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Cells rapidly remodel their proteomes to align their cellular metabolism to environmental conditions. Ubiquitin E3 ligases enable this response, by facilitating rapid and reversible changes to protein stability, localization, or interaction partners. In Saccharomyces cerevisiae, the GID E3 ligase regulates the switch from gluconeogenic to glycolytic conditions through induction and incorporation of the substrate receptor subunit Gid4, which promotes the degradation of gluconeogenic enzymes. Here, we show an alternative substrate receptor, Gid10, which is induced in response to changes in temperature, osmolarity, and nutrient availability, regulates the ART-Rsp5 ubiquitin ligase pathway, a component of plasma membrane quality control. Proteomic studies reveal that the levels of the adaptor protein Art2 are elevated upon GID10 deletion. A crystal structure shows the basis for Gid10-Art2 interactions, and we demonstrate that Gid10 directs a GID E3 ligase complex to ubiquitinate Art2. Our data suggest that the GID E3 ligase affects Art2-dependent amino acid transport. This study reveals GID as a system of E3 ligases with metabolic regulatory functions outside of glycolysis and gluconeogenesis, controlled by distinct stress-specific substrate receptors.

N-degron E3 ubiquitin ligases recognize specific residues at the N-termini of substrates. Although molecular details of N-degron recognition are known for several E3 ligases, the range of N-terminal motifs that can bind a given E3 substrate binding domain remains unclear. Here, we discovered capacity of Gid4 and Gid10 substrate receptor subunits of yeast "GID"/human "CTLH" multiprotein E3 ligases to tightly bind a wide range of N-terminal residues whose recognition is determined in part by the downstream sequence context. Screening of phage displaying peptide libraries with exposed N-termini identified novel consensus motifs with non-Pro N-terminal residues binding Gid4 or Gid10 with high affinity. Structural data reveal that conformations of flexible loops in Gid4 and Gid10 complement sequences and folds of interacting peptides. Together with analysis of endogenous substrate degrons, the data show that degron identity, substrate domains harboring targeted lysines, and varying E3 ligase higher-order assemblies combinatorially determine efficiency of ubiquitylation and degradation. (c) 2021 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Summary How are E3 ubiquitin ligases configured to match substrate quaternary structures? Here, by studying the yeast GID complex (mutation of which causes deficiency in glucose-induced degradation of gluconeogenic enzymes), we discover supramolecular chelate assembly as an E3 ligase strategy for targeting an oligomeric substrate. Cryoelectron microscopy (cryo-EM) structures show that, to bind the tetrameric substrate fructose-1,6-bisphosphatase (Fbp1), two minimally functional GID E3s assemble into the 20-protein Chelator-GIDSR4, which resembles an organometallic supramolecular chelate. The Chelator-GIDSR4 assembly avidly binds multiple Fbp1 degrons so that multiple Fbp1 protomers are simultaneously ubiquitylated at lysines near the allosteric and substrate binding sites. Importantly, key structural and biochemical features, including capacity for supramolecular assembly, are preserved in the human ortholog, the CTLH E3. Based on our integrative structural, biochemical, and cell biological data, we propose that higher-order E3 ligase assembly generally enables multipronged targeting, capable of simultaneously incapacitating multiple protomers and functionalities of oligomeric substrates., Graphical abstract, Highlights • GID E3 ligase assembly resembles a behemoth organometallic supramolecular chelate • Multipronged targeting of oligomeric structure and metabolic function of substrate • Yeast Gid7, human WDR26, and MKLN1 mediate supramolecular assembly of GID/CTLH E3s • Human CTLH E3 structural and mechanistic features parallel yeast GID E3 ligase, Structural, biochemical, and cellular data reveal supramolecular assembly of yeast GID E3 ligase. GID E3 forms a chelate-like structure tailored to target the oligomeric structure and metabolic function of the gluconeogenic enzyme substrate Fbp1. The orthologous human CTLH E3 contains a GID-like core assembly that forms a supramolecular chelate and utilizes a GID-like ubiquitin ligase mechanism.

SUMMARYCells respond to environmental changes by toggling metabolic pathways, preparing for homeostasis, and anticipating future stresses. For example, inSaccharomyces cerevisiae, carbon stress-induced gluconeogenesis is terminated upon glucose availability, a process that involves the multiprotein E3 ligase, GIDSR4, recruiting N-termini and catalyzing ubiquitylation of gluconeogenic enzymes. Here, genetics, biochemistry, and cryo electron microscopy define molecular underpinnings ofglucose-induceddegradation. Unexpectedly, carbon stress induces an inactive anticipatory complex (GIDAnt), which awaits a glucose-induced substrate receptor to form the active GIDSR4. Meanwhile, other environmental perturbations elicit production of an alternative substrate receptor assembling into a related E3 ligase complex. The intricate structure of GIDAntenables anticipating and ultimately binding various N-degron targeting (i.e. “N-end rule”) substrate receptors, while the GIDSR4E3 forms a clamp-like structure juxtaposing substrate lysines with the ubiquitylation active site. The data reveal evolutionarily conserved GID complexes as a family of multisubunit E3 ubiquitin ligases responsive to extracellular stimuli.