Figure 5.

GST-tagged Sar1–GTP supports vesicle budding from salt-washed microsomal membranes. (A) Salt-washed microsomes prepared as described in Materials and Methods were incubated in a budding reaction with cytosol on ice (a) or for 30 min at 32°C without (b) or with wild-type Sar1 (1.5 μg) (c), Sar1-GTP (4 μg) (d), or a GST-tagged Sar1–GTP (10 μg) (e) as indicated. The amount of VSV-G (% of total) released from the ER into the HSP was determined as described (Rowe et al., 1996). (B) Salt-washed microsomes were incubated in a budding reaction supplemented with a GST-tagged Sar1–GTP (4 μg) for 30 min at 32°C. The HSP of seven budding reactions was collected and then subjected to immunoisolation with magnetic beads alone (a) or beads coupled with a monoclonal antibody to the VSV-G tail (P5D4) as described (Rowe et al., 1996) (b). GST–Sar1–GTP bound to beads was detected by Western blotting using a polyclonal antibody to Sar1. Molecular weight markers are indicated on the left. The typical results of three independent experiments are shown.