Figure 2. ALG-2 attenuates in vitro reconstituted COPII vesicle budding in a reversible manner.

Budding investigated using p58/ERGIC as a marker after incubation of HeLa cell donor membranes with recombinant Sar1A, Sec23A/24D, Sec13/31A and 0–2 ug of recombinant ALG-2. A. Budding efficiency was measured at increasing concentrations of either ALG-2 or Ca²⁺. B. Attenuation of budding was dependent on EF hand 1 of ALG-2. Binding of ALG-2 wildtype (wt) and mutants to Sec31A was tested by incubating either wt ALG-2 (wt), ALG-2.1 (2.1), ALG-2mutEF1 (EF-1), ALG-2mutEF3 (EF-3) or ALG-2mutEF1&3 (EF-1–3) with Sec31A-coated beads. Eluates were separated by SDS-PAGE and visualized using SyproRed. C. In vitro reconstituted budding using p58 as a budding marker. Mutant versions of ALG-2 were added to the budding reaction in equimolar amounts. D. ALG-2/Ca²⁺ budding attenuation was reversed by chelation of Ca²⁺. In vitro reconstituted vesicle budding using p58 as a cargo protein marker. Budding was performed for 45 min in the presence of Ca²⁺ only (control), Ca²⁺/ALG-2 (ALG-2) or in absence of nucleotides. EGTA was added to budding reactions at T = 15 and T = 30 min and reactions were incubated in the presence of 1 mM EGTA for the indicated time periods (min). Background budding levels were found by omitting nucleotides (no nuc). SEM of three independent experiments. E. 10–40% Nycodenz gradient was layered on top of the product of the budding reactions performed in the absence (upper panel) or presence (lower panel) of recombinant ALG-2.