Fig 2: Structural basis of orphan recognition by the ZNRD2-HERC2 complex(A) AlphaFold-predicted model of a putative ternary complex consisting of CCT4 (pink), ZNRD2 (blue), and the RLD3 domain of HERC2 (green). The position of a C-terminal two-helix domain of ZNRD2 was not predicted with high confidence, so this element, the flexible linker attaching it to the N-terminal domain, and the unstructured N-terminal tail, are not shown.(B) Interface between CCT4 and ZNRD2 in the predicted model. CCT4 is shown in surface representation and colored by hydrophobicity. ZNRD2 is in cartoon representation and colored by conservation. The close-up view shows four highly conserved residues of ZNRD2 that occupy a hydrophobic patch of CCT4 also found in other CCT subunits.(C) Interface between ZNRD2 and RLD3 of HERC2 in the predicted model. RLD3 is shown in surface representation and colored by electrostatic potential, while ZNRD2 is shown as in (B). The close-up views show three highly conserved residues of ZNRD2 that mediate this predicted interaction.(D) ZNRD2-3xFLAG constructs containing point mutations in the residues, highlighted in (B) and (C), were transiently expressed in ZNRD2-KO cells and analyzed for interactions with endogenous HERC2, CCT4, and CCT7 by non-denaturing IP with anti-FLAG affinity resin. Input and IP samples were subjected to immunoblotting as indicated.(E) CCT4 reporter degradation was analyzed by flow cytometry in WT cells (filled gray), ZNRD2-KO cells (blue), and ZNRD2-KO cells transiently re-expressing WT (red) or mutant ZNRD2-3xFLAG constructs. The mutants are the same as those analyzed in (D).See also Figures S6 and S7.

Fig 3: Degradation of unassembled chaperonin subunits requires the E3 ligase HERC2(A) Cells treated with control or HERC2-targeting siRNAs were transfected with the dual-color CCT4 reporter and analyzed by immunoblotting (left) and flow cytometry (right).(B) Cells treated with the indicated siRNAs were transfected with the CCT4 reporter and siRNA-resistant 3xFLAG-HERC2 (wild type [WT]) or its catalytically inactive mutant (C4762S), as indicated. Cells were analyzed by immunoblotting (left) and flow cytometry (right).(C) Cells were transfected with CCT2-targeting siRNAs, together with or without HERC2-targeting siRNAs, as indicated. Total cell lysates were prepared at 72 h post-transfection and analyzed by immunoblotting for the indicated proteins.(D) Flp-In T-REx 293 cells stably integrated with an inducible 3xFLAG-HERC2 construct (WT), its catalytically inactive mutant (C4762S), or empty vector as a control (-) were treated with doxycycline to induce the expression of each HERC2 construct (see Figure S3E). Cytosolic extracts (input) were separated on a 5%–45% sucrose gradient and analyzed by immunoblotting for CCT4 and CCT7. Unassembled CCT subunits migrate in fractions 2–4, whereas assembled CCT migrates in fractions 7–9.(E) MCF7 cells treated with control or HERC2-targeting siRNAs were subjected to a pulse-chase experiment with 35S-methionine to monitor endogenous CCT7 degradation (see Figure S3G). Top panels show immunoblots of cell lysates for HERC2 and ß-actin. The graph shows the proportion of radiolabeled CCT7 degraded after a 4-h chase. Data show mean ± SD, along with individual data points of 3 replicates. p = 0.005 by unpaired two-tailed t test.(F) Cytosolic extracts of MCF7 cells treated with control or HERC2-targeting siRNAs were separated by native PAGE and immunoblotted for CCT7. Two exposures of the blot are shown.See also Figure S3.

