Ubiquitination is a key regulatory mechanism for synaptic development, signaling, and plasticity. The covalent attachment of the 76 aa peptide ubiquitin to target proteins is a rapid and reversible modification that regulates protein stability, activity and localization. As such, it is a potent mechanism for sculpting the synapse. We have uncovered a ubiquitination complex composed of the E3 ubiquitin ligase Highwire (Hiw) and the F-Box protein DFsn that plays a central role in controlling synaptic growth and function at the Drosophila neuromuscular junction (NMJ). A highly homologous ubiquitination complex has also been identified in the mammalian brain, where it plays a critical role in regulating axon guidance and synaptogenesis. However, the molecular architecture and molecular action of this ubiquitination complex is not well understood. We propose that Hiw and DFsn form a non-SCF ubiquitin complex where Hiw functions as an E3 ligase and a scaffolding protein to facilitate multi-subunit interaction, and the combination of different co-factors and ubiquitin substrates confers time- and cell type-specific regulation of neuronal functions. Thus identifying other components and novel ubiquitin targets of this ubiquitination complex is key to understanding how the hiw-mediated ubiquitin pathway specifically regulates synaptic development. We have taken two independent approaches to address this question. Biochemically, we identified Hiw/DFsn interacting proteins through tandem affinity purification using fly brains that express affinity-tagged Hiw and DFsn proteins, respectively. Studying the role of two of the Hiw- binding proteins, NSF and Rae1, in synaptic development (aim1), and how they work together with Hiw and DFsn to modulate the ubiquitin ligase activity (aim2) will define an essential ubiquitination machinery that controls synaptic growth. Genetically, we identified 5 hiw enhancer complementation groups through a hiw enhancer screen. Studying these genetic hiw interactors will allow us to identify other molecular pathways that work together with the Hiw/DFsn ubiquitin pathway to shape the structure and strength of synaptic connections formed during development (aim3).