Generation and Validation of a Panel of Epitope-Tagged UAS-Ubiquitin Transgenes for Tissue-specific Analysis of Ubiquitination in Drosophila

Ubiquitination is a highly regulated post-translational modification that controls protein stability, activity, localization, and interactions, and its disruption process contributes to a broad range of human diseases, including cancer, neurodegeneration, and immune dysfunction. The genetic model organism Drosophila melanogaster offers powerful in vivo tools for studying ubiquitination, including the GAL4/UAS, Split-GAL4, GeneSwitch, and MARCM system, which can be combined with imaging and biochemical approaches to achieve precise spatial and temporal analyses. Realizing this potential, however, requires epitope-tagged UAS-Ubiquitin transgenes-both unmodified and chain-restricted-that behave like endogenous ubiquitin. Here we report a screen to generate and validate such a panel. We first defined the endogenous ubiquitination pattern at larval neuromuscular junctions (NMJs) as a bench mark, then compared it with a series of transgenically expressed, tagged ubiquitin in muscle and neurons. His-Myc-, HA-, and His-Biotin-tagged ubiquitins reproduced endogenous postsynaptic ubiquitination pattern and, by anti-tag immunoprecipitation followed by anti-ubiquitin immunoblotting, were shown to be covalently incorporated into ubiquitin conjugates in vivo. In contrast, a larger NTAP-tagged ubiquitin failed to conjugate at the postsynaptic density. Expression of the validated transgenes did not alter NMJ bouton number. We describe the practical uses of the validated transgenes for tissue-specific imaging and affinity purification, together with their limitations, to help investigators select appropriate tools for studying ubiquitination across diverse cellular and developmental contexts.