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 alpha-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 alpha-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.
Highlights
Vesicle targeting and fusion in the endomembrane system are mediated by several conserved protein families
In vitro SNARE assembly studies involving the Rab1 effector p115 and ER/Golgi SNAREs suggested that during Golgi reassembly p115 first mediated membrane tethering through its interactions with other Golgi tethers, and facilitated trans-SNARE complex formation via direct p115-SNARE interactions [8]
Our data demonstrate that SNAREs play an unanticipated role upstream of fusion in recruiting GM130 and p115 to complex II (COPII) vesicles for function in homotypic tethering
Summary
Golgi vesicle protein Giantin are proposed to work in complex with p115 as tethers and are required for anterograde ER-toGolgi transport at a late step as well as intra-Golgi transport (19 –21). ER-to-Golgi cargo transport must involve at least two membrane fusions as follows: one to transfer cargo from COPII vesicles into VTCs in the periphery of the cell, and a second to transfer cargo from Golgi-centric VTCs into the cis-Golgi (or alternatively to create the cis-Golgi de novo). This complexity of fusion events has not been dissected using in vitro reconstitutions of ER-to-Golgi transport [1, 31, 32]. Whether p115, GM130, or other Rab effectors are involved in homotypic COPII vesicle fusion has not been determined
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