Fig 2: 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 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: Multiple types of orphaned proteins depend on the UBR4-KCMF1 complex for efficient degradation(A) HEK293T cells treated with control, KCMF1, UBR4, HERC2, or HUWE1-targeting siRNAs were transiently transfected with CCT4, NCOA4, or UBL4A fluorescent reporters, as indicated. Cells were then analyzed by flow cytometry (top) or immunoblot (bottom).(B) WT and ΔHERC2 cells treated with control or UBR4-targeting siRNAs were co-transfected with the CCT4-GFP reporter and HERC2, as indicated, and analyzed by flow cytometry.(C) Cells treated with control, HERC2, or UBR4-targeting siRNAs were transfected with the CCT4-GFP reporter and analyzed by flow cytometry (left) or immunoblot (right).(D) HEK293T cells treated with control, HERC2, KCMF1, or UBR4-targeting siRNA were transfected with CCT4-3xFLAG, then subjected to anti-FLAG IP under native conditions. Input and IP samples were analyzed by immunoblot.(E) HEK293T cells were transfected with CCT2-targeting siRNA together with or without KCMF1-targeting siRNAs as indicated. Total cell lysates were prepared at 72 h post-transfection and analyzed by immunoblotting for the indicated proteins.(F) HEK293T cells were transfected with CCT2-targeting siRNAs together with or without UBR4-targeting siRNAs. Total cell lysates were analyzed as in (E). See also Figure S6.