The chaperonin CCT promotes the formation of fibrillar aggregates of γ-tubulin
Luis Pouchucq, Pablo Lobos-Ruiz, Gissela Araya, José María Valpuesta and Octavio Monasterio.
Supplementary figure 1. γ-tubulin renaturation and its interaction with CCT. CCT effect over γ-tubulin aggregation. A) γ-tubulin equilibrium renaturation. Denatured γ-tubulin in 6 M GdmCl was renatured at equilibrium by fast dilution in reaction buffer. Renaturation was followed by light scattering (350 nm) and tryptophan intrinsic fluorescence in order to measure the aggregation and structure formation, respectively. At low denaturant concentrations, the γ-tubulin protein aggregated forming a high scattering precipitate. B) γ-tubulin-CCT interaction revealed by two-dimension electrophoresis assay. γ-tubulin, CCT and putative CCT:γ-tubulin complexes were resolved in a native gel (first dimension). Bands were excised at the level of CCT migration, and their content loaded onto an SDS gel (second dimension). The second dimension is shown. 1) molecular mass marker. 2) γ-tubulin control (-); human recombinant γ-tubulin was loaded in the first dimension. 3) γ-tubulin control (+); the same concentration of human recombinant γ-tubulin was loaded directly in the second dimension. 4) CCT control (-); purified bovine CCT complexes loaded in the first dimension in absence of recombinant γtubulin. 5) CCT plus γ-tubulin; the putative complexes were loaded in the first
dimension. A black arrow indicates the band of γ-tubulin. The optical density relation between the γ-tubulin control and CCT + γ-tubulin indicates that the monomeric γ-tubulin bound to CCT corresponds to not more than 5% of the total γtubulin added to the reaction; the rest was probably trapped in the first dimension due to its high state of aggregation. CCT and γ-tubulin concentrations were 0.3 µM and 6 µM respectively. C) Light scattering binding assay. A fixed amount of γtubulin (1: 0.5 µM; 2: 1 µM; and 3: 2 µM) was titrated by increasing concentrations of CCT. γ-tubulin aggregation was followed by light scattering at 350 nm. All measurements were performed at equilibrium and the CCT scattering contribution was subtracted from the data. The data were normalized as fractional change. The black arrows point to the equimolar γ-tubulin:CCT ratio. D) CCT effect over the apparent critical concentration (Ccapp) of γ-tubulin aggregation. Increasing concentrations of γ-tubulin were induced to aggregate in the presence of a fixed concentration of CCT. All reactions were done at equilibrium and followed by light scattering at 350 nm. The CCT scattering contribution was subtracted from the data and fitted to a linear regression equation.
Supplementary figure 2. CCT-ATP binding. 0.03 µM CCT was titrated with increasing ATP concentrations. The reaction was followed by circular dichroism (CD) and its value at 220 nm was plotted and fitted to a one-site ligand binding equation. The CCT sample were dialyzed three times (1:1000 v/v) against 20 mM potassium phosphate buffer pH 7.0 and filtered (0.2 µm) prior to the CD measurements (JASCO J-600 Spectropolarimeter), at room temperature.
Supplementary figure 3. CCT-γ-tubulin aggregation, with and without ATP, followed by thioflavin T (ThT) and Congo Red (CR) binding. A) ThT binding experiment.1 µM γ-tubulin was induced to aggregate alone or in the presence of 0.03 µM CCT and CCT + 1 mM ATP. 50 µM ThT was added to the reactions, incubated for 1 min at room temperature and the ThT fluorescence emission spectra were registered (λex = 450 nm; λem = 485 nm). CCT alone was used as non-ThT binding control. B) CR binding experiment. 1 µM γ-tubulin was induced to aggregate alone or in the presence of 0.03 µM CCT and CCT + 1 mM ATP. 0.17 mM CR was added to the reactions, and incubated for 30 min at room temperature. The protein aggregates were precipitated by centrifugation at 15.000 x g and the absorption at 540 nm of the supernatant (free CR) was registered. The negative control of CR without protein was set as 100%.
Supplementary figure 4. γ-tubulin mutants design to affect the binding with CCT and its effects on the aggregation. A) γ-tubulin consensus sequence. Loop T7 and loop M regions are highlighted in green. The red asterisks depict M248 and D278 positions. The secondary structure of γ-tubulin is represented by a blue and red color code (red/blue α-helix/β-strand) and labeled by a numbering code [58]. B) γ-tubulin structural model obtained using 1Z5V (Protein Data Bank) as a template. Loops T7 and M were modeled using MODELLER. Reconstructed T7 and M loops are indicated and the M248 and D278 residues depicted by sphere representation. C) Purification of wild type (WT) γ-tubulin and mutants DN, ME and MEDN. A representative Coomassie stained SDS-PAGE is shown in order to show that the purity of proteins used was greater than 98%. D) Aggregation Ccapp of WTγ-tubulin and DN, ME and MEDN mutants. Ccapp was calculated in the absence and presence of CCT and normalized with respect to the WT apparent critical concentration.
Supplementary figure 5. Percentage of survival of γ-tubulin injected zebrafish embryos. Single-cell zebrafish embryos were injected with WT and ME γ-tubulin (5 nl at 200 µM). Survival of injected embryos was monitored two hours post injection. Embryos were incubated at 28°C in M3 medium. Control (CTL) corresponds to embryos injected with the same volume of vehicle (5 mM phosphate buffer; 1 M urea). n corresponds to the number of injected embryos. Error bars indicate standard deviation of three independent biological replicas.
Supplementary figure 6. Effect of CCT on the aggregation of rhodanase, α/βtubulin and BSA. Co-precipitation experiment revealed by SDS-PAGE Coomassie blue staining of 1 µM rhodanase, 1 µM α/β tubulin heterodimer purified from chicken brain and 1 µM BSA with 0.02 µM CCT.
