Beta-strand recombination in tricalbin evolution and the origin of synaptotagmin-like C2 domains

Two protein families involved in membrane traffic, tricalbins and synaptotagmins, contain several copies of C2 domains and are related based on their sequence and domain architecture. Paradoxically, tricalbin and synaptotagmin C2 domains belong to different structural types with apparent circular permutation of terminal beta-strands. To understand whether a topological switch took place, we analyzed tricalbin and synaptotagmin-like C2 domains using two-dimensional structural analysis. We found that yeast tricalbins contain five to six C2 domains. One of these C2 domains possesses many features of synaptotagmin-like C2 domains and also carries a conserved C-terminal strand that is similar to its structural equivalent in synaptotagmin-like C2 domains, suggesting a structural permutation event. Indeed, among higher eukaryotes, animal tricalbins have evolved a C2 domain with synaptotagmin-like topology indicating that the structural conversion has taken place. Investigation of plant synaptotagmins, however, proves that they are direct tricalbin orthologs. Our analysis shows that beta-strand recombination is a possible evolutionary mechanism to generate new structural topologies with altered functional properties.     

Calcium-Dependent Self-Association of Synaptotagmin I

Abstract: Synaptotagmin I, an integral membrane protein of secretory vesicles, appears to have an essential role in calcium-triggered hormone and neurotransmitter release. The large cytoplasmic domain of synaptotagmin I has two C2 domains that are thought to mediate calcium and phospholipid binding. A recombinant protein (p65 1–5) comprised of the cytoplasmic domain was previously shown to aggregate purified chromaffin granules and artificial phospholipid vesicles in a calcium-dependent manner. p65 1–5 may be able to aggregate membrane vesicles by a self-association reaction. This hypothesis led us to investigate the ability of synaptotagmin I protein fragments to multimerize in vitro. We found that p65 1–5, in the absence of membranes, was able to self-associate to form large aggregates in a calcium-dependent manner as shown by light-scattering assays and electron microscopy. In addition, a recombinant protein comprised of only the second half of the cytoplasmic domain, including the second C2 domain, was also able to self-associate and aggregate phospholipid vesicles in a calcium-dependent manner. A recombinant protein comprised of only the first C2 domain was not able to self-associate or aggregate vesicles. These results suggest that synaptotagmin I is able to bind calcium in the absence of membranes and that the second half of the cytoplasmic domain is able to bind calcium and mediate its multimerization in a calcium-dependent manner. The ability of synaptotagmin I protein fragments to multimerize in a calcium-dependent manner in vitro suggests that multimerization may have an important function in vivo.     

Mechanism of calcium-independent synaptotagmin binding to target SNAREs

Synaptic vesicle exocytosis requires three SNARE (soluble N-ethylmaleimide-sensitive-factor attachment protein receptor) proteins: syntaxin and SNAP-25 on the plasma membrane (t-SNAREs) and synaptobrevin/VAMP on the synaptic vesicles (v-SNARE). Vesicular synaptotagmin 1 is essential for fast synchronous SNARE-mediated exocytosis and interacts with the SNAREs in brain material. To uncover the step at which synaptotagmin becomes linked to the three SNAREs, we purified all four proteins from brain membranes and analyzed their interactions. Our study reveals that, in the absence of calcium, native synaptotagmin 1 binds the t-SNARE heterodimer, formed from syntaxin and SNAP-25. This interaction is both stoichiometric and of high affinity. Synaptotagmin contains two divergent but conserved C2 domains that can act independently in calcium-triggered phospholipid binding. We now show that both C2 domains are strictly required for the calcium-independent interaction with the t-SNARE heterodimer, indicating that the double C2 domain structure of synaptotagmin may have evolved to acquire a function beyond calcium/phospholipid binding.     

