MECHANISM OF FORMATION OF POST-GOLGI VESICLES FROM TGN MEMBRANES - ARF-DEPENDENT COAT ASSEMBLY AND PKC-REGULATED VESICLE SCISSION

We have developed an experimental system that utilizes purified Golgi fractions obtained from virus infected infected MDCK cells to reproduc e in vitro the process of vesicle generation in the trans Golgi networ k, an important site for the sorting of proteins addressed to the plas ma membrane, secretory vesicles, or lysosomes. Using an integrated bio chemical and electron microscopic approach, we have shown that the for mation of post Golgi vesicles carrying proteins destined to both plasm a membrane domains of epithelial cells requires the activation of an A rf-like GTP-binding protein that serves to promote the assembly of the protein coat necessary to deform the donor membrane and generate a ve sicle. The formation of the post Golgi vesicles also requires the part icipation of a Golgi membrane-associated Protein Kinase C, but not its phosphorylating activity. Other authors have shown that this is also the case for the PKC activation of the enzyme phospholipase D, which g enerates phosphatidic acid from phosphatidyl choline and may be involv ed in remodeling of membranes. We have been able to dissect the proces s of post Golgi vesicle generation into two sequential stages, one of coat assembly and bud formation, and a subsequent one of vesicle sciss ion. The first stage can occur at 20 degrees C and requires the activa tion of the Arf protein necessary for coat assembly. The second stage does not require nucleotides or an energy supply, but requires cytosol ic proteins, and in particular, an NEM sensitive membrane scission pro moting activity that operates only at a higher temperature of incubati on. Because various PKC inhibitors blocked vesicle scission without pr eventing bud formation, we propose that the PKC is required for the ac tivation of a PLD in the TGN, which leads to remodeling of the donor m embrane and the severing of connections between the emerging vesicles and the membranes.