Interaction of synaptotagmin with lipid bilayers, analyzed by single-molecule force spectroscopy.
Synaptotagmin I is the major Ca²(+) sensor for membrane fusion during neurotransmitter release. The cytoplasmic domain of synaptotagmin consists of two C2 domains, C2A and C2B. On binding Ca²(+), the tips of the two C2 domains rapidly and synchronously penetrate lipid bilayers. We investigated the forces of interaction between synaptotagmin and lipid bilayers using single-molecule force spectroscopy. Glutathione-S-transferase-tagged proteins were attached to an atomic force microscope cantilever via a glutathione-derivatized polyethylene glycol linker. With wild-type C2AB, the force profile for a bilayer containing phosphatidylserine had both Ca²(+)-dependent and Ca²(+)-independent components. No force was detected when the bilayer lacked phosphatidylserine, even in the presence of Ca²(+). The binding characteristics of C2A and C2B indicated that the two C2 domains cooperate in binding synaptotagmin to the bilayer, and that the relatively weak Ca²(+)-independent force depends only on C2A. When the lysine residues K189-192 and K326, 327 were mutated to alanine, the strong Ca²(+)-dependent binding interaction was either absent or greatly reduced. We conclude that synaptotagmin binds to the bilayer via C2A even in absence of Ca²(+), and also that positively charged regions of both C2A and C2B are essential for the strong Ca²(+)-dependent binding of synaptotagmin to the bilayer.
Takahashi
Hirohide
H
Kyoto University, Japan.
Shahin
Victor
V
Henderson
Robert M
RM
Takeyasu
Kunio
K
Edwardson
J Michael
JM
D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism*; D018625:Microscopy, Atomic Force; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D050857:Synaptotagmins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D008051:Lipid Bilayers; D050857:Synaptotagmins; D002118:Calcium