Real-time assay for monitoring membrane association of lipid-binding domains

The C2 domain is a common protein module which mediates calcium-dependent phospholipid binding. Several assays have previously been developed to measure membrane association. However, these assays either have technical drawbacks or are laborious to carry out. We now present a simple solution-based turbidity method for rapidly assaying membrane association of single lipid-binding domains in real time. We used the first C2 domain of synaptotagmin1 (C2A) as a model lipid-binding moiety. Our use of the common dimeric glutathione-S-transferase (GST) fusion tag allowed two C2A domains to be brought into close proximity. Consequently, calcium-triggered phospholipid binding by this artificially dimerized C2A resulted in liposomal aggregation, easily assayed by following absorbance of the solution at 350 nm. The assay is simple and sensitive and can be scaled up conveniently for use in a multiwell plate format, allowing high-throughput screening. In our screens, we identified nickel as a novel activator of synaptotagmin1 C2A domain membrane association. Finally, we show that the turbidity method can be applied to the study of other GST-tagged lipid-binding proteins such as epsin, protein kinase C-β, and synaptobrevin.     

The Ca2+ Affinity of Synaptotagmin 1 Is Markedly Increased by a Specific Interaction of Its C2B Domain with Phosphatidylinositol 4,5-Bisphosphate

The synaptic vesicle protein synaptotagmin 1 is thought to convey the calcium signal onto the core secretory machinery. Its cytosolic portion mainly consists of two C2 domains, which upon calcium binding are enabled to bind to acidic lipid bilayers. Despite major advances in recent years, it is still debated how synaptotagmin controls the process of neurotransmitter release. In particular, there is disagreement with respect to its calcium binding properties and lipid preferences. To investigate how the presence of membranes influences the calcium affinity of synaptotagmin, we have now measured these properties under equilibrium conditions using isothermal titration calorimetry and fluorescence resonance energy transfer. Our data demonstrate that the acidic phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂), but not phosphatidylserine, markedly increases the calcium sensitivity of synaptotagmin. PI(4,5)P₂ binding is confined to the C2B domain but is not affected significantly by mutations of a lysine-rich patch. Together, our findings lend support to the view that synaptotagmin functions by binding in a trans configuration whereby the C2A domain binds to the synaptic vesicle and the C2B binds to the PI(4,5)P₂-enriched plasma membrane.Calcium-dependent secretion of neurotransmitter-loaded synaptic vesicles is at the heart of synaptic transmission. The underlying membrane fusion reaction between vesicle and plasma membrane has been intensively studied and found to be promoted by both protein-protein as well as protein-lipid interactions. From the multitude of proteins involved in this membrane fusion event, the Ca²⁺-binding protein synaptotagmin 1 is one of its central regulating factors (for review, see Refs. 1–6). Synaptotagmin 1 is anchored in the membrane of synaptic vesicles via a single transmembrane region. Its N-terminal region comprises a short luminal domain, whereas the larger cytoplasmic C-terminal region consists of tandem C2 domains, termed C2A and C2B, tethered to each other via a short linker (7) (a schematic outline of the structural features of synaptotagmin 1 is given in Fig. 1A). Several isoforms with similar domain structure have been identified (8).Open in a separate windowFIGURE 1.Structure of synaptotagmin 1. Synaptotagmin 1 protein consists of two C2 domains, C2A and C2B, that coordinate three and two calcium ions, respectively (16). The acidic residues that coordinate calcium binding is shown schematically, with the residues mutated in the calcium binding mutants (i.e. C2Ab*, C2a*B, and C2a*b*) shown in red. The Lys-rich patch is represented as a ball-and-stick model colored blue with the single cysteine site for the FRET assay (S342C) colored in green (A). The different mutants and constructs used in the study are schematically depicted (B).C2 domains are Ca²⁺ binding modules of ∼130 amino acids, first described as the second conserved region of protein kinase C (PKC)² (9). The C2A domain of synaptotagmin 1 was the first C2 domain structure to be determined (10). In subsequent studies other C2 domains, including the C2B domain of synaptotagmin, were shown to exhibit very similar three-dimensional structures. They have a conserved eight-stranded anti-parallel β-sandwich connected by surface loops. C2 modules are most commonly found in enzymes involved in lipid modifications and signal transduction (PKC, phospholipases, phosphatidylinositol 