The best understood substrate receptor is Gid4, which binds GID Ant to generate GID SR4. A bound substrate receptor is indicated by the nomenclature GID SR#, with # referring to its Gid subunit number. The core GID Ant complex comprises the Gid1, Gid2, Gid5, Gid8, and Gid9 subunits. These assemblies are denoted with “Ant” in superscript because they “anticipate” a switch in conditions that triggers expression of a substrate receptor. Some complexes are incompletely assembled, and are inactive due to lack of a substrate-binding receptor subunit. Another mutant, GID3, encodes the GID E3’s dedicated E2 enzyme partner, Ubc8 20.īiochemical and structural studies have shown that the GID E3 is not a singular complex, but a collection of assemblies that vary in activity 21, 22, 23, 24. The term GID derives from Fbp1 being Glucose- induced degradation deficient in mutants, a majority of which are now recognized to encode subunits (Gid1, Gid2, Gid4, Gid5, Gid7, Gid8, and Gid9) of the multiprotein E3 10, 11, 12, 19. In yeast, the gluconeogenesis enzymes fructose 1,6-biphosphatase (Fbp1), malate dehydrogenase (Mdh2), isocitrate lyase (Icl1) and phosphoenolpyruvate carboxykinase (Pck1) are subject to degradation depending on ubiquitylation by the GID E3 ligase 11, 19. However, when a fermentable carbon source is available, gluconeogenesis is superfluous and terminated through transcriptional and post-transcriptional mechanisms 17, 18. Yeast grown on non-fermentable carbon sources depend on gluconeogenic enzymes to produce glucose. Molecular functions are best understood for the yeast GID E3, which regulates nutrient-dependent control of carbon metabolism 10, 11, 12, 13, 14, 15, 16. Moreover, the GID E3 in soybean legumes is critical for rhizobial nodulation required for nitrogen fixation 9. cerevisiae GID E3 (also called “CTLH complex” in higher eukaryotes) are essential for erythropoiesis, organ development and embryogenesis, respectively 2, 3, 4, 5, 6, 7, 8.
Genetic studies have shown that mammalian, frog, and fly orthologs of the budding yeast S. We set out to explore E3 ligase regulatory mechanisms through studies of the multiprotein GID complex. Despite great progress in understanding how some ubiquitin ligases are controlled, for most E3s, the factors modulating activity, and their mechanisms-of-action remain unknown. For example, multiprotein E3 ligases are often activated by timely incorporation of their substrate-binding subunits, or inhibited by factors that block E3 ligase assembly or activity. In some cases, regulation is achieved by a substrate modification triggering binding to an E3 ligase. Proteins are selected for degradation by E3 ligases, which recruit specific substrate “degron” motifs and promote ubiquitylation 1. Our analysis of the role of Gid12 establishes principles that may more generally underlie E3 ligase regulation.Ī major eukaryotic mechanism controlling the timing of protein expression is ubiquitin-mediated proteolysis. Gid12 also sterically blocks a recruited Fbp1 or Mdh2 from the ubiquitylation active sites. 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. Here, we structurally and biochemically characterize Gid12 as a modulator of the GID E3 ligase complex. However, knowledge of additional cellular factors directly regulating GID-type E3s remains rudimentary. “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. 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.
Protein degradation, a major eukaryotic response to cellular signals, is subject to numerous layers of regulation.
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