Neurons are highly polarized cells characterized by specific structural and functional regions. The soma, or cell body, serves as a primary site for protein synthesis, while synaptic terminals located at the ends of axons are responsible for synaptic transmission. The precise transport of presynaptic proteins from the soma to these terminals is vital for the development, maturation, and maintenance of synapses. Presynaptic precursor biogenesis is a fundamental process for synaptic function and plasticity; however, its molecular mechanisms remain incompletely understood. Our research identifies Rab2 and its effector RUND1 as key regulators of presynaptic precursor vesicle (PV) formation at the trans-Golgi network (TGN). Rab2, a small GTPase, coordinates the biogenesis, sorting, and maturation of vesicles carrying presynaptic proteins essential for synaptic vesicle (SV) recycling and active zone (AZ) organization. Loss of Rab2 results in the accumulation of synaptic material— including scaffold proteins, SV proteins, and lysosomal markers—within motoneuronal cell bodies in Drosophila larval ventral nerve cords (VNCs). This mislocalization depletes presynaptic protein levels at synaptic terminals, leading to impaired neurotransmission. Electron microscopy revealed that these aggregates likely correspond to Golgi-derived transport vesicles, suggesting that Rab2 is crucial for efficient precursor export. Our RNAi-mediated screen identified RUND1 as a critical Rab2 effector that regulates presynaptic protein sorting, maturation, and trafficking. The RUN domain of RUND1 facilitates interactions with Rab2 and other regulatory proteins, including ICA69, Trabuco, and Golgin104. ICA69, a Rab2 effector, participates in secretory vesicle biogenesis and synaptic organization, while Golgin104 (CCDC186) is implicated in vesicle tethering at the TGN. Notably, the loss of RUND1produces a phenotype reminiscent of Rab2 mutants, supporting its role in the same pathway of presynaptic precursors biogenesis. Interestingly, ultrastructural analysis of vesicles in rund1−/−mutant backgrounds revealed a striking increase in elongated and tubular structures. These vesicles exhibited two distinct populations: clear and dense-core vesicles, potentially indicating a disruption in fusion or maturation processes. The presence of these heterogeneous vesicle populations suggests that RUND1 is essential for the proper segregation and functional refinement of vesicles before their transport to synaptic terminals. Overall, our findings highlight the intricate regulation of presynaptic precursor trafficking by Rab2 and RUND1, reinforcing the importance of Golgi-associated sorting mechanisms in synaptic development and function. Disruptions in this pathway impair synaptic protein localization and neurotransmission, with potential implications for understanding synaptic disorders.
