Disruption of Golgi structure and function in mammalian cells expressing amutant dynamin

The large GTPase dynamin is a mechanoenzyme that participates in the scissi on of nascent vesicles from the plasma membrane. Recently, dynamin has been demonstrated to associate with the Golgi apparatus in mammalian cells by m orphological and biochemical methods. Additional studies using a well chara cterized, cell-free assay have supported these findings by demonstrating a requirement for dynamin function in the formation of clathrin-coated, and n on-clathrin-coated vesicles from the trans-Golgi network (TGN), Tn this stu dy, we tested if dynamin participates in Golgi function in living cells thr ough the expression of a dominant negative dynamin construct (K44A), Cells co-transfected to express this mutant dynamin and a GFP-tagged Golgi reside nt protein (TGN38) exhibit Golgi structures that are either compacted, vesi culated, or tubulated, Electron microscopy of these mutant cells revealed l arge numbers of Golgi stacks comprised of highly tubulated cisternae and an extraordinary number of coated vesicle buds. Cells expressing mutant dynam in and GFP-tagged VSVG demonstrated a marked retention (8- to 11-fold) of t he nascent viral G-protein in the Golgi compared to control cells. These ob servations in living cells are consistent with previous morphological and i n vitro studies demonstrating a role for dynamin in the formation of secret ory vesicles from the TGN.