A hierarchical assembly pathway directs the unique subunit arrangement of TRiC/CCT

Abstract

How the essential eukaryotic chaperonin TRiC/CCT assembles from eight distinct subunits into a unique double-ring architecture remains undefined. We show TRiC assembly involves a hierarchical pathway that segregates subunits with distinct functional properties until holocomplex (HC) completion. A stable, likely early intermediate arises from small oligomers containing CCT2, CCT4, CCT5, and CCT7, contiguous subunits that constitute the negatively charged hemisphere of the TRiC chamber, which has weak affinity for unfolded actin. The remaining subunits CCT8, CCT1, CCT3, and CCT6, which comprise the positively charged chamber hemisphere that binds unfolded actin more strongly, join the ring individually. Unincorporated late-assembling subunits are highly labile in cells, which prevents their accumulation and premature substrate binding. Recapitulation of assembly in a recombinant system demonstrates that the subunits in each hemisphere readily form stable, noncanonical TRiC-like HCs with aberrant functional properties. Thus, regulation of TRiC assembly along a biochemical axis disfavors the formation of stable alternative chaperonin complexes.

Keywords: TRiC/CCT; actin folding; assembly intermediates; chaperonin; complex assembly; orphan subunits; protein folding.

Figure 1.. TRiC assembles hierarchically from defined sets of CCT subunits.

A. Unique arrangement of eight CCTs within the TRiC double ring complex and schematic of the knockdown experiment to probe assembly. B. SDS-PAGE immunoblots for each TRiC subunit in HeLa cell lysate following siRNA treatments. NT = non-transfected; siSCR = scrambled siRNA control. Subunits with similar depletion patterns are grouped together. Bottom text indicates knockdown efficiencies. C. Heat map of Pearson correlation coefficients between CCT pairs after knockdowns. D. Fraction of CCT protein remaining after each knockdown (top) normalized to NT control (n=3, errors bars represent SEM, *** = p < 0.0001 using two-way ANOVA and Dunnett’s multiple comparisons test). E. Summary of the TRiC subunit depletion patterns incorporated into a proposed assembly order. See also Figure S1.

Figure 2.. Unintegrated CCTs form selective subcomplexes in cells.

A. NativePAGE immunoblots of HeLa cell lysates after individual CCT knockdowns. Low exposure blots (top) show TRiC depletion; higher exposure blots (bottom) highlight subcomplexes (CCT^sc) or monomers (CCT^M). Representative blots from each CCT subset with detectable species smaller than TRiC are shown; Figure S2 contains blots for remaining subunits. NT = non-transfected. B. Left, summary of results from A informing right, immunoblots of HeLa cell lysate depleted of CCT3 compared to control lysate after fractionation by size exclusion chromatography. TRiC elutes near the void volume V₀; subcomplexes elute in later fractions. Due to clearance mechanisms and signal dispersion, 4-fold higher amounts of CCT3 knockdown lysates compared to controls were loaded to detect CCT subcomplexes. C. Subcomplexes between assembly partners CCT5-CCT7 and CCT2-CCT4 detected by NativePAGE after co-overexpression in HeLa cells. The mCherry tag hinders higher-order assembly. D. NativePAGE immunoblots for CCT2 in HeLa cells treated with siRNA targeting CCT3 or scrambled control after treatment with proteasome inhibitor MG132 and/or autophagy inhibitor NH₄Cl. E. Summary of in vivo CCT subcomplex capacities based on knockdown and overexpression experiments. N indicates higher-order oligomerization. See also Figure S2.

Figure 3.. Recombinant CCTs assemble into various complexes via preferentially but not exclusively canonical contacts.

A. Schematic approach to express recombinant TRiC or alternative CCT combinations in an insect cell (Hi5) system using dual cassette baculovirus vectors. B. Total protein stain (left) and ³⁵S radiograph (right) of Hi5 cell lysates expressing all CCT subunits (TRiC) or pairs. Only the radiograph samples were metabolically labeled with ³⁵S-methionine. Arrows indicate CCT complexes. C. NativePAGE immunoblots for all recombinant subunits in Hi5 cells expressing each CCT pair. Arrows and Roman numerals indicate distinct complexes containing each subunit. The non-adjacent pair CCT1-CCT8 show the least co-migration. D. NativePAGE immunoblot for CTT8 in Hi5 cells expressing all TRiC subunits or pairs mixed post-lysis. TRiC does not assemble in the mixture. HeLa cell lane shows endogenous TRiC. E. SDS-PAGE immunoblots for CCT subunits after GFP immunoprecipitation from Hi5 cells co-expressing the colored subunits above each lane. F. SDS-PAGE immunoblots for CCT subunits after affinity purification of CCT7^His from Hi5 cells. Colored subunits to the right of each set of blots were co-expressed and beads were washed with stringencies of 100 mM NaCl, 500 mM NaCl, and 500 mM NaCl with 0.5% NP-40 (a, b, c). Broad CCT recovery secondary to the subunit pulled down indicates that complexes form in the absence of complete TRiC, with canonical ring contacts mediating recovery more than noncanonical contacts. See also Figure S3.