3-kinases, etc.) and proteins involved in membrane trafficking (synaptotagmins, rabphilin, DOC2, etc.) (11).Calcium ions bind in a cup-shaped depression formed by the N- and C-terminal loops of the C2 key motifs of C2 domains. Notably, the coordination spheres for the Ca²⁺ ions are incomplete (12, 13). In canonical C2 domains, this incomplete coordination sphere can be occupied by anionic and neutral (14, 15) phospholipids, enabling the C2 domain to be attached to the membrane. Hence, it is thought that the general function of C2 domains is to mediate Ca²⁺-triggered binding of the protein to a membrane. In fact, upon rise of the intracellular calcium level, C2 domain-containing enzymes are translocated to the membrane so that the catalytic domains can interact with lipids or membrane-anchored protein substrates (11). Yet synaptotagmin 1 does not contain such a catalytic domain, suggesting that the properties of its tandem C2 domains are the sole key to understanding its molecular function. In neurotransmission, synaptotagmin is thought to transmit the Ca²⁺ signal onto the core membrane fusion machinery, composed of the three SNARE (soluble N-ethylmaleimide sensitive factor attachment receptor) proteins syntaxin 1, SNAP-25 (Q-SNAREs, residing on the plasma membrane), and synaptobrevin 2 (also referred to as VAMP2 (vesicle-associated membrane protein) (R-SNARE, residing on the synaptic vesicle)). So far the multifarious interplay between the SNARE machinery, the two fusing membranes, and synaptotagmin 1 is not well understood. The crystal structure of the entire cytosolic domain of synaptotagmin in the absence of Ca²⁺ has revealed an interesting domain arrangement with the two C2 domains facing in opposite directions (16), hinting at the possibility that the molecule might interact with two opposing membranes upon rise of intracellular Ca²⁺.Although the underlying processes of Ca²⁺ binding and Ca²⁺-dependent membrane binding of synaptotagmin 1 have been studied by a multitude of structural and biochemical investigations, they have not revealed features of synaptotagmin C2 domains that are different from those of other C2 domain-containing proteins. Calcium binding to synaptotagmin in the absence of membranes has been studied by NMR. These studies showed that the isolated C2A domain of synaptotagmin 1 binds three calcium ions with an apparent affinity of ∼60–75 μm, ∼400–500 μm, and more than 1 mm (17). The isolated C2B domain binds two calcium ions with similar calcium affinities in the range of ∼300–600 μm (18). The relatively low intrinsic Ca²⁺ affinities of both C2 domains are difficult to reconcile with the role of synaptotagmin 1 as the Ca²⁺ sensor for fast and synchronous neurotransmitter release, suggesting that interaction with phospholipids contributes to its Ca²⁺ sensitivity. Indeed, Ca²⁺-triggered binding of isolated C2 domains to lipid membranes was first shown in an in vitro study of synaptotagmin 1 using a fluorescence-based approach (19). Subsequent equilibrium fluorescence studies have shed more light on the molecular process underlying membrane binding of synaptotagmin 1, for example by demonstrating that the isolated C2A domain dips into the membrane bilayer upon Ca²⁺ binding (20). This penetration was corroborated by electro-paramagnetic resonance (EPR) spectroscopy studies, which also showed that the penetration depth increased when both C2 domains of synaptotagmin 1 were attached to each other (21) as compared with the single domains (22, 23). However, a variety of different Ca²⁺ and lipid preferences for the individual C2 domains of synaptotagmin has been reported (3, 5, 6).To resolve these discrepancies and to shed more light on the molecular interactions of synaptotagmin 1, we have now used quantitative approaches to study the Ca²⁺ concentration and the lipid composition needed for synaptotagmin to bind to membranes. We employed isothermal titration calorimetry (ITC) to measure the intrinsic calcium binding affinities of synaptotagmin 1 C2 domains both as isolated domains as well as in the context of the tandem C2AB protein. Then, we investigated whether the intrinsic calcium affinity is modulated in the presence of lipids using a newly developed fluorescence resonance energy transfer (FRET) approach. In addition, we investigated how Ca²⁺ and phospholipid binding of synaptotagmin is affected when the Ca²⁺ binding sites in both C2 domains and the putative phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂)-interacting site in the C2 domain are inactivated. We found that the two C2 domains bind calcium largely independently but cooperate in membrane binding. Furthermore, we confirmed that the C2B domain interacts specifically with PI(4,5)P₂. Remarkably, in the presence of PI(4,5)P₂, drastically lower amounts of calcium were needed for membrane binding.     

