The human GID complex engages two independent modules for substrate recruitment

Abstract

The human GID (hGID) complex is a conserved E3 ubiquitin ligase regulating diverse biological processes, including glucose metabolism and cell cycle progression. However, the biochemical function and substrate recognition of the multi-subunit complex remain poorly understood. Using biochemical assays, cross-linking mass spectrometry, and cryo-electron microscopy, we show that hGID engages two distinct modules for substrate recruitment, dependent on either WDR26 or GID4. WDR26 and RanBP9 cooperate to ubiquitinate HBP1 in vitro, while GID4 is dispensable for this reaction. In contrast, GID4 functions as an adaptor for the substrate ZMYND19, which surprisingly lacks a Pro/N-end degron. GID4 substrate binding and ligase activity is regulated by ARMC8a, while the shorter ARMC8b isoform assembles into a stable hGID complex that is unable to recruit GID4. Cryo-EM reconstructions of these hGID complexes reveal the localization of WDR26 within a ringlike, tetrameric architecture and suggest that GID4 and WDR26/ Gid7 utilize different, non-overlapping binding sites. Together, these data advance our mechanistic understanding of how the hGID complex recruits cognate substrates and provides insights into the regulation of its E3 ligase activity.

Structural and Functional Insights into GID/CTLH E3 Ligase Complexes

“…Firstly, muskelin is not encoded in the yeast genome and there is only one gene, instead of the two paralogues, for RanBP9/10 and RMND5A/B [ 11 ]. Second, in contrast to the S. cerevisiae complex, interchangeable substrate receptors with GID4 have yet to be identified in other eukaryotes, although evidence of substrate engagement independent of GID4 has emerged [ 20 ]. Finally, although yeast Gid5 is not a true homologue of ARMC8 [ 21 ], structurally they are similar [ 15 ].…”

“…Finally, although yeast Gid5 is not a true homologue of ARMC8 [ 21 ], structurally they are similar [ 15 ]. An important difference, however, is that two ARMC8 isoforms (α and β), instead of one Gid5 in yeast, associate with the human complex [ 10 , 22 , 23 ], but only the α isoform can bind GID4 [ 20 ]. The presence of these differing components in other species likely gives the CTLH complex distinct functionalities as compared with the S. cerevisiae GID complex.…”

“…In general, these repeats fold together to form a superhelix of helices that serves as a versatile protein interaction surface ( Figure 2 f) [ 31 ]. In the GID/CTLH complex, one-half of the Gid5/ARMC8 ARM repeats engages Gid1/RanBP9–GID8/TWA1 in the scaffold, whereas the other half anchors GID4 [ 12 , 15 , 20 ].…”

“…At present, however, a substrate recruitment function has only been demonstrated for the yeast Gid4/Gid10 β-barrel. Interestingly, the WDR26 β-propeller has been proposed to act as a substrate receptor for the complex binding target HBP1 [ 20 ], although this awaits structural validation. Perhaps muskelin also recruits substrates, either via its kelch repeat β-propeller or its discoidin domain, which bears resemblance to the Gid4/Gid10 β-barrel.…”

“…For human proteins, the monomeric complex architecture is similar to the yeast complex where a RanBP9–GID8–ARMC8 scaffold replaces Gid1–Gid8–Gid5, the RMND5A-MAEA RING module replaces the Gid2–Gid9 RING heterodimer, and human GID4 is a presumed substrate receptor ( Figure 3 c) [ 15 , 20 ]. The human complex can also oligomerize and adopt a ring or oval-like shape with WDR26, the homologue of Gid7, mediating dimerization interfaces and being required for higher-order assemblies of the CTLH complex in vivo ( Figure 3 d) [ 15 , 20 ]. Unique to the human complex, tetrameric muskelin directly binds RanBP9–GID8, similarly to WDR26, although currently it is unclear in vivo if there are WDR26- or muskelin-exclusive complexes ( Figure 3 e) [ 15 ].…”