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

PC12 cells contain synaptic vesicle-sized organelles that are rich in the integral membrane protein, synaptophysin (p38), (Cliff-O'Grady et al., 1990; Wiedenmann et al ., 1988) . We estimate that >95 % of the synaptic vesicle-sized organelles escaped when the cells were permeabilized, without requiring further incubation in vitro (data not shown) . This method of mechanical permeabilization, therefore, must tear holes in the plasma membrane big enough to release synaptic vesicles . Release of small intermediate organelles involved in the exocytotic pathways would therefore be expected if they are not attached in some way to the cells . . Equilibrium density separation of vesicles that escaped from cells labeled and permeabilized as in Fig. 2 . TCA-precipitable 35S radioactivity (-a-, S1 cpm) of membranes that escaped from semi-intact cells, during an incubation with ATP, after separation on a sucrose equilibrium density gradient. Unlabeled synaptophysin (--0-, p38) released from the cell was detected using a Western blot (see Materials and Methods) and quantified by phosphorimaging . Also shown is TCA-precipitable 35S radioactivity from cell ghost membranes from the same experiment after incubation at 37°C with ATP (-ü-, PIM) . The density of each fraction was measured by refractometry and the quantities plotted as a function of density. The samples were centrifuged for 16 h at 32,500 rpm .