Sr2+ binding to the Ca2+ binding site of the synaptotagmin 1 C2B domain triggers fast exocytosis without stimulating SNARE interactions

Sr(2+) triggers neurotransmitter release similar to Ca(2+), but less efficiently. We now show that in synaptotagmin 1 knockout mice, the fast component of both Ca(2+)- and Sr(2+)-induced release is selectively impaired, suggesting that both cations partly act by binding to synaptotagmin 1. Both the C(2)A and the C(2)B domain of synaptotagmin 1 bind Ca(2+) in phospholipid complexes, but only the C(2)B domain forms Sr(2+)/phospholipid complexes; therefore, Sr(2+) binding to the C(2)B domain is sufficient to trigger fast release, although with decreased efficacy. Ca(2+) induces binding of the synaptotagmin C(2) domains to SNARE proteins, whereas Sr(2+) even at high concentrations does not. Thus, triggering of the fast component of release by Sr(2+) as a Ca(2+) agonist involves the formation of synaptotagmin/phospholipid complexes, but does not require stimulated SNARE binding.     

Cross-linking of phospholipid membranes is a conserved property of calcium-sensitive synaptotagmins

Synaptotagmins are vesicular proteins implicated in many membrane trafficking events. They are highly conserved in evolution and the mammalian family contains 16 isoforms. We now show that the tandem C2 domains of several calcium-sensitive synaptotagmin isoforms tested, including Drosophila synaptotagmin, rapidly cross-link phospholipid membranes. In contrast to the tandem structure, individual C2 domains failed to trigger membrane cross-linking in several novel assays. Large-scale liposomal aggregation driven by tandem C2 domains in response to calcium was confirmed by the following techniques: turbidity assay, dynamic light-scattering and both confocal and negative stain electron microscopy. Firm cross-linking of membranes was evident from laser trap experiments. High-resolution cryo-electron microscopy revealed that membrane cross-linking by tandem C2 domains results in a constant distance of ∼9 nm between the apposed membranes. Our findings show the conserved nature of this important property of synaptotagmin, demonstrate the significance of the tandem C2 domain structure and provide a plausible explanation for the accelerating effect of synaptotagmins on membrane fusion.     

The cooperative response of synaptotagmin I C2A. A hypothesis for a Ca2+-driven molecular hammer

In the current understanding of exocytosis at the nerve terminal, the C2 domain of synaptotagmin (C2A) is presumed to bind Ca2+ and the membrane in a stepwise fashion: cation then membrane as cation increases the affinity of protein for membrane. Fluorescence spectroscopy data were gathered over a variety of lipid and Ca2+ concentrations, enabling the rigorous application of microscopic binding models derived from partition functions to differentiate between Ca2+ and phosphatidylserine contributions to binding. The data presented here are in variance with previously published models, which were based on the Hill approximation. Rather, the data are consistent with two forms of cooperativity that modulate the responsiveness of C2A: in Ca2+ binding to a network of three cation sites and in interaction with the membrane surface. We suggest synaptotagmin I C2A is preassociated with the synaptic vesicle membrane or nerve terminal. In this state, upon Ca2+ influx the protein will bind the three Ca2+ ions immediately and with high cooperativity. Thus, membrane association creates a high-affinity Ca2+ switch that is the basis for the role of synaptotagmin I in Ca2+-regulated exocytosis. Based on this model, we discuss the implications of protein-induced phosphatidylserine demixing to the exocytotic process.     

