Complexes of syndapin II with dynamin II promote vesicle formation at the trans-Golgi network

Abstract

The role of dynamin and so-called accessory proteins in endocytosis is well established. However, molecular details of the function(s) of dynamin II at the Golgi are largely unclear. We demonstrate that the ubiquitously expressed syndapin II isoform interacts with the proline-rich domain (PRD) of dynamin II through its Src-homology 3 (SH3) domain. Co-immunoprecipitation of endogenous syndapin II and dynamin II, and successful reconstitutions of such complexes at membranes in COS-7 cells, show the in vivo relevance of the interaction. Syndapin II can associate with Golgi membranes and this association increases upon Golgi exit block. Brefeldin A treatment clearly shows that the observed perinuclear localization of syndapin II co-localizing with syntaxin 6 reflects the Golgi complex and that it requires functional integrity of the Golgi. Syndapins are crucial for Golgi vesicle formation because anti-syndapin antibodies, used either in in vitro reconstitutions or in living cells, inhibited this process. Both types of assays additionally revealed the essential role of syndapin II SH3 interactions with the dynamin II PRD in vesicle formation. An excess of the syndapin SH3 domain strongly inhibited budding from Golgi membranes in vitro. Likewise, overexpression of the syndapin SH3 domain or of a dynamin II variant incapable of associating with syndapin II (dynamin IIΔPRD) impaired trafficking of vesicular stomatitis virus glycoprotein (VSVG)-GFP in vivo. By contrast, full-length syndapin II-l had no negative effect, and instead promoted VSVG-GFP export from the Golgi. Importantly, a cytosolic fraction containing endogenous syndapin-dynamin complexes was sufficient to promote vesicle formation from Golgi membranes in a syndapin-dependent manner. Thus, syndapin-dynamin complexes are crucial and sufficient to promote vesicle formation from the trans-Golgi network.

LIM Kinase 1 and Cofilin Regulate Actin Filament Population Required for Dynamin-dependent Apical Carrier Fission from theTrans-Golgi Network

“…Note that Golgi markers are excluded from these transporters. (2) p75GFP-containing tubules extend along MT drawn by a kinesin motor; syndapin 2, via its BAR-domain, binds to the membrane curvature of the Golgi (Kessels et al, 2006) and facilitates membrane remodeling necessary for tubulation. (3) p75-GFP tubules undergo fission from the TGN or into smaller transport vesicles; this process depends on actin filaments (regulated by LIMK 1-cofilin), which recruit cortactin, as well as syndapin 2.…”

“…These proteins form Golgi complexes with dynamin 2 via their SH3 domains (Cao et al, 2005;Kessels et al, 2006) and may be expected to regulate Golgi trafficking via their interactions with the actin cytoskeleton in the vicinity of this organelle Kelly, 2000, Cao et al, 2003;Kessels et al, 2006). Overexpression of LIMK1-KD decreased the levels of syndapin 2 and cortactin at the Golgi ( Figure 5D).…”

“…In contrast, it has been shown that recruitment of dynamin to the Golgi is dependent on actin and cortactin (Cao et al, 2005), as well as on syndapin 2 (Kessels et al, 2006). A unifying interpretation of these data are that the role of LIMK in the TGN is to promote the assembly of a population of actin filaments that are required for generation and dynamin-mediated fission of post-Golgi carriers for p75.…”

“…Interestingly, dynamin 2 mediates fission from the TGN of vesicular stomatitis virus G protein carriers in a cell-specific manner (Cao et al, 2000(Cao et al, , 2005Bonazzi et al, 2005). The cargo/cellspecific TGN fission activity of dynamin 2 may reflect cargo/cell specific requirements for its partners, cortactin and syndapin 2, involved in its recruitment to the TGN (Cao et al, 2005;Kessels et al, 2006;Yang et al, 2006). Additionally, these dynamin partners may contribute to the generation of post-Golgi routes through dynamin-independent functions, e.g., syndapin 2's abilities to promote actin filament assembly via Wiskott-Aldrich syndrome protein (WASP) or to bend membranes via its BAR domain (Kessels et al, 2006).…”

“…The cargo/cellspecific TGN fission activity of dynamin 2 may reflect cargo/cell specific requirements for its partners, cortactin and syndapin 2, involved in its recruitment to the TGN (Cao et al, 2005;Kessels et al, 2006;Yang et al, 2006). Additionally, these dynamin partners may contribute to the generation of post-Golgi routes through dynamin-independent functions, e.g., syndapin 2's abilities to promote actin filament assembly via Wiskott-Aldrich syndrome protein (WASP) or to bend membranes via its BAR domain (Kessels et al, 2006). The latter function of syndapin 2 could be critically needed, e.g., to shape tubular carriers, such as those used by apical cargo proteins to exit the TGN (Kreitzer et al, 2000(Kreitzer et al, , 2003.…”