Close membrane-membrane proximity induced by Ca2+-dependent multivalent binding of synaptotagmin-1 to phospholipids

Abstract

Synaptotagmin acts as a Ca(2+) sensor in neurotransmitter release through its two C(2) domains. Ca(2+)-dependent phospholipid binding is key for synaptotagmin function, but it is unclear how this activity cooperates with the SNARE complex involved in release or why Ca(2+) binding to the C(2)B domain is more crucial for release than Ca(2+) binding to the C(2)A domain. Here we show that Ca(2+) induces high-affinity simultaneous binding of synaptotagmin to two membranes, bringing them into close proximity. The synaptotagmin C(2)B domain is sufficient for this ability, which arises from the abundance of basic residues around its surface. We propose a model wherein synaptotagmin cooperates with the SNAREs in bringing the synaptic vesicle and plasma membranes together and accelerates membrane fusion through the highly positive electrostatic potential of its C(2)B domain.

A Quaternary SNARE–Synaptotagmin–Ca2+–Phospholipid Complex in Neurotransmitter Release

“…It is actually plausible that the C 2 B-domain interacts with the SNARE complex through the side of the β-sandwich and with both membranes through the Ca 2+ -binding loops and the opposite tip of the β-sandwich, which is positively charged (Figure 6a; see also ref. 25 ). Note that the previously proposed role of the C 2 B domain in bending the membranes to initiate fusion 25 , which has been supported by theoretical calculations 60 , could be played in a similar fashion and even more efficiently by the SSCAP complex, as the positive charge of the C-termini of the SNAREs adds to that of the C 2 B domain to form a continuous, highly positive surface (Figure 6a).…”

“…However, measuring such an increase directly is hindered by the very high affinity of the C 2 AB fragment for negatively charged phospholipids in the presence of Ca 2+ (in the low nanomolar range; see refs. 25,48 ). As an alternative, we sought to design a partition experiment that could reveal whether the C 2 AB fragment binds preferentially to membranes containing SNARE complexes compared to protein-free membranes.…”

“…A potential explanation for these findings was provided by reports of Ca 2+ -dependent synaptotagmin 1 oligomerization mediated by the C 2 B domain (see ref. 33 ), but extensive evidence has shown that a fragment including both synaptotagmin 1 C 2 domains (C 2 AB fragment) does not oligomerize in solution or on membranes 25,34,35 . A property that is more likely to underlie the preponderant role of the C 2 B domain in release is its ability to bind to two membranes simultaneously upon Ca 2+ binding, which is not shared by the C 2 A domain and could cooperate with the SNAREs in promoting membrane fusion 25 .…”

Minimal membrane docking requirements revealed by reconstitution of Rab GTPase-dependent membrane fusion from purified components

“…Moreover, temperature-sensitive SNARE mutants that permit docking of ER-derived vesicles after incubation at a restrictive temperature that blocks membrane fusion (32) may be defective for fusion, but not for docking, at restrictive temperatures. Finally, a golgin protein acting in consort with an Arf GTPase (66), and several synaptotagmin isoforms (67,68), can induce SNARE-independent, but also Rab-and Rab effector-independent, liposome clustering.…”

Prevalent mechanism of membrane bridging by synaptotagmin-1

“…Mutation of two arginine residues at the bottom of the C 2 B domain (R398 and R399) strongly impairs neurotransmitter release, the liposome-clustering activity, and the ability to stimulate SNARE-dependent lipid mixing between liposomes (7). These findings led to the proposal that synaptotagmin-1 cooperates with the SNAREs in inducing membrane fusion by helping to bring the two membranes together in a Ca 2+ -dependent manner, directly bridging the two membranes via the Ca 2+ -binding loops at the top and the arginines at the bottom of the C 2 B domain (7,20,23).Though EPR data agreed with this model of membrane bridging (24), and increasing evidence supports the notion that the membrane-bridging activity of synaptotagmin-1 is critical for its function (7, 25-31), a recent study concluded that the bridging occurs by a fundamentally different mechanism that requires trans interactions between synaptotagmin-1 oligomers bound to each membrane, without binding of the bottom of the C 2 B domain to the lipids (29). This mechanism, which we refer to as the oligomerization model, is compared in Fig.…”