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

The integrity of the Golgi complex was maintained in the permeabilized cells . 87 % of the Golgi marker galactosyl transferase remained with the ghost membranes (PIM), migrating near the bottom of a sucrose velocity gradient (25,000 g, 1 h) similar to that used by . Only 13 % of the galactosyl transferase activity was detected in membranes (P2) that escaped the permeabilized cells, when the cells were warmed to 37°C in the presence of an ATP regenerating system ( Fig . 2 A) . We conclude that this method of tearing holes in the plasma membrane does not dislodge the Golgi apparatus. To characterize the sulfatelabeling membrane compartment in the cell ghosts, PC12 cells were pulse labeled for 5 min with 35 SO4z -and the membranes remaining with the ghosts (PlM) were applied to a sucrose velocity gradient identical to that in Fig . 2 A (25,000 g, 1 h) . The peak of sulfate label coincided with the galactosyl transferase marker in agreement with (data not shown) . To quantify the fraction of sulfate label in fast sedimenting materials, the experiment was repeated using higher sedimentation forces (100,000 g, 1 h) that allow small vesicles (see below) to enter the gradient, but which move the sulfate-labeled membranes nearer to the bottom of the tube (Fig . 2 B) . Without a subsequent reaction or ifcells were warmed in the absence of ATP, at least 80 % of the TCAprecipitable radioactivity was recovered at the bottom of the velocity gradient ( Fig. 2 B) . Since sulfate incorporation is primarily into Golgi compartments, the data imply that these compartments are not significantly released from semiintact cells .