Figure 5.

VAMP3, an endosomal SNARE protein, is ubiquitinated by Godzilla. (A) Schematic of identification of ubiquitiylated proteins by anti-GlyGly antibody purification (Xu et al, 2010). Cell extracts containing ubiquitylated proteins were digested by trypsin, resulting in generation of di-glycine conjugated peptides on the ε-amine group of lysine. Peptides were immuno-purified by anti-GlyGly antibody then subjected to nLC-MS/MS analysis. (B) Sequence alignment of VAMP3 and its Drosophila homologue, Synaptobrevin (Syb, dVAMP) and neuronal-Synaptobrevin (n-Syb). Identically conserved residues are highlighted in grey and the transmembrane domain and linker region are marked. Three ubiquitinated lysine residues (Lys⁶⁶, Lys⁶⁸ and Lys⁷⁷) identified in VAMP3 are shown in red. These ubiquitinated lysine residues are completely conserved between human and Drosophila VAMP3 homologues. (C) Localization of VAMP3 on Godzilla-induced giant endosomes. Wild-type or ligase-dead Godzilla expressing HEK293 cells were stained with anti-VAMP3 antibody (red). (D) Rab11-mediated recycling is disrupted in wild-type Godzilla expressing cells, but not in ligase dead. (E) VAMP3 knockdown rescues the formation of giant endosomes induced by Godzilla, quantified in (F). Quantified results are given as mean±s.d. for four independent experiments. Bar 10 μm. (G) Formation of Rab5-positive giant endosomes in godzilla loss-of-function wing discs is suppressed upon knockdown of the Drosophila VAMP3 homologue Synaptobrevin (Syb). Upper panel: late third instar larvae wing disc expressing UAS-godzilla RNAi driven by MS1096-Gal4; bottom panel: MS1096-Gal4 driving both UAS-godzilla RNAi and UAS-syb RNAi wing disc. Wing discs were visualized with Rab5 (red) and phalloidin to visualize F-actin (green). Dotted box shows an expanded view. (H) Formation of FYVE-positive giant endosomes in Godzilla-overexpressing wing discs is suppressed upon knockdown of Syb. Giant endosomes observed upon Godzilla overexpression (upper panel, arrowheads) are suppressed in a Syb knockdown background (bottom panel).