Fig 2: ARMC8α but not ARMC8β recruits GID4 to the core complex in an assembly that does not prevent binding of the WDR26/RanBP9 module Schematic representation of ARMC8α (Q8IUR7‐1), ARMC8β (Q8IUR7‐6), and GID4 (Q8IVV7‐1) proteins. The binding site of GID4 and the C‐terminus of ARMC8α is indicated.Western blot analysis showing the levels of ARMC8α and ARMC8β in HeLa Kyoto cells treated for 72 h with control siRNA or siRNA pools against ARMC8 (n = 3).Transiently expressed FLAG‐GID4 and HSS‐tagged ARMC8 isoforms (α or β) in HEK‐293T cells. The presence of GID4 in isoform‐specific ARMC8 immunoprecipitates was visualized by immunoblotting (left panels). The right panel shows a Western blot of transiently expressed and immunoprecipitated HSS‐WDR26 from HEK‐293T cells, and the presence of endogenous ARMC8 isoforms (α or β) was probed by immunoblotting (n = 2).Baculoviral co‐expression in Sf9 cells of the 5‐subunit hGID complex (5mer; His‐RanBP9, His‐WDR26, FLAG‐MAEA, His‐RMND5a, and His‐TWA1) along with His‐GID4 in the presence of Strep‐ARMC8α or Strep‐ARMC8β. Strep‐ or His‐pulldowns revealed the presence of GID4 in ARMC8α, but not in ARMC8β, complexes (n = 3).Immunoblot analysis of in vitro ubiquitinated GID4‐hGID complexes containing either ARMC8α or ARMC8β (6mer; ARMC8, RanBP9, WDR26, MAEA, RMND5a, and TWA1). Where indicated, the reaction was carried out in the presence of 20‐fold molar excess of the PGLV GID4‐binding peptide (n = 3).Western blot analysis of in vitro ubiquitinated HBP1 by the hGID 5‐subunit complex in the presence of the different ARMC8 isoforms (α or β; n = 2).Schematic representation illustrating that in contrast to ARMC8α, incorporation of ARMC8β prevents hGID activity toward GID4 substrates.XL‐MS analysis of the 6‐subunit hGID complex (RanBP9, WDR26, RMND5a, MAEA, TWA1, and ARMC8β). Cross‐links within the different complex subunits are indicated by green lines and cross‐links within the same subunit by purple lines. The predicted domain boundaries within the different subunits are colored as follows: LisH domain in light orange, CTLH domain in dark orange, RING domains in blue, TWA1's CRA domain in light blue, RanBP9's CRA domain in light gray, WD40 in dark cyan, ARMC8β in dark red, and the SPRY domain in light magenta.Comparison of the cryo‐EM maps of the 5‐subunit hGID complex (RanBP9, WDR26, RMND5a, MAEA, and TWA1) and the 6‐subunit hGID complex (RanBP9, WDR26, RMND5a, MAEA, TWA1, and ARMC8β).A difference map (red) shows the extra density in the 6‐subunit hGID complex corresponding to ARMC8β. Source data are available online for this figure.

Fig 3: Inter‐subunit interactions within the hGID sub‐complexes The fitted homology model of RanBP9 (SPRY and LisH domain, magenta), TWA1 (blue), ARMC8β (dark red), and WD40 (dark cyan) in the 9 Å map of the 5‐subunit hGID complex (gray; left panel). The observed cross‐links between the different residues are indicated by black lines. Schematic architecture and domain representation of the 5 subunits (RanBP9, WDR26, RMND5a, MAEA, and TWA1) are shown to the right.SDS–PAGE showing in vitro pull‐down assays using baculoviral Sf9 co‐expression of Strep‐WDR26 and His‐TWA1. Note that WDR26 and TWA1 directly interact (n = 2).SDS–PAGE showing in vitro pull‐down assays using baculoviral Sf9 co‐expression of Strep‐ARMC8α, which forms a complex with His‐TWA1 and His‐GID4 (lanes 1 and 2) but not with FLAG‐MAEA and His‐RMND5a (lanes 3 and 4). His‐RanBP9 does not bind Strep‐ARMC8α and His‐GID4 (lanes 5 and 6; n = 2).His‐RanBP9 does not interact with FLAG‐MAEA and Strep‐RMND5a (lanes 1 and 2), but Strep‐RanBP9 forms a complex with His‐TWA1 (lanes 3 and 4; n = 3).FLAG immunoprecipitation (FLAG IP) of extracts prepared from HEK‐293T cells transiently transfected with an empty control plasmid or a plasmid overexpressing FLAG‐GID4. The presence of endogenous WDR26 in the extract (input) or bound to GID4 was probed by immunoblotting. GAPDH was used as a specificity control (n = 2). Source data are available online for this figure.

