Isolation of exocytic carrier vesicles from BHK cells.
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.
de Curtis
I
I
European Molecular Biology Laboratory, Heidelberg, Federal Republic of Germany.
Simons
K
K
D000818:Animals; D001692:Biological Transport; D002458:Cell Fractionation; D002460:Cell Line; D002474:Cell-Free System; D006224:Cricetinae; D003594:Cytoplasmic Granules / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D005089:Exocytosis; D005700:Galactosyltransferases / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism*; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000378:metabolism*; D007668:Kidney / Q000648:ultrastructure; D008562:Membrane Glycoproteins / Q000378:metabolism*; D011499:Protein Processing, Post-Translational
D008562:Membrane Glycoproteins; D005700:Galactosyltransferases