Calcium-sensitive phospholipid binding properties of normal and mutant ferlin C2 domains

Mutations in dysferlin, a novel membrane protein of unknown function, lead to muscular dystrophy. Myoferlin is highly homologous to dysferlin and like dysferlin is a plasma membrane protein with six C2 domains highly expressed in muscle. C2 domains are found in a variety of membrane-associated proteins where they have been implicated in calcium, phospholipid, and protein-binding. We investigated the pattern of dysferlin and myoferlin expression in a cell culture model of muscle development and found that dysferlin is expressed in mature myotubes. In contrast, myoferlin is highly expressed in elongated "prefusion" myoblasts and is decreased in mature myotubes where dysferlin expression is greatest. We tested ferlin C2 domains for their ability to bind phospholipid in a calcium-sensitive manner. We found that C2A, the first C2 domain of dysferlin and myoferlin, bound 50% phosphatidylserine and that phospholipid binding was regulated by calcium concentration. A dysferlin point mutation responsible for muscular dystrophy was engineered into the dysferlin C2A domain and demonstrated reduced calcium-sensitive phospholipid binding. Based on these data, we propose a mechanism for muscular dystrophy in which calcium-regulated phospholipid binding is abnormal, leading to defective maintenance and repair of muscle membranes.     

Phosphatidylinositol phosphates as co-activators of Ca2+ binding to C2 domains of synaptotagmin 1

Ca2+-dependent phospholipid binding to the C2A and C2B domains of synaptotagmin 1 is thought to trigger fast neurotransmitter release, but only Ca2+ binding to the C2B domain is essential for release. To investigate the underlying mechanism, we have compared the role of basic residues in Ca2+/phospholipid binding and in release. Mutations in a polybasic sequence on the side of the C2B domain beta-sandwich or in a basic residue in a top Ca2+-binding loop of the C2A domain (R233) cause comparable decreases in the apparent Ca2+ affinity of synaptotagmin 1 and the Ca2+ sensitivity of release, whereas mutation of the residue homologous to Arg233 in the C2B domain (Lys366) has no effect. Phosphatidylinositol polyphosphates co-activate Ca2+-dependent and -independent phospholipid binding to synaptotagmin 1, but the effects of these mutations on release only correlate with their effects on the Ca2+-dependent component. These results reveal clear distinctions in the Ca2+-dependent phospholipid binding modes of the synaptotagmin 1 C2 domains that may underlie their functional asymmetry and suggest that phosphatidylinositol polyphosphates may serve as physiological modulators of Ca2+ affinity of synaptotagmin 1 in vivo.     

Kinase associated-1 domains drive MARK/PAR1 kinases to membrane targets by binding acidic phospholipids

Phospholipid-binding modules such as PH, C1, and C2 domains play crucial roles in location-dependent regulation of many protein kinases. Here, we identify the KA1 domain (kinase associated-1 domain), found at the C terminus of yeast septin-associated kinases (Kcc4p, Gin4p, and Hsl1p) and human MARK/PAR1 kinases, as a membrane association domain that binds acidic phospholipids. Membrane localization of isolated KA1 domains depends on phosphatidylserine. Using X-ray crystallography, we identified a structurally conserved binding site for anionic phospholipids in KA1 domains from Kcc4p and MARK1. Mutating this site impairs membrane association of both KA1 domains and intact proteins and reveals the importance of phosphatidylserine for bud neck localization of yeast Kcc4p. Our data suggest that KA1 domains contribute to "coincidence detection," allowing kinases to bind other regulators (such as septins) only at the membrane surface. These findings have important implications for understanding MARK/PAR1 kinases, which are implicated in Alzheimer's disease, cancer, and autism.     

