Figure 1.

Goliath and Godzilla encode endosomally localized PA-TM-RING E3 ligases. (A) Schematic domain structure of Drosophila Goliath and Godzilla. SP, signal peptide; PA, protease-associated domain; TM, transmembrane, and RING domain. (B) Sequence alignment of related RING domains. RNF167, RNF128 (also known as GRAIL) and RNF130 (also known as hGoliath) are human sequences. The RING domain (blue) contains six conserved Cys (C1–C6) and two His (H1 and H2) residues, which coordinate Zn²⁺ and are important for ligase activity. *For ligase-dead mutants, two His residues were substituted to Arg, corresponding to His³²³ and His³²⁶ in Goliath and His²⁵⁵ and His²⁵⁸ in Godzilla. (C) Phylogenic relationship of PA-TM-RING E3-ligase genes. (D) Subcellular localization of Goliath and Godzilla. Goliath-C-GFP (green, left) or Godzilla-C-GFP (green, right) transfected HEK293 cells were co-stained with cell organelle markers (red). EEA1, early endosome; LC3B, autophagosome; LysoTracker, lysosome; MitoTracker, mitochondria. Goliath-C-GFP and Godzilla-C-GFP-induced enlarged vesicle-like structures colocalize with EEA1 (arrowheads), and partially with LC3B (arrowheads); indicating that Goliath and Godzilla localize on an enlarged endosome membranes, but not on lysosomes or mitochondria. Bar 10 μm. The results shown here are typical images from at least four independent experiments. (E) Endogenous Drosophila protein is localized on endosomes in vivo. Endosomes were enlarged by expression of constitutively active Rab5 (Rab5Q88L^YFP) in the Drosophila wing disc with MS1096-Gal4. Endogenous Godzilla is accumulated on the resultant enlarged Rab5-positive endosomes. The results shown here are typical images from at least four independent experiments.