Fig. 6: Gid12 biochemically modulates both assembly of Gid4 into the GID E3 ligase complex and its activity toward its substrates.
a Strep-tagged Gid4 in solution can replace untagged Gid4 within GIDSR4 but not in the Gid12-bound complex. A mutant (mut) lacking the C-terminal four residues 359–362 required for incorporation into GIDSR4 is used as a control for specificity of Strep-Gid4 incorporation into the complex (n = 3 biologically independent experiments). b Pulse chase assay examining effects of Gid12 on GID ubiquitin transferase activity in substrate-independent manner. Ubc8 is C-terminally fluorescently labeled with TAMRA for fluorescence detection of the free E2 versus the version thioester-linked to Ub. First, the thioester-linked Ubc8-TAMRA~Ub intermediate was generated in a pulse reaction. Second, the chase reaction monitors formation of the faster-migrating Ubc8-TAMRA upon Ub discharge to free lysine, stimulated by GIDSR4 with or without a bound Gid12 (n = 3 biologically independent experiments). c In vitro ubiquitylation assay of model peptide substrates (with an N-terminal substrate degron, a flexible linker, a single lysine at optimal position to accept ubiquitin, and a C-terminal fluorescein for fluorescent detection) testing effects of adding purified Gid4 alone or Gid4 co-expressed with Gid12 to GIDAnt (n = 3 biologically independent experiments). d 18.9 Å-resolution cryo-EM density map of Chelator-GIDSR4-Mdh2. Mdh2 dimer encapsulated in the oval center of Chelator-GIDSR4 is color-coded in black. e Experiment as in (c), except with the indicated full-length metabolic enzyme substrates, each appended to a C-terminal fluorescein for fluorescent detection (n = 3 biologically independent experiments).