Interaction of synaptotagmin with lipid bilayers, analyzed by single-molecule force spectroscopy

Synaptotagmin I is the major Ca²⁺ sensor for membrane fusion during neurotransmitter release. The cytoplasmic domain of synaptotagmin consists of two C2 domains, C2A and C2B. On binding Ca²⁺, the tips of the two C2 domains rapidly and synchronously penetrate lipid bilayers. We investigated the forces of interaction between synaptotagmin and lipid bilayers using single-molecule force spectroscopy. Glutathione-S-transferase-tagged proteins were attached to an atomic force microscope cantilever via a glutathione-derivatized polyethylene glycol linker. With wild-type C2AB, the force profile for a bilayer containing phosphatidylserine had both Ca²⁺-dependent and Ca²⁺-independent components. No force was detected when the bilayer lacked phosphatidylserine, even in the presence of Ca²⁺. The binding characteristics of C2A and C2B indicated that the two C2 domains cooperate in binding synaptotagmin to the bilayer, and that the relatively weak Ca²⁺-independent force depends only on C2A. When the lysine residues K189-192 and K326, 327 were mutated to alanine, the strong Ca²⁺-dependent binding interaction was either absent or greatly reduced. We conclude that synaptotagmin binds to the bilayer via C2A even in absence of Ca²⁺, and also that positively charged regions of both C2A and C2B are essential for the strong Ca²⁺-dependent binding of synaptotagmin to the bilayer.     

Crystal structure of the cytosolic C2A-C2B domains of synaptotagmin III. Implications for Ca(+2)-independent snare complex interaction.

Synaptotagmins are synaptic vesicle-associated, phospholipid-binding proteins most commonly associated with Ca(+2)-dependent exocytotic and Ca(+2)- independent endocytotic events. Synaptotagmin III is a 63.2-kD member of the synaptotagmin homology group; one of its characteristic properties is the ability to bind divalent cations and accessory proteins promiscuously. In the cytosolic portion of this protein, a flexible seven-amino acid linker joins two homologous C2 domains. The C2A domain binds to phospholipid membranes and other accessory proteins in a divalent cation-dependent fashion. The C2B domain promotes binding to other C2B domains, as well as accessory proteins independent of divalent cations. The 3.2 A crystal structure of synaptotagmin III, residues 295-566, which includes the C2A and C2B domains, exhibits differences in the shape of the Ca(+2)-binding pocket, the electrostatic surface potential, and the stoichiometry of bound divalent cations for the two domains. These observations may explain the disparate binding properties of the two domains. The C2A and the C2B domains do not interact; synaptotagmin, therefore, covalently links two independent C2 domains, each with potentially different binding partners. A model of synaptotagmin's involvement in Ca(+2)-dependent regulation of membrane fusion through its interaction with the SNARE complex is presented.     

Examining synaptotagmin 1 function in dense core vesicle exocytosis under direct control of Ca2+

We tested the long-standing hypothesis that synaptotagmin 1 is the Ca2+ sensor for fast neurosecretion by analyzing the intracellular Ca2+ dependence of large dense-core vesicle exocytosis in a mouse strain carrying a mutated synaptotagmin C2A domain. The mutation (R233Q) causes a twofold increase in the KD of Ca2+-dependent phospholipid binding to the double C2A-C2B domain of synaptotagmin. Using photolysis of caged calcium and capacitance measurements we found that secretion from mutant cells had lower secretory rates, longer secretory delays, and a higher intracellular Ca2+-threshold for secretion due to a twofold increase in the apparent KD of the Ca2+ sensor for fast exocytosis. Single amperometric fusion events were unchanged. We conclude that Ca2+-dependent phospholipid binding to synaptotagmin 1 mirrors the intracellular Ca2+ dependence of exocytosis.     

