Synaptotagmin isoforms couple distinct ranges of Ca2+, Ba2+, and Sr2+ concentration to SNARE-mediated membrane fusion

Abstract

Ca2+-triggered exocytosis of synaptic vesicles is controlled by the Ca2+-binding protein synaptotagmin (syt) I. Fifteen additional isoforms of syt have been identified. Here, we compared the abilities of three syt isoforms (I, VII, and IX) to regulate soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated membrane fusion in vitro in response to divalent cations. We found that different isoforms of syt couple distinct ranges of Ca2+, Ba2+, and Sr2+ to membrane fusion; syt VII was approximately 400-fold more sensitive to Ca2+ than was syt I. Omission of phosphatidylserine (PS) from both populations of liposomes completely abrogated the ability of all three isoforms of syt to stimulate fusion. Mutations that selectively inhibit syt.target-SNARE (t-SNARE) interactions reduced syt stimulation of fusion. Using Sr2+ and Ba2+, we found that binding of syt to PS and t-SNAREs can be dissociated from activation of fusion, uncovering posteffector-binding functions for syt. Our data demonstrate that different syt isoforms are specialized to sense different ranges of divalent cations and that PS is an essential effector of Ca2+.syt action.

Figure 1.

Syt I, VII, and IX stimulate SNARE-mediated membrane fusion in response to divalent cations. (A) Illustration showing the different components of the in vitro fusion assay. Fusion of v-SNARE (v) vesicles, containing a donor (D) and acceptor (A) FRET pair, with unlabeled t-SNARE vesicles (t), results in an increase in donor fluorescence. The cytoplasmic domain of syt was assayed for its ability to stimulate fusion in the presence of different divalent cations. (B) Syt I, VII, and IX differentially stimulate fusion in response to divalent cations. The indicated syt isoform (7 μM) was incubated with v- and t-SNARE vesicles in the presence of 1 mM Ca²⁺ (open circles), Ba²⁺ (open squares), Sr²⁺ (open diamonds), or Mg²⁺ (closed triangles), or in the presence of 0.2 mM EGTA (open triangles). Rounds of fusion were plotted as a function of time. (C) Divalent cation dependency of syt I-, VII-, and IX-stimulated membrane fusion. v-SNARE and t-SNARE vesicles were incubated in the presence of the indicated syt isoforms (7 μM) and [divalent cation]. The total amount of fusion (t = 120 min) was plotted as a function of [divalent cation]. The [divalent cation]_1/2 values are listed in Table 1. (D) Ca²⁺·syt-stimulated membrane fusion was efficiently blocked by cd VAMP. The indicated syt isoform (7 μM) was mixed with v- and t-SNARE vesicles in the presence of 0.2 mM EGTA (open triangles), 1 mM Ca²⁺ (open circles), or 1 mM Ca²⁺ plus cd VAMP (10 μM; closed diamonds). Rounds of fusion were plotted as a function of time.

Figure 2.

Coflotation of syt I, VII, and IX with reconstituted t-SNARE vesicles and PS-harboring protein-free vesicles. (A) The cytoplasmic domain of syt was incubated with vesicles before mixing with the Accudenz density media. The mixture was overlaid with decreasing concentrations of Accudenz. After centrifugation, the vesicles floated to the 0/30% Accudenz interface along with any bound syt. Samples collected from the 0/30% Accudenz interface were analyzed by SDS-PAGE and stained with Coomassie Blue. Divalent cation (1 mM) was present throughout the gradient. Unbound syt remained in the bottom portion of the tube. (B) The indicated syt isoforms (10 μM) were incubated with t-SNARE vesicles (100% PC) in the presence of 1 mM [divalent cation] and applied to gradients as described in A. Syt can bind to these proteoliposomes via interactions with membrane embedded t-SNAREs. (C) The indicated syt isoforms (10 μM) were incubated with protein-free vesicles composed of either 15% PS and 85% PC or 25% PS and 75% PC in the presence of 1 mM [divalent cation] and applied to gradients as described in A. syt can bind to these liposomes via interactions with PS. (D) Syt I does not stimulate fusion in Ba²⁺ or Sr²⁺ even when PS is increased to 25%. Syt I (7 μM) was incubated with v- and t-SNARE vesicles harboring either 15% PS (left) or 25% PS (right), in the presence of 1 mM Ca²⁺ (open circles), Ba²⁺ (open squares), Sr²⁺ (open diamonds), Mg²⁺ (closed triangles), or in the presence of 0.2 mM EGTA (open triangles). Rounds of fusion were plotted as a function of time. (E) The amount of stimulation obtained after 2 h compared with control (-syt) was plotted for each condition. Samples contained either 0.2 mM EGTA or 1 mM divalent cation. Percent stimulation by syt was determined using the following equation: (fusion with syt - fusion without syt) × 100/fusion without syt. Error bars represent the SD (n = 4).

