Characterization of Endogenous and Epitope-Tagged Ubiquitin Expression and Ubiquitination Patterns at Drosophila Neuromuscular Junctions

Abstract

Ubiquitination is a highly regulated post-translational modification that controls protein stability, activity, localization, and interactions. Disruption of this process contributes to a broad range of human diseases, including cancer, neurodegeneration, and immune dysfunction. The genetic model organism Drosophila melanogaster offers powerful tools to study ubiquitination in vivo, where versatile systems such as GAL4/UAS, Split-GAL4, GeneSwitch, and MARCM can be combined with imaging and biochemical approaches to achieve precise spatial and temporal analyses. To fully exploit these advantages, experimental designs often require epitope-tagged UAS-Ubiquitin transgenes--both modified and unmodified--that accurately recapitulate the behavior of endogenous Ubiquitin. Here, we report the generation and validation of a panel of epitope-tagged UAS-Ubiquitin transgenes. We first characterize the endogenous ubiquitination pattern at larval neuromuscular junctions (NMJs), then compare that with transgenically expressed Ubiquitin in muscle. Our analysis showed that transgenic His-Myc-, HA-, and His-Biotin-tagged Ubiquitin proteins accurately reproduce endogenous postsynaptic ubiquitination patterns. These validated transgenes provide versatile resources for investigating ubiquitination dynamics across diverse cellular and developmental contexts.