Figure 4.. Heterogeneous TRiC-like complexes assemble from CCT subsets.

A. After enrichment by affinity purification from Hi5 cells, holocomplexes (HCs) are purified by size exclusion chromatography. The representative trace shown is from the purification of ^HCCCT4257. B. Coomassie-stained NativePAGE of HCs containing four or six CCTs reveal similar sizes to TRiC and monodispersity. C. Negative stain electron micrographs of ^HCCCT4257 and ^HCCCT1368 showing top (left) and side (right) views of double ring topologies. D. 2D class averages of electron micrographs of TRiC and ^HCCCT578631 with representative side views on the right. E. Reconstructed 3D maps of TRiC and ^HCCCT578631 reveal shared asymmetric structural features (red arrows). Subunits within ^HCCCT578631 are unassigned. F. Summary of cross-linking mass spectrometry of HCs. Cross-links are mapped to canonical, noncanonical, intra-ring, and inter-ring CCT-CCT interfaces. Subunit orientations in cartoons connote distinct interfaces; e.g. 4–5 and 5–4 represent two interfaces where CCT4 is positioned on either side of CCT5. Cross-links were inconsistent with a parsimonious model of holocomplex assembly via minimal noncanonical contacts, and instead indicated that multiple arrangements form. G. Native mass spectra of holocomplexes (insets) and their dissociation products. The most abundant charge state of each assigned peak series is annotated with the colors of CCT monomers, dimers, and so on. CCTxN designates charge series closely matching CCT oligomers with ambiguous sub-stoichiometry. Red spheres denote charge series with masses close to 70 kDa, likely Hsp70 isoforms based on proteomic analysis. Intensities are normalized to 100%; absolute maximum signal intensities are 4, 1.7, and 8.3 × 10⁵ from left to right, meaning differences in oligomerization could be partly concentration-dependent. H. Concentration-independent ratios of native MS signal demonstrate that holocomplexes are less stable than TRiC. See also Figure S4.

Figure 5.. CCT holocomplexes segregate TRiC functions.

A. Segregation of net charge on the inner chamber surface (left) and of ATP affinity (right) within each TRiC ring, and thus between HCs. B. Conformational cycling assay read out by SDS-PAGE. ATP triggers chamber opening (unprotected) and closure (protected). More protection is observed for HCs containing high-ATP-affinity subunits. The transition state analog ATP/AlFx stabilizes the PK-protected state. C. ATPase activity of HCs by quinaldine red assay measuring Pi release over time. Error bars represent the SD. Right, magnified view of early time points. D. Radioactive labeled actin binding and folding assay. Representative radiographs after NativePAGE are shown; additional in Figure S6. Left, 15 minute time course in presence of ATP; right, the same assay using ^HCCCT4257 and ^HCCCT1368 incubated together for 30 minutes at 30°C (+/− ATP during preincubation) prior to introducing actin (+/− ATP). E. Actin bound by HCs relative to actin bound by TRiC (n = 3). F. Actin folded as a percentage of total actin present with each HC (n = 3). Error bars represent SEM. ns = not significant if p> 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. All statistical comparisons to TRiC not indicated reached significance p < 0.01 (**). Two-way ANOVA, Tukey’s multiple comparisons test. See also Figure S5.

Figure 6.. Model of TRiC hierarchical assembly and consequences of its dysregulation.

Co- or immediately post-translationally, CCT proteins from the negatively charged hemisphere assemble into subcomplexes and form a scaffold. CCT8 joins as a monomer, followed lastly by CCT1, CCT3, and CCT6, which are deterred from subcomplex formation amongst each other. Orphaned subunits and intermediates are rapidly degraded, and can form transient lower-order off-pathway associations with each other. This keeps the free energy landscape (B) broad, kinetically separating different holocomplexes and ensuring the cell can funnel subunits toward the proper pathway. Without ordered assembly, noncanonical holocomplexes can form with improper biochemical symmetry and aberrant function.