SNARE Status Regulates Tether Recruitment and Function in Homotypic COPII Vesicle Fusion

Abstract

In mammals, coat complex II (COPII)-coated transport vesicles deliver secretory cargo to vesicular tubular clusters (VTCs) that facilitate cargo sorting and transport to the Golgi. We documented in vitro tethering and SNARE-dependent homotypic fusion of endoplasmic reticulum-derived COPII transport vesicles to form larger cargo containers characteristic of VTCs (Xu, D., and Hay, J. C. (2004) J. Cell Biol. 167, 997-1003). COPII vesicles thus appear to contain all necessary components for homotypic tethering and fusion, providing a pathway for de novo VTC biogenesis. Here we demonstrate that antibodies against the endoplasmic reticulum/Golgi SNARE Syntaxin 5 inhibit COPII vesicle homotypic tethering as well as fusion, implying an unanticipated role for SNAREs upstream of fusion. Inhibition of SNARE complex access and/or disassembly with dominant-negative ␣-soluble NSF attachment protein (SNAP) also inhibited tethering, implicating SNARE status as a critical determinant in COPII vesicle tethering. The tethering-defective vesicles generated in the presence of dominant-negative ␣-SNAP specifically lacked the Rab1 effectors p115 and GM130 but not other peripheral membrane proteins. Furthermore, Rab effectors, including p115, were shown to be required for homotypic COPII vesicle tethering. Thus, our results demonstrate a requirement for SNARE-dependent tether recruitment and function in COPII vesicle fusion. We anticipate that recruitment of tether molecules by an upstream SNARE signal ensures that tethering events are initiated only at focal sites containing appropriately poised fusion machinery.

Minimal membrane docking requirements revealed by reconstitution of Rab GTPase-dependent membrane fusion from purified components

“…Antibodies against Sec18p and removal of ATP prevent vacuole docking; both of these treatments inhibit cis-SNARE complex disassembly (5, 50) and would be expected to block formation of 3Q cis-SNARE or 3Q:1R transSNARE complexes. In an in vitro assay for formation of the vesicular tubular cluster, an intermediate in transport from the endoplasmic reticulum (ER) to the Golgi apparatus, antibodies against Syntaxin 5, a homolog of Vam3p, inhibit vesicle ''coisolation,'' as does addition of a dominant negative mutant of ␣-SNAP that blocks NSF SNARE complex disassembly activity (58,59). Docking of synaptic vesicles to the plasma membrane in Caenorhabditis elegans neuromuscular junctions also requires syntaxin (60).…”

Apoptosis-linked Gene-2 (ALG-2)/Sec31 Interactions Regulate Endoplasmic Reticulum (ER)-to-Golgi Transport

“…Antibodies and Expression Constructs-Rabbit polyconal anti-Sec31 (10) and anti-p24 (14) were produced in rabbits against synthetic peptides and affinity-purified as described previously. Monoclonal anti-Rbet1 antibodies were described before (37).…”

“…p115 appears to be packaged in budding COPII vesicles (15), is essential for their fusion (14), and rapidly cycles on and off the membrane (55) presumably as a function of SNARE availability. In triply transfected cells displaying delayed transport, p115 distribution was noticeably altered (Fig.…”

“…COPII vesicles fuse homotypically to produce vesicular tubular clusters (VTCs). This process involves multiple tethers and the ER/Golgi SNARE complex (12)(13)(14)(15). Maturing VTCs are the primary site of cargo concentration (16).…”

Coordination of Golgin Tethering and SNARE Assembly

“…This is believed to link SNAREs on apposing membranes and promote their assembly into SNAREpin complexes, thereby directly connecting tethering with downstream SNARE-mediated fusion events (27). The interaction with unassembled SNAREs also helps recruit p115 to the membrane, which may ensure that tethering only occurs at sites where active unassembled SNAREs are present (17,18). Although it is clear p115 binds to GM130, the functional role of this interaction in the context of tethering and fusion is debated.…”