GID E3 ligase supramolecular chelate assembly configures multipronged ubiquitin targeting of an oligomeric metabolic enzyme

Abstract

To achieve precise cellular regulation, E3 ubiquitin ligases must be configured to match substrate quaternary structures. Here, by studying the yeast GID complex, mutation of which is Glucose-Induced Degradation deficient, we discover supramolecular chelate assembly as an E3 ligase strategy for targeting an oligomeric substrate. Cryo EM structures show that to bind the tetrameric substrate fructose-1,6-bisphosphatase (Fbp1), two otherwise functional GID E3s assemble into a 20-protein Chelator-GIDSR4, which resembles an organometallic supramolecular chelate. The Chelator-GIDSR4 assembly avidly binds multiple Fbp1 degrons and positions Fbp1 so that its protomers are simultaneously ubiquitylated at lysines near its allosteric and substrate binding sites. Significantly, 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 underlies multipronged targeting, capable of simultaneously incapacitating multiple protomers and functionalities of oligomeric substrates.

The metabolite-controlled ubiquitin conjugase Ubc8 promotes mitochondrial protein import by enhancing assembly of the TOM complex

“…(1) On the one hand, we observed a rapid Ubc8-dependent depletion of the gluconeogenic enzymes upon glucose addition, resulting in the rapid loss of ‘old’ peptides of Fbp1, Mdh2 and Icl1in wild-type but not in Δ ubc8 cells. This is consistent with the established role of Ubc8 in catabolic degradation of gluconeogenic enzymes (Schüle et al, 2000, Chen et al, 2017, Chen et al, 2021, Karayel et al, 2020, Sherpa et al, 2021). Even though Δ ubc8 cells had considerably higher levels of gluconeogenic enzymes, their growth rates remained unaffected even when glucose levels were low.…”

“…Mitochondria isolated from Δ gid4 and Δ gid10 cells also showed reduced Tom22 levels. Gid4 and Gid10 serve as substrate-binding subunits of the GID complex (Chen et al, 2017, Melnykov et al, 2019, Sherpa et al, 2021). Thus, the GID complex, for which Ubc8 serves as the ubiquitin conjugase, increases the levels of the crucial TOM protein Tom22 in mitochondria, which is a rate-limiting factor of the mitochondrial protein import system (Schmidt et al, 2011, Zeng et al, 2019, Shiota et al, 2011, van Wilpe et al, 1999).…”

“…The components of this multi-subunit ubiquitin ligase (E3) were initially identified in a genetic screen for mutants deficient in glucose-induced Fbp1 degradation (Schüle et al, 2000, Hämmerle et al, 1998). A recently solved cryo electron microscopy structure of the GID complex showed that its 20 protein subunits resemble a large organometallic chelator with a central binding site for substrates and the ubiquitin conjugase (E2) Ubc8 (Sherpa et al, 2021). The conserved Ubc8 protein is homologous to other E2 enzymes (Qin et al, 1991) and seems to work exclusively in the context of GID-mediated protein degradation (Kong et al, 2021).…”

A GID E3 ligase assembly ubiquitinates an Rsp5 E3 adaptor and regulates plasma membrane transporters

“…Importantly, both Art2 deletion and an Art2 P2S mutant also showed delayed growth on thialysine (Fig 6A), suggesting that the Art2 deletion effect can at least partly be attributed to regulation by the GID E3 ligase. Furthermore, individual deletions of all GID core subunits, with the exception of Gid7, which is dispensable for some substrates (Negoro et al, 2020;Kong et al, 2021;Sherpa et al, 2021), as well as a double deletion of GID4 and GID10 resulted in cellular toxicity during growth on thialysine, even in the absence of an additional stress condition (Fig 6B and C). The requirement for both SRs (Fig 6C ) suggests that there may be some overlap in SR function in vivo, consistent with the low level of GID SR4 activity observed in the in vitro ubiquitination assay (Fig 4D).…”

The human GID complex engages two independent modules for substrate recruitment

“…Moreover, this interaction was further studied by in vitro ubiquitination, and indeed, this mutant shows a significantly reduced catalytic activity toward HBP1 (Fig 3E), suggesting that the WD40 domain of WDR26 is functionally important. Indeed, the yeast GID complex was shown to be monomeric in the absence of Gid7 (the yeast homologue of WDR26), and addition of Gid7 leads to its oligomeric assembly (Qiao et al, 2019;Sherpa et al, 2021). Taken together, these data demonstrate that the HBP1-degrading hGID complex composed of MAEA, RMND5a, TWA1, WDR26, and RanBP9 forms a ring-like, Fitting the available yeast GID structure (Qiao et al, 2019) into the hGID cryo-EM map confirms that the overall structural fold of the GID complex is conserved between yeast and human.…”