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

tutive vesicles (velocity gradient fractions 16-22) released when semi-intact cells were incubated with ATP was highest after a 15 min in vivo chase (Fig. 5 B) . This is probably attributable to continued sulfate incorporation during the chase (Fig. 5 A) before the bulk ofproteoglycans is secreted. After a 60-min chase, the total amount ofradioactivity in the constitutive vesicles diminished rapidly (Fig. 5 B) . Since 80% of proteoglycans are secreted by a 60-min chase (data not shown), fewer labeled proteoglycans are expected to be present in a carrier vesicle traveling between the Golgi complex and the cell surface. Velocity and density gradient analysis showed (Fig. 4 ) that the small light vesicles were still generated in the presence of ATP with an efficiency that was independent of chase time, averaging -23% (Fig. 5 B) . Secretory vesicles containing 35 S-labeled proteoglycans escaped from the permeabilized cells to a small extent, even in the absence of incubation in vitro. The sedimentation properties ofvesicles released without ATP did not resemble those generated during incubation in the presence of ATP Their sedimentation rate gradually increased with time ( Fig. 4 B and C, no reaction).