Fig 4: Compromised GID activity results in an intermittent cellular growth defect.(A) Cell proliferation of RPE control and MAEA-KO cells was quantified with MTT assays between days 6 and 9 after lentiviral transfection. Data are shown as mean of triplicates and % change in signal relative to control gRNA-treated cells ± SD, n = 3, ∗∗∗p≤0.0006. (B) In vitro autoubiquitination assay of native GID particles isolated via the stably expressed HSS-tagged RING protein Rmnd5a from HEK-293 control or MAEA-KO cells in the presence of Ube2H. Deletion of MAEA results in complete loss of the catalytic activity of the GID complex as shown by immunoblotting using an antibody directed against Ubiquitin. (C) Cell proliferation of RPE MAEA-KO and WDR26-KO cells was measured by MTT assays between days 6–9 (naïve) and 15–17 (adapted) after lentiviral transfection. Data are shown as mean of triplicates and % change in signal relative to control gRNA-treated cells ± SD, n =≥2, ∗∗p≤0.0016, ∗∗∗p≤0.0007. (D) Cell extracts prepared from naïve and adapted RPE MAEA-KO and WDR26-KO cells were analyzed for cell cycle and growth pathway markers by immunoblotting. Note that the adapted GID-KO cells overcome the proliferation defect as judged by the presence of phosphorylated Histone H3, Rb (S780 and S807/811) and restored levels of the cell cycle markers Cyclin A and Cyclin D1. (E) Untreated (naïve) control and MAEA-KO RPE cells express comparable FBP1 protein levels in contrast to the autoregulated and stabilized GID subunit YPEL5. (F and G) The murine GID complex is dispensable for degradation of the gluconeogenic enzymes FBP1 and PCK1 in HEK-293 and primary murine hepatocytes. Cells were starved after control and GID siRNA treatment before switching them back to glycolytic conditions. Samples for immunoblot analysis were taken at the indicated time points and probed for FBP1 and PCK1 protein levels (n = 2). The metabolic switch in these cells was controlled by phosphorylation of Akt (Ser473) and the efficiency of GID complex depletion was probed using an antibody directed against the subunit RanBP9 of the complex.

Fig 5: WDR26/RanBP9‐containing hGID complexes assemble ring‐shaped tetramers Chromatogram of the SEC‐MALS analysis at a flow rate of 0.5 ml/min, showing the UV curve and the Rayleigh ratio (1/cm) at a scattering angle of 90 degrees (left y‐axis), together with the molar mass (MDa) of the peaks determined by MALS (right y‐axis). The peak fraction with a homogenous size distribution at around 1.1 MDa is labeled with gray dotted lines.XL‐MS analysis of the 5‐subunit hGID complex (RanBP9, WDR26, RMND5a, MAEA, and TWA1). Cross‐links within different complex subunits are indicated by green lines and cross‐links within the same subunit with purple lines. The predicted domain boundaries of the different subunits are colored as follows: LisH domain in light orange, CTLH domain in dark orange, RING domains in blue, TWA1's CRA domain in light blue, RanBP9's CRA domain in light gray, WD40 in dark cyan, and SPRY in light magenta.Rotational views of the cryo‐EM map of the 5‐subunit hGID complex (RanBP9, WDR26, RMND5a, MAEA, and TWA1) at 11.2 Å resolution. The higher resolution cryo‐EM map at 9 Å produced by particle symmetry expansion is shown in blue. The dotted rectangle highlights the positions of the fitted WD40 domains from two tetrameric building blocks.Western blotting of samples following ectopic overexpression of HBP1 either alone, or with full‐length (FL) or WD40‐truncated WDR26 (ΔWD40) in HEK‐293T cells. HBP1 levels were monitored in cells treated with MG132 or DMSO for 12–14 h (n = 3).Western blot analysis of in vitro ubiquitinated HBP1 in the presence of wild‐type WDR26 (WT) or the WDR26 (ΔWD40) mutant (n = 2). Source data are available online for this figure.