Lipid organization regulates annexin A2 Ca(2+)-sensitivity for membrane bridging and its modulator effects on membrane fluidity

Annexin A2 (AnxA2) is a phospholipid binding protein that has been implicated in many membrane-related cellular functions. AnxA2 is able to bind different acidic phospholipids such as phosphatidylserine (PS) and phosphatidylinositol-4,5-bisphosphate (PI2P). This binding is mediated by Ca(2+)-dependent and Ca(2+)-independent mechanisms. The specific functions of annexin A2 related to these two phospholipids and the molecular mechanisms involved in their interaction remain obscure. Herein we studied the influence of lipid composition on the Ca(2+)-dependency of AnxA2-mediated membrane bridging and on membrane fluidity. Membrane models of ten different lipid compositions and detergent-resistant membranes from two cellular sources were investigated. The results show that the AnxA2-mediated membrane bridging requires 3 to 50 times less calcium for PS-membranes than for PI2P-membranes. Membrane fluidity was measured by the ratiometric fluorescence parameter generalized polarization method with two fluorescent probes. Compared to controls containing low phospholipid ligand, AnxA2 was found to reduce the membrane fluidity of PI2P-membranes twice as much as the PS-membranes in the presence of calcium. On the contrary, at mild acidic pH in the absence of calcium AnxA2 reduces the fluidity of the PS-membranes more than the PI2P-membranes. The presence of cholesterol on the bilayer reduced the AnxA2 capacity to reduce membrane fluidity. The presented data shed light on the specific roles of PI2P, PS and cholesterol present on membranes related to the action of annexin A2 as a membrane bridging molecule during exocytosis and endocytosis events and as a plasma membrane domain phospholipid packing regulator.     

The calcium binding loops of the cytosolic phospholipase A2 C2 domain specify targeting to Golgi and ER in live cells

Translocation of cytosolic phospholipase A2 (cPLA2) to Golgi and ER in response to intracellular calcium mobilization is regulated by its calcium-dependent lipid-binding, or C2, domain. Although well studied in vitro, the biochemical characteristics of the cPLA2C2 domain offer no predictive value in determining its intracellular targeting. To understand the molecular basis for cPLA2C2 targeting in vivo, the intracellular targets of the synaptotagmin 1 C2A (Syt1C2A) and protein kinase Calpha C2 (PKCalphaC2) domains were identified in Madin-Darby canine kidney cells and compared with that of hybrid C2 domains containing the calcium binding loops from cPLA2C2 on Syt1C2A and PKCalphaC2 domain backbones. In response to an intracellular calcium increase, PKCalphaC2 targeted plasma membrane regions rich in phosphatidylinositol-4,5-bisphosphate, and Syt1C2A displayed a biphasic targeting pattern, first targeting phosphatidylinositol-4,5-bisphosphate-rich regions in the plasma membrane and then the trans-Golgi network. In contrast, the Syt1C2A/cPLA2C2 and PKCalphaC2/cPLA2C2 hybrids targeted Golgi/ER and colocalized with cPLA2C2. The electrostatic properties of these hybrids suggested that the membrane binding mechanism was similar to cPLA2C2, but not PKCalphaC2 or Syt1C2A. These results suggest that primarily calcium binding loops 1 and 3 encode structural information specifying Golgi/ER targeting of cPLA2C2 and the hybrid domains.     

Structural basis for the evolutionary inactivation of Ca2+ binding to synaptotagmin 4

The neuronal protein synaptotagmin 1 functions as a Ca(2+) sensor in exocytosis via two Ca(2+)-binding C(2) domains. The very similar synaptotagmin 4, which includes all the predicted Ca(2+)-binding residues in the C(2)B domain but not in the C(2)A domain, is also thought to function as a neuronal Ca(2+) sensor. Here we show that, unexpectedly, both C(2) domains of fly synaptotagmin 4 exhibit Ca(2+)-dependent phospholipid binding, whereas neither C(2) domain of rat synaptotagmin 4 binds Ca(2+) or phospholipids efficiently. Crystallography reveals that changes in the orientations of critical Ca(2+) ligands, and perhaps their flexibility, render the rat synaptotagmin 4 C(2)B domain unable to form full Ca(2+)-binding sites. These results indicate that synaptotagmin 4 is a Ca(2+) sensor in the fly but not in the rat, that the Ca(2+)-binding properties of C(2) domains cannot be reliably predicted from sequence analyses, and that proteins clearly identified as orthologs may nevertheless have markedly different functional properties.     

