Parkinson’s disease is the second most common neurodegenerative disease, currently without any disease modifying therapy. Although α-synuclein is causatively linked to Parkinson’s pathogenesis, the primary mechanism and subcellular localisation of early cytotoxicity as well as its most damaging proteoform remain unknown. To address this issue, we spatially and temporally resolved proteomic and transcriptomic changes in human iPSC-derived dopaminergic neurons with increasing burden of pathological α-synuclein. We show that microscale α-synuclein aggregates are biochemically inert whereas nanoscale aggregates, not visible by conventional confocal microscopy, are associated with impaired Sec61A translocon function at the endoplasmic reticulum (ER). α-Synuclein blocks the cotranslational translocation of ER-processed proteins including the vacuolar-type ATPase V0a1 subunit, glucocerebrosidase, and VPS13C, causing defective organelle function, as exemplified by measurements of lysosomal acidification, secondary UFMylation and proteasomal recruitment without activation of the unfolded protein response. Reduction of nano-aggregate abundance using either CRISPRi to decrease α-synuclein expression or rolipram to activate proteasomal degradation mitigate the ER translocation defect. Our study offers a unifying mechanistic link between early α-synuclein pathology and dysregulation of diverse organelle-associated proteins that are both translocon substrates and genetic modifiers. Importantly, our data suggest that proteasomal activation with repurposed drugs should be considered for therapeutic intervention in patients with early-stage pathology.