A mutant impaired in SNARE complex dissociation identifies the plasma membrane as first target of synaptobrevin 2.
Membrane fusion depends on the formation of a complex of four SNARE motifs, three that bear a central glutamine and are localized in the target membrane (t-SNARE) and one that bears an arginine and is localized in the donor vesicle (v-SNARE). We have characterized the arginine 56 to proline mutant (R56P) of synaptobrevin-2 (Sb). SbR56P was blocked at the plasma membrane in association with the endogenous plasma membrane t-SNARE due to an inhibition of SNARE complex dissociation, suggesting that the plasma membrane is its first target. Cell surface blockade of SbR56P could be rescued by coexpression of synaptophysin, a partner of Sb. Sb was blocked at the plasma membrane but SNARE complexes were unaffected in cells expressing defective dynamin, indicating that the phenotype of SbR56P was not due to an internalization defect. When expressed in neurons, SbR56P localized both to axonal and dendritic plasma membranes, showing that both domains are initial targets of Sb. The R56P mutation affects a highly conserved position in v-SNAREs, and might thus provide a general tool for identifying their first target membranes.
Martinez-Arca
Sonia
S
Membrane Traffic and Neuronal Plasticity, INSERM U536, Institut du Fer-à-Moulin, 75005, Paris, France.
Arold
Stefan
S
Rudge
Rachel
R
Laroche
Fabrice
F
Galli
Thierry
T
D000818:Animals; D001120:Arginine / Q000235:genetics; D001369:Axons / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D003712:Dendrites / Q000378:metabolism; D006367:HeLa Cells; D006624:Hippocampus / Q000166:cytology; D006358:Hot Temperature / Q000009:adverse effects; D006801:Humans; D007477:Ions / Q000737:chemistry; D007700:Kinetics; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D011392:Proline / Q000235:genetics; D011485:Protein Binding; D050683:R-SNARE Proteins; D011817:Rabbits; D051381:Rats; D050600:SNARE Proteins; D013816:Thermodynamics; D033921:Vesicular Transport Proteins / Q000737:chemistry / Q000378:metabolism*
D007477:Ions; D008565:Membrane Proteins; D046912:Multiprotein Complexes; D050683:R-SNARE Proteins; D050600:SNARE Proteins; D033921:Vesicular Transport Proteins; D001120:Arginine; D011392:Proline