Self-assembly of minimal COPII cages

Abstract

The small G-protein Sar1 and the cytosolic complexes Sec23/24 and Sec13/31 associate sequentially on endoplasmic reticulum membranes to form a protein coat named COPII, which drives the formation of transport vesicles. Using dynamic light scattering, we show that Sec23/24 and Sec13/31 can self-assemble in a stoichiometric manner in solution to form particles with hydrodynamic radii in the range of 40-60 nm. Self-assembly is favoured by lowering the pH, the ionic strength and/or the temperature. Electron microscopy reveals the formation of spherical particles 60-120 nm in diameter with a tight, rough mesh on their surfaces. We suggest that these structures, which represent a minimal COPII cage, mimic the molecular organization of the membrane-associated COPII coat.

Figure 1

Effect of temperature on the assembly of COPII aggregates. Stock solutions of (A) Sec23/24 (1.35 μM), (B) Sec13/31 (4.15 μM) or (C) a mixture of the two solutions (Sec23 and Sec24 at 0.82 μM; Sec13 and Sec31 at 1.64 μM) were analysed by dynamic light scattering. The panels on the left show the initial intensity histograms of hydrodynamic radius (R_h) at 27 °C. The middle panels show the evolution of the change in the mean R_h (bold trace) when the temperature of the sample was decreased from 27 °C to 4 °C. The temperature ramps are shown as thin traces. The panels on the right show the final intensity histograms of R_h at 4 °C.

Figure 2

Effect of pH and ionic strength on the assembly of COPII aggregates at 27 °C. Mixtures of Sec23/24 and Sec13/31 were made by dilution of stock solutions in buffers of various pHs and potassium-acetate concentrations. After 40 min at 27 °C, the distribution of hydrodynamic radius (R_h) was determined by dynamic light scattering. In all experiments, the Sec subunit concentration was 0.30 μM. (A) Stoichiometric mixtures of Sec23/24 plus Sec13/31 in MES (2-(N-morpholino) ethanesulphonic acid)/HEPES buffers. The final pH is indicated. The final concentration of potassium acetate was 225 mM. (B) Stoichiometric mixtures of Sec23/24 plus Sec13/31 in Mes buffer containing increasing amounts of potassium acetate. The final concentration of potassium acetate is indicated. The final pH was 6.45. (C) Sedimentation analysis of Sec23/24 plus Sec13/31 mixtures prepared as in (A) in a final volume of 250 μl and centrifuged at 37,000 r.p.m. (50,000g) for 30 min at 25 °C. The percentage of Sec31, Sec23 and Sec24 in the pellet was determined by densitometry after SDS–polyacrylamide gel electrophoresis (10% acrylamide) and Coomassie-blue staining. (D) Dynamic light-scattering analysis of isolated Sec23/24, isolated Sec13/31 or a mixture of the two complexes after dilution in Mes buffer. When only one COPII complex was present, the missing complex was replaced by the same volume of the corresponding purification buffer. The final pH was 6.45 and the final potassium-acetate concentration was 225 mM.

Figure 3

Visualization of COPII aggregates by electron microscopy. (A) Negative staining of a Sec23/24 plus Sec13/31 mixture that was prepared by dilution in MES (2-(N-morpholino) ethanesulphonic acid) buffer and incubated at 25 °C. The sample contained each Sec subunit at a concentration of 0.3 μM. The final pH was 6.45 and the final potassium-acetate concentration was 230 mM. (B) An isolated Sec23/24 complex under the same conditions. (C) The same mixture as in (A) after concentration by centrifugation and fixation with glutaraldehyde. The black arrow indicates an elongated particle. (D) Thin-section image of a similar preparation embedded in Epon resin. (E) Close-up view from part of panel (C), as indicated by the dashed box and arrow. (F) Distribution of the diameters of 151 aggregates prepared as in (C). Scale bars, 100 nm.

Figure 4

Effect of pH on the diameter of COPII aggregates. Concentrated solutions of Sec23/24 and Sec13/31 were mixed to give a final concentration of 0.9 μM of each Sec subunit, dialysed at 25 °C against solutions containing 230 mM potassium acetate and 1 mM MgCl2, and buffered at pH 6.85 (A,D), pH 6.33 (B) or pH 6.00 (C) using different ratios of MES (2-(N-morpholino) ethanesulphonic acid)/HEPES. After dialysis, aliquots were analysed by dynamic light scattering (DLS). The remaining solutions were fixed with glutaraldehyde and processed for negative staining (A–C) or thin-section electron microscopy (EM) (D). (E) Comparison of the average diameter of the spherical aggregates, as observed by EM, with the hydrodynamic diameter (2 × hydrodynamic radius) of the aggregates, as measured by DLS. The error bars indicate the s.d. from the mean. Scale bars, 100 nm.