Stepwise assembly of functionally active transport vesicles.
Budding of COP-coated vesicles (the likely carriers of newly synthesized proteins from the endoplasmic reticulum through the Golgi stack) from Golgi cisternae requires ADP-ribosylation factor (ARF), coatomer proteins from the cytosol, GTP, and fatty acyl-coenzyme A (CoA). The assembly of coated buds on the membranes requires coatomer, ARF, and GTP. When palmitoyl-CoA is added, membrane fission occurs at the coated bud, releasing coated vesicles. We show that COP-coated vesicles can be generated stepwise in vitro and isolated in a functionally active form, demonstrating that the minimal set of cytosolic components required for their formation as well as principal steps in their assembly have been identified.
Ostermann
J
J
Cellular Biochemistry and Biophysics Program Memorial Sloan Kettering Cancer Center, New York, New York 10021.
Orci
L
L
Tani
K
K
Amherdt
M
M
Ravazzola
M
M
Elazar
Z
Z
Rothman
J E
JE
D020727:ADP-Ribosylation Factors; D000214:Acyl Coenzyme A / Q000378:metabolism; D000818:Animals; D001693:Biological Transport, Active; D016466:CHO Cells; D002451:Cell Compartmentation; D020756:Coatomer Protein; D006224:Cricetinae; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D009024:Morphogenesis; D015388:Organelles / Q000378:metabolism / Q000648:ultrastructure*
D000214:Acyl Coenzyme A; D020756:Coatomer Protein; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D020727:ADP-Ribosylation Factors