SNARE status regulates tether recruitment and function in homotypic COPII vesicle fusion.
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.
Bentley
Marvin
M
Division of Biological Sciences, University of Montana, Missoula, MT 59812-4824, USA.
Liang
Yingjian
Y
Mullen
Karl
K
Xu
Dalu
D
Sztul
Elizabeth
E
Hay
Jesse C
JC
D000595:Amino Acid Sequence; D000818:Animals; D022181:COP-Coated Vesicles; D002478:Cells, Cultured; D008969:Molecular Sequence Data; D051381:Rats; D050600:SNARE Proteins / Q000378:metabolism*
D050600:SNARE Proteins