posted on 2015-12-17, 06:34authored byJoseph
K. Vasquez, Kan Chantranuvatana, Daniel
T. Giardina, Matthew D. Coffman, Jefferson D. Knight
The
synaptotagmin (Syt) family of proteins contains tandem C2 domains,
C2A and C2B, which bind membranes in the presence of Ca2+ to trigger vesicle fusion during exocytosis. Despite recent progress,
the role and extent of interdomain interactions between C2A and C2B
in membrane binding remain unclear. To test whether the two domains
interact on a planar lipid bilayer (i.e., experience thermodynamic
interdomain contacts), diffusion of fluorescent-tagged C2A, C2B, and
C2AB domains from human Syt7 was measured using total internal reflection
fluorescence microscopy with single-particle tracking. The C2AB tandem
exhibits a lateral diffusion constant approximately half the value
of the isolated single domains and does not change when additional
residues are engineered into the C2A–C2B linker. This is the
expected result if C2A and C2B are separated when membrane-bound;
theory predicts that C2AB diffusion would be faster if the two domains
were close enough together to have interdomain contact. Stopped-flow
measurements of membrane dissociation kinetics further support an
absence of interdomain interactions, as dissociation kinetics of the
C2AB tandem remain unchanged when rigid or flexible linker extensions
are included. Together, the results suggest that the two C2 domains
of Syt7 bind independently to planar membranes, in contrast to reported
interdomain cooperativity in Syt1.