Figure 5.

The ATXN3 Josephin domain contains two conserved LIR motifs. (a) Schematic illustration of peptide spot assay to identify LIR motifs in the ATXN3 Josephin domain. The N‐terminal sequence of ATXN3 (1–182) was synthesized as 20‐mer peptides and spotted on cellulose membranes. (b) Binding of GST‐tagged fusions of GABARAP and LC3C to the spotted ATXN3 Josephin peptides was analyzed by immunoblotting. (c) Conservation of the identified LIR1 and LIR2 across species. (d) Catalytic activity of recombinant ATXN3 toward recombinant pre‐GABARAP‐GFP was analyzed in vitro by incubation of 250 ng pre‐GABARAP‐GFP with the indicated amounts of ATXN3 for 16 hr at 37°C. Recombinant His₆‐Atg4B was used as control