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

GTP is involved in both the budding of vesicles and their fusion . In yeast, the GTP-binding protein SARI is required for budding from the ER and the nonhydrolyzable analogue, GTPyS, slows but may not block the budding completely (Oka et al ., 1991; Rexach and Schekman, 1991) . In mammalian cell preparations, GTPyS inhibited the formation of both regulated and constitutive carrier vesicles, but only by -50% . The fusion process also involves GTP and can usually be completely inhibited by GTPyS in ER to Golgi, intra-Golgi, and Golgi to cell surface transport (Baker et al., 1988; Ruohola et al ., 1988; Beckers and Balch, 1989; Melangon et al., 1987; Miller and Moore, 1991; Rexach and Schekman, 1991; Segev, 1991) . Nonclathrin-coated vesicles that are involved in intercisternal Golgi transport accumulate in the cell in the presence of GTPyS (Malhotra et al., 1989) . When budding from the TGN ofPC12 cells was examined in the presence of GTPyS, fewer constitutive vesicles were formed and their sedimentation velocity and equilibrium density were increased, consistent with the retention ofa coat. The partial inhibition of constitutive secretory formation (Fig. 6 A constitutive vesicle fusion has an absolute requirement for GTP hydrolysis (Miller and Moore, 1991) . The second class of vesicles released from permeabilized cells contained secretogranin II, a marker for the regulated secretory pathway. Although the secretogranin 11-rich and proteoglycan-rich vesicles could be separated by density, velocity sedimentation allowed more complete resolution (Figs. 4 and 7) . This resolution has revealed several aspects of the sorting process . Immediately after a pulse label, the release of the vesicles is ATP dependent, consistent with their formation in vitro during the incubation . They are significantly lighter than Golgi fractions and have a secretogranin II to proteoglycan ratio 2-3 times the constitutive vesicles (Fig . 8 B) . After a long chase, any ATPdependent formation of the secretogranin II-rich vesicles is obscured by ATP-independent release of pre-existing secretogranin II-rich vesicles. The density of the vesicles that accumulate during the chase is greater than that of vesicles generated in vitro, about the same as the donor Golgi membranes (Fig . 3) , but still not as dense as mature secretory vesicles. Their content, however, has a secretogranin II to proteoglycans ratio that is twice that of the Golgi at 15 min chase and four times that of the Golgi at 60 min chase . The latter ratio is 15 times greater than constitutive vesicles, and approaches that of mature vesicles (Fig . 8 B) . Such properties would be predicted for immature secretory granules but are less easy to reconcile with other possibilities such as fragmented Golgi membranes.