Disruption of Golgi structure and function

The formation of nascent secretory vesicles from the transGolgi network (TGN) involves the coordinated participation of multiple enzymes, coat proteins, and cytoskeletal elements. Currently, the mechanisms that liberate nascent budding vesicles from the TGN are undefined. The large GTPase dynamin is an attractive candidate to participate in this function for several reasons. It is a mechanoenzyme that has been shown to bind and hydrolyze nucleotide while undergoing a conformational change to distort and sever membrane tubules (Sweitzer and Hinshaw, 1998) and has been referred to as a ‘molecular pinchase’ (McNiven, 1998; McNiven et al., 2000). Further, a role for dynamin in the liberation of both clathrincoated pits and caveolae from the plasma membrane during endocytosis has been reported by multiple laboratories (Henley et al., 1998; McNiven et al., 2000). The plasma membrane and Golgi apparatus utilize many of the same protein families to produce nascent vesicles during endocytosis and secretion. It seems likely that dynamin could function at both cellular locations. In support of this premise, several reports have implicated dynamin in the formation of secretory vesicles from the TGN (Henley and McNiven, 1996; Jones et al., 1998; Maier et al., 1996). These initial findings included the immunofluorescense and immunoelectron microscopic localization of dynamin to the Golgi apparatus using multiple dynamin antibodies (Henley and McNiven, 1996; Maier et al., 1996) and the immunoisolation of rat liver Golgi using dynamin antibody-coated beads (Henley and McNiven, 1996). In a recent study, a specific form of dynamin 2 coupled to green fluorescent protein, [Dyn 2(aa)-GFP] was observed to target to clathrin-coated pits at both the plasma membrane and the Golgi in cultured cells (Cao et al., 1998). Most recently, dynamin function has been shown to be required for the formation of TGN-derived secretory vesicles in vitro using a well characterized, cell-free assay (Jones et al., 1998). The combined observations described above strongly suggest that Dyn 2 associates with the Golgi apparatus in mammalian cells where it may release newly forming secretory vesicles from the TGN. In this present study we have closely monitored the morphology and function of the Golgi apparatus in cultured cells expressing a mutant dynamin protein. Specifically, we have constructed a point mutation in the first GTP-binding element (K44A) of Dyn 2(aa), the dynamin form that is normally expressed in epithelial cells and has been shown to associate with the Golgi apparatus (Cao et al., 1998). We find that epithelial cells expressing this mutant dynamin protein display either compacted, vesiculated, or tubulated Golgi structures that are defective in the transport of GFPtagged vesicular stomatitus viral glycoprotein (VSVG) to the plasma membrane. These findings are in direct support of previous studies 1993 Journal of Cell Science 113, 1993-2002(2000) Printed in Great Britain © The Company of Biologists Limited 2000 JCS1387