Dynamics of myo1c (myosin-ibeta ) lipid binding and dissociation

Myosin-I is the single-headed member of the myosin superfamily that associates with lipid membranes. Biochemical experiments have shown that myosin-I membrane binding is the result of electrostatic interactions between the basic tail domain and acidic phospholipids. To better understand the dynamics of myosin-I membrane association, we measured the rates of association and dissociation of a recombinant myo1c tail domain (which includes three IQ domains and bound calmodulins) to and from large unilamellar vesicles using fluorescence resonance energy transfer. The apparent second-order rate constant for lipid-tail association in the absence of calcium is fast with nearly every lipid-tail collision resulting in binding. The rate of binding is decreased in the presence of calcium. Time courses of myo1c-tail dissociation are best fit by two exponential rates: a fast component that has a rate that depends on the ratio of acidic phospholipid to myo1c-tail (phosphatidylserine (PS)/tail) and a slow component that predominates at high PS/tail ratios. The dissociation rate of the slow component is slower than the myo1c ATPase rate, suggesting that myo1c is able to stay associated with the lipid membrane during multiple catalytic cycles of the motor. Calcium significantly increases the lifetimes of the membrane-bound state, resulting in dissociation rates 0.001 s(-1).     

Position of synaptotagmin I at the membrane interface: cooperative interactions of tandem C2 domains

Synaptotagmin I is a synaptic vesicle associated membrane protein that appears to regulate Ca(2+)-mediated exocytosis. Here, the Ca(2+)-dependent membrane interactions of a water soluble fragment of synaptotagmin I (C2AB) that contains its two C2 domains (C2A and C2B) were determined using site-directed spin labeling. Membrane depth parameters were obtained for 19 spin-labeled mutants of C2AB when bound to phosphatidylcholine and phosphatidylserine membranes, and these distance constraints were used in combination with the high-resolution structures of C2A and C2B to generate a model for the membrane orientation and position of synaptotagmin at the bilayer interface. Both C2A and C2B bind to the membrane interface with their first and third Ca(2+) binding loops penetrating the membrane interface. The polybasic face of C2B does not interact with the membrane lipid but is available for electrostatic interaction with other components of the fusion machinery. When compared to positions determined previously for the isolated domains, both C2A and C2B have similar orientations; however, the two domains are positioned deeper into the bilayer interior when present in the tandem construct. These data indicate that C2A and C2B do not act independently but influence their mutual membrane penetration. This may explain the occurrence of multiple C2 domains in proteins that function in membrane trafficking and repair.     

The tandem C2 domains of synaptotagmin contain redundant Ca2+ binding sites that cooperate to engage t-SNAREs and trigger exocytosis

Real-time voltammetry measurements from cracked PC12 cells were used to analyze the role of synaptotagmin-SNARE interactions during Ca2+-triggered exocytosis. The isolated C2A domain of synaptotagmin I neither binds SNAREs nor inhibits norepinephrine secretion. In contrast, two C2 domains in tandem (either C2A-C2B or C2A-C2A) bind strongly to SNAREs, displace native synaptotagmin from SNARE complexes, and rapidly inhibit exocytosis. The tandem C2 domains of synaptotagmin cooperate via a novel mechanism in which the disruptive effects of Ca2+ ligand mutations in one C2 domain can be partially alleviated by the presence of an adjacent C2 domain. Complete disruption of Ca2+-triggered membrane and target membrane SNARE interactions required simultaneous neutralization of Ca2+ ligands in both C2 domains of the protein. We conclude that synaptotagmin-SNARE interactions regulate membrane fusion and that cooperation between synaptotagmin's C2 domains is crucial to its function.