Transmembrane-domain determinants for SNARE-mediated membrane fusion.
Neurosecretion involves fusion of vesicles with the plasma membrane. Such membrane fusion is mediated by the SNARE complex, which is composed of the vesicle-associated protein synaptobrevin (VAMP2), and the plasma membrane proteins syntaxin-1A and SNAP-25. Although clearly important at the point of membrane fusion, the precise structural and functional requirements for the transmembrane domains (TMDs) of SNAREs in bringing about neurosecretion remain largely unknown. Here, we used a bimolecular fluorescence complementation (BiFC) approach to study SNARE protein interactions involving TMDs in vivo. VAMP2 molecules were found to dimerise through their TMDs in intact cells. Dimerisation was abolished when replacing a glycine residue in the centre of the TMD with residues of increasing molecular volume. However, such mutations still were fully competent in bringing about membrane-fusion events, suggesting that dimerisation of the VAMP2 TMDs does not have an important functional role. By contrast, a series of deletion or insertion mutants in the C-terminal half of the TMD were largely deficient in supporting neurosecretion, whereas mutations in the N-terminal half did not display severe secretory deficits. Thus, structural length requirements, largely confined to the C-terminal half of the VAMP2 TMD, seem to be essential for SNARE-mediated membrane-fusion events in cells.
Fdez
Elena
E
Institute of Parasitology and Biomedicine López-Neyra, Consejo Superior de Investigaciones Científicas, Avda del Conocimiento s/n, 18100 Granada, Spain.
Martínez-Salvador
Mar
M
Beard
Matthew
M
Woodman
Philip
P
Hilfiker
Sabine
S
D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D008561:Membrane Fusion / Q000235:genetics; D008856:Microscopy, Fluorescence; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D009489:Neurosecretion / Q000235:genetics; D016716:PC12 Cells; D055503:Protein Multimerization / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D051381:Rats; D050600:SNARE Proteins / Q000235:genetics / Q000378:metabolism*; D050825:Synaptosomal-Associated Protein 25 / Q000378:metabolism; D050827:Syntaxin 1 / Q000378:metabolism; D050988:Vesicle-Associated Membrane Protein 2 / Q000235:genetics / Q000378:metabolism*
D046912:Multiprotein Complexes; D050600:SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; D050988:Vesicle-Associated Membrane Protein 2