Structure and dynamics of a two-helix SNARE complex in live cells.
SNAREs are clustered membrane proteins essential for intracellular fusion steps. During fusion, three to four SNAREs with a Q(a)-, Q(b)-, Q(c)- and R-SNARE-motif form a complex. The core complex represents a Q(a)Q(b)Q(c)R-SNARE-motif bundle, most certainly assembling in steps. However, to date it is unknown which intermediate SNARE complex observed in vitro also exists in vivo. Here we have applied comparative fluorescence recovery after photobleaching (FRAP)-studies as a novel approach for studying in intact cells a SNARE interaction involved in synaptic vesicle fusion [catalyzed by syntaxin 1A (Q(a)), SNAP25 (Q(b)/Q(c)) and synaptobrevin 2 (R)]. We find that the Q(b)-SNARE-motif of SNAP25 interacts reversibly with clustered syntaxin. The interaction requires most of the alpha helical Q(b)-SNARE-motif and depends on its position within the molecule. We conclude that a zippered Q(a)Q(b)-SNARE complex represents a short-lived SNARE intermediate in intact cells, most likely providing an initial molecular platform toward membrane fusion.
Halemani
Nagaraj D
ND
Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.
Bethani
Ioanna
I
Rizzoli
Silvio O
SO
Lang
Thorsten
T
D000595:Amino Acid Sequence; D000818:Animals; D036681:Fluorescence Recovery After Photobleaching; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000378:metabolism; D016716:PC12 Cells; D017433:Protein Structure, Secondary; D021381:Protein Transport; D051381:Rats; D050600:SNARE Proteins / Q000737:chemistry / Q000378:metabolism*; D013572:Synaptic Vesicles / Q000378:metabolism*; D050825:Synaptosomal-Associated Protein 25 / Q000737:chemistry / Q000378:metabolism; D050827:Syntaxin 1 / Q000737:chemistry / Q000378:metabolism; D050988:Vesicle-Associated Membrane Protein 2 / Q000737:chemistry / Q000378:metabolism
D009419:Nerve Tissue Proteins; D050600:SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; D050988:Vesicle-Associated Membrane Protein 2