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

Abstract

Synaptotagmin 1 acts as the Ca 2þ 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 fulllength 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 2þ ; 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 2þ , the Ca 2þ 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 2þ 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 2þ .

Partial metal ion saturation of C2 domains primes Syt1-membrane interactions

“…To determine how the occupancy of metal ion binding sites influences the membrane insertion of the C2 domains in full-length Syt1, we conducted EPR experiments on the protein reconstituted into anionic LUVs. Under the conditions of our EPR experiments, both C2 domains of Syt1 bind in cis with respect to the transmembrane segment that anchors the protein to the LUVs (43). The spinlabeled sidechain R1 ( Fig.…”

“…1A) constitute the membrane-binding unit of Syt1 (18,41). Their distinct electrostatic properties and preferences towards binding partners govern the Syt1-mediated vesicle fusion (15,42,43). The question of the C2AB conformational preferences has elicited some discussion in the literature.…”

“…The full length-Syt1 (FL-Syt1) (residues 1-421) with the native cysteines mutated as: C73A, C74A, C76A, C78A, C82S, and C277S was cloned into a pET-28a vector. For EPR measurements, additional cysteine mutation were introduced into the cysteine-free FL-Syt1 at either positions M173C or V304C and expressed as 6xHis-tagged proteins in the E. coli BL21(DE3)-RIL strain as described before (22,43). All mutants were prepared using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA.)…”

“…Expression and purification steps from the previously described protocols were followed (22,27,43). Briefly, all SUMO fusion proteins were purified from cell lysates using HisTrap HP columns followed by removal of 6xHis-SUMO fusion tag using SUMO protease.…”

“…For EPR measurements, FL-Syt1 was reconstituted into POPC:POPS=85:15 at a 1:200 protein to lipid ratio and dialyzed into metal-free buffer (20 mM HEPES, 150 mM KCl, pH 7.4) in the presence of Bio-Beads (Bio-Rad Laboratories, Hercules, CA) (43). Pb 2+ was then added to samples at either a 2:1 Pb 2+ :FL-Syt1 ratio or in 12to 50-fold excess.…”