Structural and Functional Insights into GID/CTLH E3 Ligase Complexes

Abstract

Multi-subunit E3 ligases facilitate ubiquitin transfer by coordinating various substrate receptor subunits with a single catalytic center. Small molecules inducing targeted protein degradation have exploited such complexes, proving successful as therapeutics against previously undruggable targets. The C-terminal to LisH (CTLH) complex, also called the glucose-induced degradation deficient (GID) complex, is a multi-subunit E3 ligase complex highly conserved from Saccharomyces cerevisiae to humans, with roles in fundamental pathways controlling homeostasis and development in several species. However, we are only beginning to understand its mechanistic basis. Here, we review the literature of the CTLH complex from all organisms and place previous findings on individual subunits into context with recent breakthroughs on its structure and function.

Keywords: CTLH complex; E3 ligase; GID complex; GID4; RMND5A; RanBP9/RanBPM; WDR26; muskelin; ubiquitination.

Figure 1

GID/CTLH subunits from yeast to human. Green checkmark or X indicates an orthologue is present or absent, respectively. Paralogues which map to the same gene have a colour-coded checkmark: blue for RanBP9/RanBP10 and yellow for RMND5A/RMND5B. For YPEL5, the light blue checkmark indicates Yipee-like proteins co-purified with RanBPM in Arabidopsis thaliana. The yeast and human protein names are indicated on the left and right, respectively. Created with Biorender.com (accessed on 21 April 2022).

Figure 2

Structure of GID/CTLH subunits. (a) Lis1 lissencephaly type-1-like homology (LisH) dimer (PDB: 1UUJ). (b) Smu1 C-terminal to LisH (CTLH) motif (PDB: 5EN8). (c) TOPLESS CT11-RanBPM (CRA) motif (PDB: 5NQV). (d) Structure of SMU1 LisH-CTLH-CRA dimer. LisH (light blue, blue), CTLH (orange, gold), and CRA (violet, pink) in each monomer are shown (PDB: 5EN8). (e) Domain organization of GID/CTLH subunits. Scale at the top reflects residue number. All proteins depicted are the human versions, except for Gid7, Gid10, and Gid11, which are S. cerevisiae. Legend below denotes the names of each domain and the corresponding symbol, which is representative of the domain structure. Created with Biorender.com (accessed on 21 April 2022). (f) Structure of the RanBP9 (blue)–GID8 (gold)-ARMC8α (red/green) scaffold in the human CTLH complex. ARMC8 is split into two colours where red represents structure shared between α and β isoforms, whereas green is only present in α. PDB: 7NSC. (g) Structure of the S. cerevisiae Gid2 (homologue of RMND5A/B) and Gid9 (homologue of MAEA) RING heterodimer. Zinc ions are coloured yellow (PDB: 7NS4). (h) Human GID4 β-barrel structure in complex with a PGLW peptide. (PDB: 6CDC). (i) S. cerevisiae Gid11 β-barrel structure in complex with a PFITSRPW peptide (7QQY) (j) Structure of the S. cerevisiae Gid7 (homologue of WDR26) dimer structure (PDB: 7NSB). (k) Structure of the N-terminus of a muskelin dimer encompassing the discoidin domain and first helix of the LisH (PDB: 4OYU).

Figure 3

Architecture and assemblies of GID/CTLH complexes. (a) Schematic representation of the architecture of the monomeric S. cerevisiae Gid complex (without Gid7) binding its cognate E2 Ubc8 and dimeric substrate Mdh2 via N-terminal proline binding the Gid4 β-barrel. (b) Supramolecular chelator S. cerevisiae GID complex (with Gid7) that encircles its tetrameric substrate Fbp1. (c) Architecture of monomeric human CTLH complex (without WDR26 or muskelin) and its cognate E2 UBE2H binding a substrate via GID4. (d) Oligomeric assembly of the human CTLH complex containing WDR26. (e,f) Examples of possible CTLH supramolecular complexes. Additional assemblies may also be formed with RanBP10 and RMND5B paralogues, and different combinations of WDR26, muskelin, and ARMC8 isoforms. For structural details, see text and Qiao et al., 2019, and Sherpa et al., 2021. Created with Biorender.com (accessed on 21 April 2022).

Figure 4

In S. cerevisiae, gluconeogenic enzymes undergo distinct glucose-induced, GID complex-dependent degradation mechanisms. Left: 1. In cells starved of glucose for 24 hours, glucose replenishment triggers Gid4 induction. 2. Gid4 associates with the GID complex via Gid5 and recruits gluconeogenic enzymes to the complex. 3. Gluconeogenic enzymes are ubiquitinated by the GID complex via the E2 enzyme Ubc8. This requires the Hsp70 chaperone Ssa1. 4. Polyubiquitinated gluconeogenic enzymes are delivered to the proteasome, which involves cdc48, Dsk2, and Rad23. 5. Gluconeogenic enzymes are degraded. Right: During long-term glucose starvation, gluconeogenic enzymes are secreted as extracellular vesicles in the periplasm (*). 1. Upon glucose replenishment, the gluconeogenic enzymes undergo endocytosis and localize at actin patches, which requires End3 and PI3 kinase Vps34. At the same time, Gid4 expression is induced. 2. At actin patches, 30–50 nm membrane-bound vesicles (named Vid vesicles) are formed with the gluconeogenic enzyme in the lumen and the Gid1–Gid4–Gid5–Sec28 complex on the periphery. Importing the gluconeogenic enzyme substrate into the lumen requires Ssa2. 3. The Vid vesicles aggregate and form endosome-like clusters of varying size that disassociate from actin. 4. The vesicles are delivered to the vacuole, which requires cAMP signaling. 5. The gluconeogenic enzymes are degraded in the vacuole, but other Vid vesicle proteins are returned to the cytosol. In both mechanisms, cells adapt back to a normal state of glycolysis. Created with Biorender.com (accessed on 21 April 2022).

Figure 5

The GID/CTLH complexes are implicated in a variety of pathways and biological processes across multiple species. Proteins that have been reported as targets of the complex are indicated with Ub. In vitro confirmed targets have a red outline. Proteins are marked without a Ub symbol if multiple complex members have been implicated in the regulation, but ubiquitination has not been tested. Proteins are marked with a proteasome and/or vacuole if the complex regulates their degradation, or a lightning bolt if the complex regulates their activity. Species in which regulation on the protein by the CTLH complex has been reported are indicated on the right. Created with Biorender.com (accessed on 21 April 2022).

Figure 6

Proposed model of GID/CTLH complex regulation and function in returning to homeostasis. Created with Biorender.com (accessed on 21 April 2022).