Fig 4: Physiological relevance of the HERC2-ZNRD2 pathway(A) ZNRD2-KO Flp-In T-REx 293 cells were complemented with either empty vector or the indicated ZNRD2 constructs at the unique FRT site in their genome. These cells were transfected with the CCT4 reporter and analyzed by immunoblotting (left) and flow cytometry (right). Wild-type (WT) Flp-In T-REx 293 cells were analyzed in parallel for comparison.(B) The exogenous ZNRD2 sequence at the FRT site was PCR-amplified from the genomic DNA of ZNRD2-KO cells complemented with ZNRD2 WT or its mutants (K82A or L42E), and the amplicons were sequenced. Shown are sequencing chromatograms for the WT and mutant cells at the regions around the K82 and K42 codons.(C) ZNRD2-KO cells expressing ZRND2 WT were mixed at a ratio of 1:1 with ZNRD2-KO cells expressing either the K82A mutant (left; WT vs. K82A) or the L42E mutant (right; WT vs. L42E) and then cultured for 4 weeks. An aliquot of each co-culture was sampled at the indicated time points, and the exogenous ZNRD2 sequences at the FRT site were amplified and sequenced as in (B). Sequencing chromatograms around the K82 and L42 codons are shown for the co-cultures containing the respective mutant cell lines.(D) Interface between ZNRD2 and RLD3 of HERC2 in the predicted structural model. RLD3 is shown in gray surface representation, while ZNRD2 is in blue cartoon representation. The close-up view highlights the E3994 residue (red) of HERC2.(E) HEK293T cells transiently expressing the indicated 3xFLAG-HERC2 constructs were subjected to anti-FLAG IP under non-denaturing conditions. Input and IP samples were analyzed by immunoblotting for the indicated proteins. The asterisk indicates a cross-reactive nonspecific band.(F) HERC2-KO cells were transfected with the CCT4 reporter (left) or the NCOA4 reporter (right), together with the indicated plasmids, and analyzed by flow cytometry.(G) Model for quality control (QC) during CCT biogenesis. Depending on the step at which biogenesis fails, qualitatively different types of QC act on the substrate. This includes ribosome-associated quality control (RQC) if CCT translation fails, protein QC if a CCT subunit fails to fold after translation, and assembly QC for several CCT subunits that fold but do not assemble into the CCT. Unassembled CCT subunits can be recognized by ZNRD2, which engages the RLD3 domain of the E3 ligase HERC2, and ubiquitinates the subunit(s) to target it for degradation. ZNRD2 can also recognize partially assembled complexes, but the triage of these complexes between degradation and assembly remains to be investigated. ZNRD2 cannot engage assembled CCT because its interaction surfaces are buried.

Fig 5: Interactors of unassembled chaperonin subunits destined for degradation(A) HEK293T cells were transfected with a GFP-RFP dual-color reporter of orphan CCT4 degradation (red; see Figure S1A) or a control GFP-RFP reporter lacking the CCT4 insert (black), then analyzed by flow cytometry. CCT4 is fused to GFP, with RFP serving as a translation control. Shown are overlaid scatter plots of individual transfected cells (top) and the corresponding histograms of the GFP:RFP ratio (bottom).(B) Cells were treated with control or CCT2-targeting small interfering RNAs (siRNAs) for 72 h, and total cell lysates were analyzed by immunoblotting for the proteins indicated on the left.(C) CCT subunits containing a C-terminal TwinStrep tag (TST) or 3xFLAG tag were translated in rabbit reticulocyte lysate (RRL) containing 35S-methionine and His-tagged ubiquitin (His-Ub). Samples were analyzed directly (total IVT, in vitro translation) or after ubiquitin pull-down under denaturing conditions via the His-tag (His-Ub PD).(D) CCT4-TST was translated in RRL, affinity-purified under native conditions, and analyzed by label-free quantitative mass spectrometry. Proteins in the upper right quadrant are significantly enriched with CCT4.(E) Cells transiently expressing 3xFLAG-tagged CCT subunits were subjected to anti-FLAG immunoprecipitation (IP) under non-denaturing conditions. Input and IP samples were analyzed by immunoblotting for the indicated proteins.See also Figures S1 and S2.