Highlights d Genetics, biochemistry, proteomics, and cryo-EM define GID E3 ligase regulation d Carbon stress induces assembly of an inactive anticipatory GID Ant complex d Environmental perturbations trigger substrate receptor assembly into active GID E3s d Structural model of N-degron substrate ubiquitylation by multisubunit RING-RING E3

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-GID SR4 , which resembles an organometallic supramolecular chelate. The Chelator-GID SR4 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.

Qiao et al. (Schulman) -Interconversion between anticipatory and active GID E3 ubiquitin ligase conformations via metabolically-driven substrate receptor assembly 2 SUMMARY Cells respond to environmental changes by toggling metabolic pathways, preparing for homeostasis, and anticipating future stresses. For example, in Saccharomyces cerevisiae, carbon stress-induced gluconeogenesis is terminated upon glucose availability, a process that involves the multiprotein E3 ligase, GID SR4 , recruiting N-termini and catalyzing ubiquitylation of gluconeogenic enzymes. Here, genetics, biochemistry, and cryo electron microscopy define molecular underpinnings of glucose-induced degradation. Unexpectedly, carbon stress induces an inactive anticipatory complex (GID Ant ), which awaits a glucoseinduced substrate receptor to form the active GID SR4 . Meanwhile, other environmental perturbations elicit production of an alternative substrate receptor assembling into a related E3 ligase complex. The intricate structure of GID Ant enables anticipating and ultimately binding various N-degron targeting (i.e. "N-end rule") substrate receptors, while the GID SR4 E3 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.

Highlights d Ede1 is an intrinsic autophagy receptor for aberrant CME protein assemblies d Aberrant CME assemblies form by liquid-liquid phase separation d Liquid-liquid phase separation of Ede1 is important for autophagic degradation d Ultrastructural view of an LLPS compartment at the PM and within autophagic bodies

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