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

In principle it should be possible to explore both the formation of secretory vesicles and their docking and fusion with the plasma membrane using gently permeabilized, semi-intact cells. In semi-intact cells from the growth hormone-secreting GH3 line, mature secretory granules of the regulated pathway do not leave the cells, but release their contents when calcium levels are raised (Martin and Walent, 1989) . To use semi-intact cells to study vesicle formation, it is essential that post-Golgi carrier vesicles be able to leak from the cells (Bennett et al., 1988) . To determine if intermediates in the secretory pathway would escape from semiintact cells, we examined the release of [ 35 S]sulfate-labeled vesicles from PC12 cells . We obtained evidence that two classes of vesicles escaped, constitutive secretory vesicles and precursors of mature secretory granules. Their formation and properties were dependent on ATP, GTP, and the time between labeling and permeabilizing the cells. The small constitutive vesicles resembled the carrier vesicles released from yeast and other cells in their homogeneous sedimentation behavior. The precursors in the regulated pathway, free to diffuse out of the permeabilized cells, were much larger and may still be engaged in the sorting process as has been suggested for exocrine cells (von Zastrow and Castle, 1987) . Cell Culture, Labeling, and Pulse-Chase Experiments Five separate clones of the rat pheochromocytoma (PC12) cell line (Green and Tischler, 1976) were tested during the course of these experiments, and gave similar results. PC12 cells were cultured in 10% C02 in DME H21 containing 10% horse serum, and 5% FCS. Six to eight 15-cm dishes of confluent PC12 cells were washed in warm PBS, then dislodged from the plates in 10-12 ml PBS by pipetting, and transferred to a 15-ml polypropylene test tube . Cells were centrifuged 2 min at 200 g, then washed once more in PBS and centrifuged as above. The cells were resuspended in 5 ml 355042labeling medium (DME H21 without cold sulfate containing 1-2 MCi/m1 35 SO42-that had been preequilibrated at 37°C and 10% C02) . The cell suspension was incubated at 37°C for 5 min, then either placed on ice or centrifuged for 2 min at 200 g at room temperature, resuspended in warm DME H21 plus 20 mM MOPS, pH 7.2, and 1 mM MgSO4, and then incubated at 37°C for various chase periods . To examine the kinetics of secretion, the cell suspension was placed on ice, then cells and media separated by centrifugation at 1,300 g for 10 min . Media samples were centrifuged 5 min at 14,000 g and the supernatant precipitated in 10 % TCA for analysis by PAGE . Cell pellets were resuspended in 10 mM Tris, pH 9.5, 20 mM DTI', and protease inhibitor cocktail (0.1 kg/ml each pepstatin, chymostatin, leupeptin, and aprotinin added from a 1,000X stock in DMSO ; 1 mM PMSF, 10 AM benzamidine, and 1 Pg/ml O-phenanthroline added from a 100x stock in ethanol) . After freezing and thawing three times, samples were centrifuged at 14,000 g for 10 min and the supernatants added to an equal volume of 2x sample buffer for PAGE. We found that 80% of proteoglycans (total of media and cells) was secreted constitutively by 1 h, while a smaller fraction of secretogranin II was secreted constitutively (30% of total by 1 h) .