Shuai QiaoCHIA-WEI LEEDawafuti SherpaJakub ChrustowiczJingdong ChengMaximilian DuennebackeBarbara SteigenbergerOzge KarayelDuc Tung VuSusanne von GronauMatthias MannFlorian WilflingBrenda A. Schulman2023-07-242023-07-242022-06-012041-1723https://scholars.lib.ntu.edu.tw/handle/123456789/634056Protein degradation, a major eukaryotic response to cellular signals, is subject to numerous layers of regulation. In yeast, the evolutionarily conserved GID E3 ligase mediates glucose-induced degradation of fructose-1,6-bisphosphatase (Fbp1), malate dehydrogenase (Mdh2), and other gluconeogenic enzymes. "GID" is a collection of E3 ligase complexes; a core scaffold, RING-type catalytic core, and a supramolecular assembly module together with interchangeable substrate receptors select targets for ubiquitylation. However, knowledge of additional cellular factors directly regulating GID-type E3s remains rudimentary. Here, we structurally and biochemically characterize Gid12 as a modulator of the GID E3 ligase complex. Our collection of cryo-EM reconstructions shows that Gid12 forms an extensive interface sealing the substrate receptor Gid4 onto the scaffold, and remodeling the degron binding site. Gid12 also sterically blocks a recruited Fbp1 or Mdh2 from the ubiquitylation active sites. Our analysis of the role of Gid12 establishes principles that may more generally underlie E3 ligase regulation.enCryo-EM structures of Gid12-bound GID E3 reveal steric blockade as a mechanism inhibiting substrate ubiquitylationjournal article10.1038/s41467-022-30803-9356502072-s2.0-85131081824https://doi.org/10.1038/s41467-022-30803-9113861167