Cis - and trans -membrane interactions of synaptotagmin-1

Abstract

In neurotransmission synaptotagmin-1 tethers synaptic vesicles to the presynaptic plasma membrane by binding to acidic membrane lipids and SNAREs and promotes rapid SNARE-mediated fusion upon Ca 2+ triggering. However, recent studies suggested that upon membrane contact synaptotagmin may not only bind in trans to the target membrane but also in cis to its own membrane. Using a sensitive membrane tethering assay we have now dissected the structural requirements and concentration ranges for Ca 2+ -dependent and -independent cis-binding and trans-tethering in the presence and absence of acidic phospholipids and SNAREs. Using variants of membrane-anchored synaptotagmin in which the Ca 2+ -binding sites in the C2 domains and a basic cluster involved in membrane binding were disrupted we show that Ca 2+ -dependent cis-binding prevents trans-interactions if the cis-membrane contains 12-20% anionic phospholipids. Similarly, no trans-interactions were observable using soluble C2AB-domain fragments at comparable concentrations. At saturating concentrations, however, tethering was observed with soluble C2AB domains, probably due to crowding on the vesicle surface and competition for binding sites. We conclude that trans-interactions of synaptotagmin considered to be essential for its function are controlled by a delicate balance between cis-and trans-binding, which may play an important modulatory role in synaptic transmission. . This increase is sensed by synaptotagmin-1, a 65-kDa protein anchored to synaptic vesicles (1, 2). Synaptotagmin-1 then triggers fusion of the synaptic vesicles with the plasma membrane resulting in release of neurotransmitter. Fusion itself is mediated by the vesicular R-SNARE synaptobrevin-2 and the plasma membrane Q-SNAREs SNAP-25 and syntaxin-1A. These SNAREs assemble in trans between the membranes and form a tight coiled-coil complex which overcomes the energy barrier of membrane fusion. Synaptotagmin-1 consists of an N-terminal transmembrane helix connected by a long (61-residue) unstructured linker to two C2 domains, called C2A and C2B. The C2A and C2B domains bind three and two Ca 2+ ions, respectively (3, 4). They also bind to both individual Q-SNAREs and assembled SNARE complexes (1, 5-7) and to anionic membranes (3,(8)(9)(10)(11)(12)(13)(14)(15)(16)). Both of these interactions are modulated by Ca 2+ and have been implicated in the mechanism of synaptotagmin-1 action (1, 2). In addition, synaptotagmin-1 possesses a polybasic stretch in the C2B domain that is structurally separated from the calcium-binding domain and that mediates calcium-independent binding to acidic phospholipids, particularly phosphatidylinositol-4,5-bisphosphate (PiP 2 ) (8, 9, 15-17).Despite intense research over the past two decades, it is still unclear by which molecular mechanism synaptotagmin-1 is capable of accelerating exocytosis by more than four orders of magnitude (18). Two types of models are presently discussed that are not necessarily exclusive. The first proposes a direct action of synaptotagmin-1 on th...

PtdInsP2 and PtdSer cooperate to trap synaptotagmin-1 to the plasma membrane in the presence of calcium

“…No aggregation of vesicles was observed in our conditions, even when lipid mixtures with the highest affinity to C2AB fragment were used. (Inset) A scale-up of the traces showing that there is no vesicle aggregation in our conditions in concordance to the results of Vennekate et al (2012). ( d ) K d values, calculated as the ratio of k off / k on, ( e ) k on and ( f ) k off of C2AB fragment binding to liposomes containing different concentrations of PtdSer and PtdIns(4,5)P 2 .…”

“…In our approach, we used a low protein concentration to avoid binding of the C2AB fragment to opposing membranes in the presence of Ca 2+ (resulting in vesicle clustering) (Vennekate et al, 2012) and other cooperative effects (Figure 1c). Our conditions allowed us to measure reliably the binding kinetics and affinity of the C2AB fragment to liposomes containing various molar ratios of PtdSer (0, 10 and 20%) and PtdIns(4,5)P 2 (0, 2.5 and 5%).…”

Prevalent mechanism of membrane bridging by synaptotagmin-1

“…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)(26)(27)(28)(29)(30)(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.…”

The Synaptic Vesicle Release Machinery

“…Subsequent studies have provided abundant data supporting the notion that the membrane-bridging activity is key for the stimulatory effect of Syt1 in reconstitution experiments (e.g., 67,112,157). This activity may be performed by parallel or antiparallel orientations, or both, of the two C 2 domains (64, 131) and depends on a delicate balance between interactions in cis with the Synaptobrevin membrane and in trans with the Syntaxin-1-SNAP-25 membrane (84,159).…”