Essential role of the chaperonin folding compartment in vivo

Abstract

The GroEL/GroES chaperonin system of Escherichia coli forms a nano-cage allowing single protein molecules to fold in isolation. However, as the chaperonin can also mediate folding independently of substrate encapsulation, it remained unclear whether the folding cage is essential in vivo. To address this question, we replaced wild-type GroEL with mutants of GroEL having either a reduced cage volume or altered charge properties of the cage wall. A stepwise reduction in cage size resulted in a gradual loss of cell viability, although the mutants bound non-native protein efficiently. Strikingly, a mild reduction in cage size increased the yield and the apparent rate of green fluorescent protein folding, consistent with the view that an effect of steric confinement can accelerate folding. As shown in vitro, the observed acceleration of folding was dependent on protein encapsulation by GroES but independent of GroES cycling regulated by the GroEL ATPase. Altering the net-negative charge of the GroEL cage wall also strongly affected chaperonin function. Based on these findings, the GroEL/GroES compartment is essential for protein folding in vivo.

Figure 1

In vivo functionality of GroEL cavity-mutants. Constructs encoding the proteins indicated were transformed into E. coli MC4100 SC3 Kan^R cells. Cells were grown in the presence of arabinose for expression of WT-GroEL/GroES. Serial dilutions corresponding to cell numbers indicated were plated on arabinose-containing plates for continued expression of WT-GroEL/GroES or IPTG-containing plates for expression of GroEL-mutants/GroES at 37°C as described in Materials and methods.

Figure 2

Substrate binding and trans-folding of GroEL cavity-mutants. (A) Prevention of rhodanese (Rho) aggregation in vitro was measured at an equimolar ratio of GroEL/rhodanese or at a twofold molar excess of GroEL (see Materials and methods). Aggregation after 10 min of rhodanese dilution from denaturant in the absence of chaperonin was set to 1. (B) Binary complexes of GroEL and GroEL-mutants with rhodanese, METK or SYT, produced by dilution of the denatured substrate proteins into GroEL-containing buffer, were analysed by size-exclusion chromatography. GroEL-bound substrates were quantified by immunoblotting with the loading control (L) set to 100%. Fractionation of GroEL and free native substrate proteins is indicated. (C) Capacity of GroEL cavity-mutants to support the soluble expression of the trans-folding substrate, yeast aconitase. Aconitase was overexpressed in E. coli BL21 cells with or without co-overexpression of WT-GroEL and GroEL-mutants with or without GroES at 37°C as indicated. Total (T), supernatant (S) and pellet (P) fractions of cells were analysed on SDS–PAGE and aconitase was quantified by densitometry. Standard deviations of at least three independent experiments are shown.

Figure 3

Encapsulation efficiency of endogenous substrates by GroEL cavity size-mutants. (A) Schematic representation of complexes of GroEL with His₆-tagged MmGroES arrested in the ADP state with substrate enclosed within the cis-cavity. (B) E. coli MC4100 cells overexpressing GroEL cavity size-mutants and His₆-tagged MmGroES were lysed, and chaperonin complexes containing endogenous substrates were isolated as described in Materials and methods. The substrate proteins indicated were detected by immunoblotting. GroEL was detected with the anti-serum against XYLA, which has strong GroEL cross-reactivity. To control for specificity, the same isolation procedure was performed with cells overexpressing WT-GroEL and non-His₆-tagged MmGroES.

Figure 4

Enhanced folding of GFP upon mild reduction of GroEL cavity size. Solubility (A) and fluorescence (B) of WT-GFP upon co-overexpression with GroEL cavity size-mutants and GroES in E. coli MC4100 cells at 30°C. Cells were grown and analysed as in Figure 2C (see Materials and methods). Total (T), supernatant (S) and pellet (P) fractions from equal amounts of cells were analysed by SDS–PAGE and Coomassie staining. GFP fluorescence was measured in cell lysates containing equal amounts of total protein with the activity in the vector only control set to 1. Standard deviations of at least three independent experiments are shown. (C) and (D) Kinetics and yield of GFP refolding with GroEL cavity size-mutants and GroES in vitro. Refolding yields are plotted with the native GFP control set to 1. Refolding traces were fitted to a double exponential equation and apparent rates are plotted as the weighted average of the slow and fast rates based on their relative amplitudes. Standard deviations of at least three independent experiments are shown.

Figure 5

Motility and protease protection of substrate protein upon encapsulation in SR-EL cavity size-mutants. Steady-state fluorescence anisotropy of GFP (A) and DHFR–GFP fusion protein (B) upon addition of GroES and AMP-PNP to binary SR-EL-substrate complexes. Anisotropy values of native and spontaneously refolded proteins are shown as controls (see Materials and methods). Standard deviations of at least three independent experiments are shown. (C) and (D) Protease protection of DHFR–GFP upon addition of GroES and AMP-PNP to binary SR-EL-substrate complexes. Proteinase K (PK) protected DHFR–GFP was detected by immunoblotting with anti-GFP antibody and quantified by densitometry. Standard deviations of three independent experiments are shown.

Figure 6

Accelerated folding of GFP and rhodanese in cavity size-mutants is independent of ongoing ATP-hydrolysis. Kinetics and yield of WT-GFP refolding (A, B) and of rhodanese refolding (C, D) with SR-EL cavity size-mutants and GroES/ATP in vitro. Refolding yields are plotted with the native GFP and rhodanese control set to 1, respectively. Refolding traces for GFP were fitted as in Figure 4D and refolding traces for rhodanese were fitted to a single exponential equation. Note that there is essentially no spontaneous renaturation of rhodanese under the experimental conditions. Standard deviations of at least three independent experiments are shown.

Figure 7

Accelerated folding by GroEL cavity size-mutants is independent of the rate of ATP hydrolysis. Steady-state ATPase activities of GroEL cavity size-mutants (A) and ATPase-deficient GroEL(D398A) cavity size-mutants (B) at 25°C. ATPase rates are indicated in ATP hydrolyzed per GroEL tetradecamer per min (see Materials and methods). Refolding yields and rates of WT-GFP (C) and rhodanese (D) with GroEL(D398A) cavity size-mutants and GroES/ATP. Refolding traces for GFP were fitted as in Figure 4D and refolding traces for rhodanese were fitted to a single exponential equation. The refolding yield obtained with WT-GroEL/GroES was set to 1. Standard deviations of at least three independent experiments are shown. (E) Simulation of rhodanese folding kinetics dependent on GroEL/GroES cycling rate at excess chaperonin over substrate. The binding rate of unfolded protein to GroEL was set to 2 × 10⁷ M⁻¹ s⁻¹ (Rye et al, 1999) and binding of GroES to GroEL-substrate complexes was set to 1 × 10⁶ M⁻¹ s⁻¹ (KC and SS, unpublished data, 2007). The rate of rhodanese refolding with WT-GroEL/GroES or SR-EL/GroES was set to 2.5 × 10⁻³ s⁻¹ (Figure 7D). The normal ATPase-induced cycling rate was fixed at 0.07 s⁻¹ with an approximate half-time of 10 s. The simulation was performed using chemical kinetics simulator (CKS) (http://www.almaden.ibm.com/st/computational_science/ck). The ATPase-induced cycling rate was varied between 0.1- and 20-fold of the normal rate and refolding rates are plotted. The concentrations used for the simulation were GroEL 1 μM, GroES 2 μM and rhodanese 0.5 μM (see Supplementary Figure S5 for a kinetic model for the simulation).