Figure 3.

Full-length syt I and IX bind syntaxin in a divalent cation promoted manner. (A) Ca²⁺, Ba²⁺, and Sr²⁺, but not Mg²⁺, promote the coIP of native syt I with the t-SNARE syntaxin. Syntaxin was IPed from a rat brain detergent extract using HPC-1 as described in Materials and Methods in either 1 mM [divalent cation] or 0.2 mM EGTA. Samples were subjected to SDS-PAGE and immunoblot analysis using α-syt I (41.1) or α-syntaxin (HPC-1) antibodies. Total represents 3 μg of the rat brain extract; 25% of the IPed material was analyzed. (B) Bound syt I from A is quantified by densitometry. Error bars represent the SD (n = 5); ^**p < 0.05. (C) Full-length syntaxin and syt IX coIP in response to Ca²⁺ and Sr²⁺. syt IX-c-myc and syntaxin were cotransfected into HEK-293 cells. Detergent extracts from transfected cells were incubated with a α-c-myc antibody to IP syt IX. Samples (50% of the IPed material) were subjected to SDS-PAGE and immunoblot analysis using α-c-myc (syt IX) or α-syntaxin antibodies. Total represents 3 μg of the extract. (D) Syntaxin bound to syt IX (C) was quantified by densitometry. Error bars represent the SD (n = 3); ^**p < 0.05, ^*p = 0.05.

Figure 4.

Role of Ca²⁺ and t-SNARE binding activity in syt regulation of fusion. (A) Increasing the length of the linker that connects the C2-domains of syt I results in graded reductions in Ca²⁺-triggered binding to membrane embedded t-SNAREs. Wt, linker mutants, and Ca²⁺-ligand mutant forms of syt I, (10 μM) were incubated with protein-free (pf; 100% PC) or t-SNARE vesicles (100% PC) in the presence (+) or absence (-; 0.2 mM EGTA) of 1 mM Ca²⁺ as described in Figure 2A. (B) Bound protein from A was quantified by densitometry (n = 3, error bars represent the SD). (C) Increasing the length of the linker between the C2A and C2B domain of syt reduces syt-stimulated membrane fusion. Wild-type and linker mutant forms of syt I (10 μM) were incubated with v- and t-SNARE vesicles in the presence of 1 mM Ca²⁺ (open symbols). Error bars represent the SD (n = 4). (D) Role of Ca²⁺ binding to the C2A and C2B domain of syt in syt-stimulated membrane fusion. Fusion assays were carried out as described in C using a mutant forms of syt I that fail to bind Ca²⁺ via the C2A (C2A_MB) or C2B (C2AB_M) domain. These experiments were carried out using liposomes that contain 15% PS. Error bars represent the SD (n = 3). (E) Fusion assays were carried out as described in D except that the amount of PS in both vesicle populations was increased to 25%. Error bars represent the SD (n = 3). In C–E, the data obtained in EGTA were omitted for clarity; in EGTA, slight inhibition of fusion by syt was observed in all experiments. Percent stimulation by syt was determined as defined in Figure 2E.

Figure 5.

PS is an essential effector of Ca²⁺·syt action. (A) The indicated isoforms of syt (7 μM) were mixed with v-SNARE (v) and t-SNARE (t) vesicles in either 0.2 mM EGTA (open triangles) or 1 mM Ca²⁺ (open circles). Vesicles that harbored PS are indicated by (+) and vesicles that lacked PS are indicated by (-); all combinations were tested. Rounds of fusion were plotted as a function of time. (B) Data from A were quantified (n ≥4; error bars represent SD). Data obtained in the absence of syt were omitted for clarity. Total rounds of fusion at 120 min were plotted for each condition.