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

In regulated secretory cells, there are two types of carrier vesicles that leave the TGN and fuse with the plasma membrane, the constitutive secretory vesicle and the regulated secretory granule (for review see Kelly, 1991) . The two carrier vesicles differ in the types of protein they transport and whether or not their fusion with the cell surface is regulated by cytoplasmic second messengers. Isolation ofthe two types of carrier vesicles ought to help clarify how protein sorting and regulated fusion are mediated. The pioneering study of suggested that isolation of the two classes of vesicles might be possible. To label secreted proteins in PC12 cells, they were pulse labeled with [11S]sulfate. Radioactive sulfate is incorporated into the intermediate phosphoadenosine phosphosulfate (PAPS), which is transported into the lumen of the Golgi network (Milla and Hirschberg, 1989 ) then transferred to carbohydrate chains (Rodén, 1980) and to tyrosine residues of proteins (Huttner, 1982) . Since tyrosine sulfation of proteins occurs concomitant with or after sialic acid addition in hybridoma cells, it is thought to occur in a late trans-Golgi compartment (Baeuerle and Huttner, 1987) . The site of sulfation of proteoglycans is less well localized. showed that intact cells, labeled briefly with [3IS]sulfate, secreted only three major "S-labeled species. Two tyrosine sulfated proteins, secretogranin II and chromogranin B, are markers for the regulated pathway while sulfated proteoglycans are a marker for the constitutive . When isolated membranes were incubated in the presence of ATP, sulfatelabeled vesicles budded from the Golgi fractions . Although vesicles containing the constitutive marker largely overlapped vesicles containing the regulated protein markers on equilibrium density gradients, enough separation was achieved to suggest that sorting was indeed occurring in vitro.