Phosphatidylinositol 4,5 Bisphosphate Controls the cis and trans Interactions of Synaptotagmin 1.

Synaptotagmin 1 acts as the Ca sensor for synchronous neurotransmitter release; however, the mechanism by which it functions is not understood and is presently a topic of considerable interest. Here, we describe measurements on full-length membrane-reconstituted synaptotagmin 1 using site-directed spin labeling in which we characterize the linker region as well as the cis (vesicle membrane) and trans (cytoplasmic membrane) binding of its two C2 domains. In the full-length protein, the C2A domain does not undergo membrane insertion in the absence of Ca; however, the C2B domain will bind to and penetrate in trans to a membrane containing phosphatidylinositol 4,5 bisphosphate, even if phosphatidylserine (PS) is present in the cis membrane. In the presence of Ca, the Ca binding loops of C2A and C2B both insert into the membrane interface; moreover, C2A preferentially inserts into PS-containing bilayers and will bind in a cis configuration to membranes containing PS even if a phosphatidylinositol 4,5 bisphosphate membrane is presented in trans. The data are consistent with a bridging activity for synaptotagmin 1 in which the two domains bind to opposing vesicle and plasma membranes. The failure of C2A to bind membranes in the absence of Ca and the long unstructured segment linking C2A to the vesicle membrane indicates that synaptotagmin 1 could act to significantly shorten the vesicle-plasma membrane distance with increasing levels of Ca.