Evidence for a lipochaperonin: association of active protein-folding GroESL oligomers with lipids can stabilize membranes under heat shock conditions

Abstract

During heat shock, structural changes in proteins and membranes may lead to cell death. While GroE and other chaperone proteins are involved in the prevention of stress-induced protein aggregation and in the recovery of protein structures, a mechanism for short-term membrane stabilization during stress remains to be established. We found that GroEL chaperonin can associate with model lipid membranes. Binding was apparently governed by the composition and the physical state of the host bilayer. Limited proteolysis of GroEL oligomers by proteinase K, which removes selectively the conserved glycine- and methionine-rich C terminus, leaving the chaperonin oligomer intact, prevented chaperonin association with lipid membranes. GroEL increased the lipid order in the liquid crystalline state, yet remained functional as a protein-folding chaperonin. This suggests that, during stress, chaperonins can assume the functions of assisting the folding of both soluble and membrane-associated proteins while concomitantly stabilizing lipid membranes.

Figure 1

Effect of chaperonins on the surface pressure of lipid monolayers. GroEL₁₄ or GroES₇ oligomers were injected underneath the monolayer and the surface pressure was measured. (A) At time points indicated by arrows, 3.5 μM GroEL, 1.5 mM ATP, and 7 μM GroES were added. (B) The same as in A, with reverse order of events. (C) Surface pressure increases as a function of GroEL concentration in the subphase. Different amounts of GroEL (—), GroES (- - -), or MDH (– – –) were injected into the subphase. The increase in surface pressure was measured after pressure equilibration.

Figure 2

Effect of proteinase K treatment on the interaction of GroEL with lipid monolayers. (A) GroEL was added to the subphase at 5 min at a final concentration of 0.1 μM. Proteinase K (final concentration, 30 μg/ml) was injected at the time indicated by the arrow. (B) GroEL or GroELΔC, prepared as detailed in Materials and Methods, was injected underneath the monolayer at 5 min. (Inset) Analysis of the samples injected into the subphase by SDS/PAGE on an 8% gel (lane 1, GroEL; lane 2, GroELΔC).

Figure 3

Effect of pH on GroEL–monolayer interaction. (A) At the indicated time, 3.5 μM GroEL was added to the subphase. After pressure equilibration, the pH of the subphase was lowered to 4 with HCl. Where indicated, the pH was readjusted to 7.5 with NaOH. (B) pH-dependent surface pressure increase caused by the addition of 0.35 μM GroEL.

Figure 4

Binding of chaperonins to LUVs. (A) Binding experiments were carried out as described in the text. GroEL at 3.5 μM and GroES at 7 μM were incubated in the presence of 1.5 mM ADP (lane 0). This mixture was added to LUVs made of DOPG at increasing lipid concentrations. After incubation at 23°C for 30 min samples were centrifuged at 250,000 × g. Supernatants and pellets were analyzed by SDS/PAGE (15% gel). Lipid concentrations were 0, 140, 170, 225, 420, and 560 μM in samples 1–6, respectively. (B) Membrane-bound GroEL is the native 14-mer. GroEL (3.5 μM) was added to 0 μM (lane 1) or 400 μM (lane 2) DOPG vesicles in buffer A. After 30 min of incubation at 23°C, samples were centrifuged. Pellets were analyzed by nondenaturing PAGE (6% gel).

Figure 5

Effect of GroEL on the membrane physical state tested on LUVs. (A and B) Steady-state fluorescence anisotropy of TMA-DPH (squares, curves 1 and 2) or DPH (triangles, curves 3 and 4) embedded in LUVs made of DMPC (A) or DPPC (B) was measured as a function of temperature in the presence (open symbols, curves 1 and 3) or in the absence (filled symbols, curves 2 and 4) of 1.5 μM GroEL in buffer A. (C and D) Effect of proteinase K treatment on the fluorescence anisotropy of TMA-DPH (squares, curves 1 and 2) or DPH (triangles, curves 3 and 4) in the presence (open symbols, curves 1 and 3) or in the absence (filled symbols, curves 2 and 4) of 1.5 μM GroEL preincubated with 50 μM LUVs of DMPC (C) or DPPC (D), at 35.6°C and 49°C, respectively. Proteinase K (30 μg/ml, final concentration) was added at the indicated time.