Figure 5.
Reconstitution of tER site assembly in vitro from semi-intact cell membranes and cytosol. (A) Budding from washed semi-intact membranes (CBY740) containing translocated [35S]gpαf after incubation at 23°C with a Sec23-GFP cytosol (CBY1860) as described under Materials and Methods. Reactions were supplemented with wild-type Sar1p or Sar1p T37N as indicated. Cytosol was replaced with buffer 88 in the “no addition” reaction (N/A). (B) Washed semi-intact membranes (CBY740) were incubated at 4°C with the same components as in A. After 30 min, DAPI was added and the reactions were imaged in the DIC, DAPI, and GFP channels. Note that the efficiency of Sec23p-GFP assembly into puncta and signal intensity correlate with COPII-dependent vesicle budding in A. (C) Average total cell fluorescence per reaction condition was determined from the images in B. (D) Percentage of semi-intact cells that formed fluorescent puncta was quantified from the images in B. Approximately 40 cells were analyzed per condition in C and D.