Recognition of the CCT5 di-glu degron by CRL4(DCAF12) is incompatible with TRiC assembly

The molecular processes within all living organisms rely on the correct functioning of proteins, most of which must assemble into multimeric complexes of defined architecture and composition. In eukaryotes, assembly quality control (AQC) E3 ubiquitin ligases target incomplete or incorrectly assembled protein complexes for degradation to ensure protein complex functionality and proteostasis. The CUL4-RBX1-DDB1-DCAF12 (CRL4(DCAF12)) E3 ubiquitin ligase induces the proteasomal degradation of proteins with a C-terminal double glutamate (di-Glu) motif. Putative CRL4(DCAF12) substrates include CCT5, a subunit of the eukaryotic TRiC chaperonin. TRiC is responsible for the folding of around 10% of the human proteome. Its functionality relies on the correct arrangement of its eight subunits, but how TRiC assembly is ensured has not yet been investigated. Furthermore, how DCAF12 recognizes its substrates is unknown. Here the cryo-EM structure of the CCT5-bound DDB1-DCAF12 complex at 2.8 Å resolution is presented. DCAF12 serves as a canonical WD40 DCAF substrate receptor and uses a positively charged pocket at the center of its β-propeller to bind the C-terminus of CCT5. DCAF12 specifically reads out the CCT5 di-Glu side chains, and contacts other visible degron amino acids through weaker Van der Waals interactions, explaining the flexibility in substrate recognition. The CCT5 C-terminus is inaccessible in an assembled TRiC complex, and functional assays demonstrate that CRL4(DCAF12) binds and ubiquitinates monomeric CCT5, but not TRiC. The presented results suggest a previously unanticipated AQC role for the CRL4(DCAF12) E3 ligase towards TRiC, and likely other complexes.