Intermediates in the constitutive and regulated secretory pathways released in vitro from semi-intact cells

Cells were placed on ice and centrifuged at 500 g for 2 min at 4°C, then washed in cold PBS containing 1 mM EDTA, 1 mM EGTA . A buffer was used (buffer C) that mimics the composition of the cytoplasm (Bennett et al ., 1988 , Meister, 1988 . Buffer C contains 38 mM potassium aspartate (DL), 38 mM potassium glutamate (L), 38 mM potassium gluconate (D), 20 mM potassium MOPS, pH 7.2, 5 mM reduced glutathione, 5 mM sodium carbonate, 2 .5 mM magnesium sulfate, 2 mM EGTA, and 0.1% BSA . The final pH at 37°C was 7.05. Cells were washed in 5 ml buffer C, then resuspended in 0.8 ml buffer C containing protease inhibitor cocktail . This suspension was passed once slowly through a cell cracker on ice, 8 .01-mm body with a 8 .006-mm ball (Balch and Rothman, 1985) . The cell suspension was aliquoted into separate tubes for different reaction conditions and incubated 15 min at 37°C or on ice . The following reagents were added from 100x stocks to these final concentrations : ATP regenerating system-8 mM creatine phosphate, 1 mM ATP, 5 kg/ml creatine kinase ; 0.2 mM PAPS, 1 mM GTP, 20 AM GTP-yS. Apyrase was added as a solid to 15 mg/ml . Control reactions received an equal quantity of water. After incubation at 37°C, the cell suspension was placed on ice and kept cold through all of the following manipulations to halt further membrane traffic . The suspension of permeabilized cells was centrifuged at 1,000 g for 10 min. The supernatant (Sl) was removed and the cell ghost pellet was further homogenized by resuspending in 0 .25 ml cold 0 .25 M sucrose, 20 mM MOPS, pH 7.2, 1 mM EGTA plus the protease inhibitor cocktail mentioned above (buffered sucrose) . After 10-12 passages through a 25-gauge needle, this homogenate was centrifuged 10 min at 70 g to give a supernatant of "cell ghost" membranes (PlM) and a white nuclear pellet .