a neuropathology observed in Parkinson’s disease (PD) and other related neurodegenerative disorders. Oligomeric species formed during a-syn aggregation process have been linked to impairments caused by PD, but the normal physiological function of a-syn and dysregulation leading to PD remain ambiguous. Much of our knowledge concerning a-syn stems from in-vitro studies focused on the aggregation process and on membrane binding properties. Despite the development of cell and animal models, the functional consequence of a-syn’s structural interactions remain poorly defined. By using multiple mammalian cell models, including RBL cells, effects of a-syn on specific cell processes can be measured and analyzed. Considering the localization of a-syn to nerve terminals and its affinity for binding phospholipid vesicles, modulation of the trafficking of vesicles, including synaptic vesicles is a likely function of a-syn. We investigated phenotypes of a-syn overexpression in RBL and PC-12 cells by monitoring stimulated exocytosis of recycling endosomes (REs) as a proxy for synaptic vesicles that are similar in size. We demonstrated that low expression levels of a-syn inhibit exocytosis of REs while higher expression levels lead to some enhancement. Using NMR spectroscopy, we demonstrated that membrane binding properties of a-syn variants correlate with the functional outcomes. In particular, perturbation in helix 1 or helix 2 of a-syn N-terminal region by proline mutations, specifically affects both participation in RE exocytosis and binding affinity. Conversely C-terminal truncation and PD-related mutations behave similarly to wt a-syn at low expression levels in the exocytosis assay. Furthermore, we found that wt a-syn binds weakly to mitochondria unlike the proline mutants and that the binding becomes considerably stronger upon mitochondrial stress. Building on our findings that a-syn binds to mitochondria, we demonstrated that expression of a-syn enhances stimulated mitochondrial Ca2+ uptake in RBL cells. We validated this observation in the HEK293 cell line which is commonly used as a model for neurons. We found that proline mutations disrupting helix 1 or 2 of a-syn nullifies this enhancing effect, consistent with the N-terminal membrane binding portion of a-syn facilitating the increase in mitochondrial Ca2+ uptake. By controlling the source of Ca2+ we found that the Ca2+ for mitochondrial uptake comes mainly from the endoplasmic reticulum (ER). Structured illumination microscopy showed that increase in mitochondrial Ca2+ uptake correlates with increase in contacts between ER and mitochondria. We further established that a-syn enhances the mitochondrial Ca2+ uptake in a neuronal cell line (differentiated dopaminergic N2a cells). We discovered and investigated a novel inhibitory effect of a-syn on recovery from mitochondrial stress caused by PD inducing toxins. Interestingly this inhibitory effect appears to depend on the unstructured C-terminal tail of a-syn. These results highlight the significance of specific structural features of a-syn in regulating vesicle release and modulating mitochondrial Ca2+ uptake, and they reveal a novel pathological function for a-syn under mitochondrial stress related to PD.