Neuronal Proteomic Analysis of the Ubiquitinated Substrates of the Disease-Linked E3 Ligases Parkin and Ube3a

Figure 3

BioUb strategy to identify ubiquitin substrates of E3 ligases in Drosophila neurons. Scheme of the strategy used to identify proteins ubiquitinated by Parkin and Ube3a in Drosophila neurons. Flies were engineered to express endogenously precursor capable of biotinylating ubiquitin in Drosophila photoreceptors using the GMR-GAL4 driver. This biotin modified ubiquitin () is then incorporated within the pool of endogenous ubiquitin, in flies that also overexpress wild-type E3 ligases (E3-WT), Parkin or Ube3a, and in their respective mutant or ligase-dead negative controls (E3-LD). Ubiquitinated material can then be purified using Neutravidin beads and isolated material analyzed by mass spectrometry (MS). Ubiquitinated proteins enriched in Parkin or Ube3a WT overexpressing neurons can then be identified based on both protein LFQ levels and peptide intensities.