Fig 2: Biochemical characterization of the hGID sub‐complexes A–FSize‐exclusion profiles of the indicated hGID sub‐complexes. In the schematic representation, the catalytic core subunits composed of MAEA, RMND5a, and TWA1 is colored in blue, WDR26 in green, RanBP9 in light magenta, ARMC8 in dark red, and ZMYND19 and HBP1 in gray.G In vitro pull‐down assay of Strep‐RanBP9 and His‐WDR26 co‐expressed in baculoviral Sf9 cells, demonstrating the formation of a stable complex (n = 2).HWestern blot analysis of HBP1 in vitro ubiquitination in the presence of a hGID complex lacking the RanBP9‐WDR26 module (n = 3).ISize‐exclusion profiles of the different endogenous hGID subunits in HeLa cells analyzed by the SEC‐Explorer web platform (Heusel et al, 2019). Source data are available online for this figure.

Fig 3: 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 4: Biochemical and structural characterization of the 6‐subunit hGID complex and its ability to recruit GID4 subunit In vitro pull‐down assay of His‐GID4 and His‐ARMC8α from baculoviral Sf9 extracts co‐expressing Strep‐RanBP9, His‐WDR26, FLAG‐MAEA, His‐RMND5a, and His‐TWA1. ARMC8α is required to recruit GID4 into the hGID complex (n = 3).Conservation between human ARMC8α and yeast Gid5 in the region required for GID4 binding using Jalview (Waterhouse et al, 2009) and following the Clustalx color scheme (http://www.jalview.org/help/html/colourSchemes/clustal.html).Baculoviral co‐expression in Sf9 cells of Strep‐GID4 and His‐ARMC8α or His‐ARMC8β. Note that ARMC8α but not ARMC8β forms a stable complex with GID4 (n = 3).Comparison of the size‐exclusion chromatograms from Superose 6 column (Cytiva) of the 5‐subunit (RanBP9, WDR26, RMND5a, MAEA, and TWA1) and the 6‐subunit (RanBP9, WDR26, RMND5a, MAEA, TWA1, and ARMC8β) hGID complexes.Single particle image processing scheme used to determine the difference map between 5‐subunit and ARMC8β‐containing 6‐subunit GID complexes. The processing steps of the GID 5‐subunit (left) and GID‐ARMC8β 6‐subunit complexes (right) were carried out with identical settings. The FSC plot shows the masked (blue), masked corrected (black), and phase randomized mask FSC (red).2D class averages of the 5‐subunit GID (top) and GID‐ARMC8β complexes (bottom). The 10 most populated classes (of 100) are shown, ordered by occupancy. Scale bar: 20 nm. Additional density is visible in the 2D classes of the 6‐subunit hGID corresponding to ARMC8β (white arrows). Source data are available online for this figure.

Fig 5: The hGID complex uses distinct substrate modules to target different substrates AImmunoblot of cell extracts following depletion of WDR26, RanBP9, ARMC8, and GID4 using pools of siRNAs for 72 h in HeLa Kyoto cells. Endogenous levels of the indicated proteins were monitored by Western blotting (n = 3).B, CWestern blotting of samples after ectopic overexpression of HBP1 (B) or ZMYND19 (C) alone, or together with WDR26 or GID4 in HEK‐293T cells. HBP1 and ZMYND19 levels were monitored after treatment of MG132 or DMSO for 10–12 h (n = 3).DSchematic representation visualizing the hGID E3 ligase complex using two distinct modules for substrate recruitment. Source data are available online for this figure.