Isolation of exocytic carrier vesicles from BHK cells

Abstract

Newly synthesized cell surface glycoproteins are transported from the trans-Golgi network (TGN) to the plasma membrane in vesicular carriers. Here we describe a cell-free system in which the formation of these carrier vesicles is reconstituted. Vesicle formation and release occurred specifically from the TGN and were dependent on ATP and cytosol. The released vesicles were isolated by density gradient sedimentation and specific immunoadsorption. Electron microscopy demonstrated that the vesicles had a diameter of 84 +/- 6 nm. The immunoisolated vesicles had a highly simplified protein pattern on two-dimensional gel electrophoresis.