Figure 6.

ALG-2–dependent coat retention primarily inhibits via a prefusion mechanism. (A) Outline of experiment to distinguish between pre- or postfusion mechanisms by which ALG-2 inhibits heterotrimer assay (see text for explanation). Cargos VSV-G-myc and radioactive VSV-G* are depicted in green and red, respectively. COPII coat is represented by thick black lines. (B) Heterotrimer data from experiment depicted in A. Heterotrimers are normalized with 100% representing the amount produced during the first incubation in the absence of recombinant ALG-2, before addition of BAPTA and second incubation. BAPTA was omitted from the conditions plotted at time 0; BAPTA was added to all other conditions, while on ice, just before beginning the second incubation at 32°C. ALG-2 added during the first incubation had a dramatic consequence on the subsequent heterotrimer signal (red vs. blue lines), but ALG-2 added only during the second incubation had no influence (green square). Plotted are means of duplicate reactions, with error bars to represent SE where they exceeded symbol size.