The organization of endoplasmic reticulum export complexes

To examine the role of COPII in VSV-G concentration and the appearance of VTCs in export complexes, we used semi-intact cells infected with a temperature-sensitive form of VSV-G whose transport is blocked at the restrictive temperature (39.5°C) (Lafay, 1974; Plutner et al., 1992) . Transfer of cells to the permissive temperature (32°C) results in the migration of a synchronous wave of ts045 VSV-G from the ER to VTCs and subsequent Golgi compartments (Plutner et al., 1992; Balch et al., 1994) . After incubation at 32°C in vitro, cells were fixed and stained with an antibody specific for the cytoplasmic tail of VSV-G using the immunodiffusion protocol . As shown in Table III , incubation in the presence of wildtype Sarl had little effect on ER export as judged by the abundance of VTCs detectable in vitro. In contrast, the guanosine diphosphate (GDP)-restricted form of Sarl (Sarl [T39N] ) drastically reduced the formation of vesicles and VTCs (Table III) , demonstrating the essential need to activate Sarl to promote membrane flow from the ER through export complexes. In contrast, incubation in the presence of the GTP-restricted mutant (Sarl[H79G]) caused a dramatic accumulation of vesicles in clusters and an approximate twofold increase in the apparent number of clusters per section that could be detected (Table III) .