Distinct domains of complexins bind SNARE complexes and clamp fusion in vitro.
In regulated exocytosis, the core membrane fusion machinery proteins, the SNARE proteins, are assisted by a group of regulatory factors in order to couple membrane fusion to an increase of intracellular calcium ion (Ca(2+)) concentration. Complexin-I and synaptotagmin-I have been shown to be key elements for this tightly regulated process. Many studies suggest that complexin-I can arrest the fusion reaction and that synaptotagmin-I can release the complexin-I blockage in a calcium-dependent manner. Although the actual molecular mechanism by which they exert their function is still unknown, recent in vivo experiments postulate that domains of complexin-I produce different effects on neurotransmitter release. Herein, by using an in vitro flipped SNARE cell fusion assay, we have identified and characterized the minimal functional domains of complexin-I necessary to couple calcium and synaptotagmin-I to membrane fusion. Moreover, we provide evidence that other isoforms of complexin, complexin-II, -III, and -IV, can also be functionally coupled to synaptotagmin-I and calcium. These correspond closely to results from in vivo experiments, providing further validation of the physiological relevance of the flipped SNARE system.
Giraudo
Claudio G
CG
Department of Physiology and Cellular Biophysics, Columbia University, College of Physicians and Surgeons, 1150 St. Nicholas Avenue, New York, NY 10032, USA.
Garcia-Diaz
Alejandro
A
Eng
William S
WS
Yamamoto
Ai
A
Melia
Thomas J
TJ
Rothman
James E
JE
D033942:Adaptor Proteins, Vesicular Transport; D000595:Amino Acid Sequence; D002118:Calcium / Q000737:chemistry*; D002462:Cell Membrane / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007477:Ions; D008954:Models, Biological; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry; D018377:Neurotransmitter Agents / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D050600:SNARE Proteins / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D050863:Synaptotagmin I / Q000737:chemistry
D033942:Adaptor Proteins, Vesicular Transport; D007477:Ions; D009419:Nerve Tissue Proteins; D018377:Neurotransmitter Agents; D011994:Recombinant Proteins; D050600:SNARE Proteins; D050863:Synaptotagmin I; C095992:complexin I; D002118:Calcium