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: Recognition of orphan CCT subunits by ZNRD2, related to Figures 5 and 6(A) The CCT1 (top), CCT4 (middle), and CCT7 (bottom) reporters were assessed for degradation by flow cytometry in WT cells, ZNRD2-KO cells (transfected with empty vector), and ZNRD2-KO cells transiently re-expressing WT or mutant ZNRD2 constructs, as indicated.(B) Cells were transfected with CCT2-targeting siRNA together with or without ZNRD2-targeting siRNA, as indicated. The following day, cells were transfected with GFP together with or without the indicated ZNRD2 constructs and then cultured for another 2 days. After sorting GFP-positive cells to enrich the transfected cells, total cell lysates were prepared and analyzed by immunoblotting for the indicated proteins.(C) Interface between CCT4 and ZNRD2 in the predicted CCT4-ZNRD2-RLD3 model. CCT4 is shown as black cartoon with transparent surface representation colored by hydrophobicity. ZNRD2 is shown in blue cartoon representation. Highlighted are the residues on the hydrophobic patch in CCT4 that forms part of the predicted ZNRD2 interaction surface.(D) The indicated CCT4-TST constructs were translated in RRL, affinity-purified under native conditions, and analyzed by immunoblotting as indicated. Three amounts of total RRL were analyzed in parallel. The AAA mutant contains I77A, M81A, and V83A mutations.(E) The indicated CCT4 reporter constructs were expressed in WT cells and assessed for degradation by flow cytometry.(F) Cartoon representation of CCT4 in the predicted CCT4-ZNRD2-RLD3 model. The inset shows a close-up view highlighting the residues deleted or mutated in the misfolding CCT4 mutants used in (G) and (H).(G) The indicated CCT4-TST constructs were translated in RRL, affinity-purified under native conditions, and analyzed by immunoblotting as indicated. Three amounts of total RRL were analyzed in parallel.(H) The indicated CCT4 reporter constructs were assessed for degradation by flow cytometry in WT and ZNRD2-KO cells.(I) AlphaFold-predicted model of the CCT4-ZNRD2-RLD3 complex was docked onto an experimentally determined structure of the CCT complex (PDB: 7lum) by aligning CCT4 in the prediction with one of the two CCT4 chains in the experimental structure. CCT1 (light green), CCT2 (light yellow), and CCT4 (light coral) from the experimentally determined structure, and ZNRD2 (blue) from the predicted model, are shown in cartoon representation. A close-up view shows that the N-terminal tail of CCT1 (magenta) might clash with ZNRD2 if it were to remain in the position seen in fully assembled CCT. Given the minimal interactions made by this tail (and the adjacent C-terminal tail), we posit that they could easily move out of the way to permit ZNRD2 binding to CCT4, even when CCT4 is flanked by adjacent CCT subunits.

Fig 5: HERC2 recognizes chaperonin subunits using the adaptor ZNRD2(A) Cells treated with siRNAs against HERC2 or ZNRD2 were transfected with CCT4-3xFLAG, followed by non-denaturing IP using anti-FLAG affinity resin. Input and IP samples were analyzed by immunoblotting for the indicated proteins.(B) Cytosolic extracts from cells treated with siRNAs targeting HERC2, ZNRD2, or control were fractionated by sucrose gradient centrifugation to quantify the proportion of unassembled CCT4 and CCT7 subunits (see Figures S4A and S4B). Data are mean ± SD of data points (dots) from three independent experiments (color-coded). **p < 0.01 and ***p < 0.001 vs. control siRNA by one-way ANOVA with Dunn’s multiple comparisons test.(C) Cells treated with the indicated siRNAs were transfected with the dual-color CCT4 reporter and analyzed by immunoblotting (left) and flow cytometry (right).(D) Cells treated with siRNAs against ZNRD2 were transfected with the CCT4 reporter and siRNA-resistant ZNRD2, followed by immunoblotting (left) and flow cytometry (right).(E) Wild-type (WT) and ZNRD2-knockout (KO) Flp-In T-REx 293 cells were transfected with the dual-color CCT4 reporter and either 3xFLAG-HERC2, ZNRD2, or empty vector. Cells were analyzed by immunoblotting (top) and flow cytometry (bottom).(F) Diagram illustrating the domain structure of human HERC2 (4,834 amino acids) and overlapping HERC2 fragments tested for interaction with ZNRD2 in Figure S4G. HERC2 contains three RCC1-like domains (RLD1–3) and a C-terminal HECT E3 ligase catalytic domain (HECT). The HERC2 fragment containing RLD3 (F5; highlighted in red) interacts with ZNRD2 (see Figure S4G).(G) Cells treated with HERC2-targeting siRNA were transfected with the CCT4 reporter together with empty vector, siRNA-resistant 3xFLAG-HERC2 (WT), or its deletion mutant lacking RLD3 (?RLD3). Cells were analyzed by immunoblotting (left) and flow cytometry (right).See also Figure S4.