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

The nonhydrolyzable GTP analogue, GTPyS, has major effects on in vitro membrane traffic, affecting both the formation and fusion of carrier vesicles. The presence of GTPyS during the in vitro reaction reduced the yield of constitutive secretory vesicles by about a third (63 % t 3 in five experiments), and those that were produced migrated with increased sedimentation velocity (Fig. 6 A) which is probably because of increased density (Fig. 6 B) . The proteoglycans in the less dense peak (Fig. 6 B density as constitutive secretory vesicles (Fig . 4) . The new peak that appeared in GTPyS had a density close to 1 .19 g/ml, suggesting coated intermediates similar to those purified by Malhotra et al . (1989) . We conclude that three separate classes of vesicles containing proteoglycans can be detected . (a) A homogeneous population of small vesicles whose accumulation depends on an in vitro incubation with ATP, which we call constitutive secretory vesicles ; (b) a small, dense class that accumulated in vitro in the presence of ATP and GTPyS, tentatively classified as coated constitutive vesicles ; and (c) a minor class of heterogeneous rapidly sedimenting vesicles that were released without an in vitro incubation with ATP and that changed their sedimentation rate with time. This third class of vesicles has properties in common with immature regulated secretory granules .