Exocytic transport vesicles generated in vitro from the trans-Golgi network carry secretory and plasma membrane...

We have developed a cell-free assay that reproduces vesicular budding during exit from the Golgi complex. The starting preparation for the in vitro system was a rat liver stacked Golgi fraction immobilized on a magnetic solid support by means of an antibody against the cytoplasmic domain of the polymeric IgA receptor. Vesicular budding was ATP, cytosol, and temperature dependent and was inhibited by 1 mM N-ethylmaleimide. Budding was maximum within 10 min and originated preferentially from the trans-Golgi. Exocytic transport vesicles immunoisolated from the total budded population were enriched in the mature forms of secretory and membrane proteins destined to the basolateral plasma membrane and were depleted in lysosomal enzymes and galactosyl-transferase activity. The finding that a major proportion (greater than 70%) of newly synthesized, siaylated secretory and transmembrane proteins is contained in a single population of post-Golgi transport vesicles implies that, in a constitutively secreting cell, basolaterally destined proteins are sorted and packaged together into the same exocytic transport vesicles.