synaptotagmin mutants reveal essential functions for the C2B domain in Ca2+-triggered fusion and recycling of synaptic vesicles in vivo.
Synaptotagmin has been proposed to function as a Ca(2+) sensor that regulates synaptic vesicle exocytosis, whereas the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex is thought to form the core of a conserved membrane fusion machine. Little is known concerning the functional relationships between synaptotagmin and SNAREs. Here we report that synaptotagmin can facilitate SNARE complex formation in vitro and that synaptotagmin mutations disrupt SNARE complex formation in vivo. Synaptotagmin oligomers efficiently bind SNARE complexes, whereas Ca(2+) acting via synaptotagmin triggers cross-linking of SNARE complexes into dimers. Mutations in Drosophila that delete the C2B domain of synaptotagmin disrupt clathrin AP-2 binding and endocytosis. In contrast, a mutation that blocks Ca(2+)-triggered conformational changes in C2B and diminishes Ca(2+)-triggered synaptotagmin oligomerization results in a postdocking defect in neurotransmitter release and a decrease in SNARE assembly in vivo. These data suggest that Ca(2+)-driven oligomerization via the C2B domain of synaptotagmin may trigger synaptic vesicle fusion via the assembly and clustering of SNARE complexes.
Littleton
J T
JT
Department of Physiology and Entomology, University of Wisconsin, Madison, Wisconsin 53706, USA.
Bai
J
J
Vyas
B
B
Desai
R
R
Baltus
A E
AE
Garment
M B
MB
Carlson
S D
SD
Ganetzky
B
B
Chapman
E R
ER
D033965:Adaptor Protein Complex alpha Subunits; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D001704:Biopolymers / Q000096:biosynthesis / Q000737:chemistry; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002135:Calcium-Binding Proteins; D019281:Dimerization; D004330:Drosophila; D004705:Endocytosis / Q000502:physiology; D005089:Exocytosis / Q000502:physiology; D046911:Macromolecular Substances; D008561:Membrane Fusion / Q000187:drug effects / Q000502:physiology*; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011233:Precipitin Tests; D011487:Protein Conformation; D017434:Protein Structure, Tertiary / Q000235:genetics; D051381:Rats; D050600:SNARE Proteins; D013329:Structure-Activity Relationship; D013572:Synaptic Vesicles / Q000378:metabolism*; D050857:Synaptotagmins; D033921:Vesicular Transport Proteins
D033965:Adaptor Protein Complex alpha Subunits; D033942:Adaptor Proteins, Vesicular Transport; D001704:Biopolymers; D002135:Calcium-Binding Proteins; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050600:SNARE Proteins; D033921:Vesicular Transport Proteins; D050857:Synaptotagmins; D002118:Calcium