Josep Rizo

Synaptotagmin I functions as a calcium regulator of release probability

In all synapses, Ca2+ triggers neurotransmitter release to initiate signal transmission. Ca2+ presumably acts by activating synaptic Ca2+ sensors, but the nature of these sensors—which are the gatekeepers to neurotransmission—remains unclear. One of the candidate Ca2+ sensors in release is the synaptic Ca2+-binding protein synaptotagmin I. Here we have studied a point mutation in synaptotagmin I that causes a twofold decrease in overall Ca2+ affinity without inducing structural or conformational changes. When introduced by homologous recombination into the endogenous synaptotagmin I gene in mice, this point mutation decreases the Ca2+ sensitivity of neurotransmitter release twofold, but does not alter spontaneous release or the size of the readily releasable pool of neurotransmitters. Therefore, Ca2+ binding to synaptotagmin I participates in triggering neurotransmitter release at the synapse.

C2-domains, structure and function of a universal Ca2+-binding domain.

A vast amount of protein sequence data accumulated over recent years has revealed that protein modules are widespread in nature. Many intracellular and extracellular proteins consist, in part or fully, of combinations of protein modules. C2-domains, together with SH2, PTB, PH, SH3, WW, and PDZ domains, are typical examples of intracellular protein modules. These modules form independently folding domains of 80–160 residues with characteristic binding properties; C2-domains bind Ca 21 and phospholipids, SH2 and PTB domains phosphotyrosine-containing sequences, PH domains phosphatidylinositol phosphates, SH3 and WW domains proline-rich sequences, and PDZ domains C-terminal sequences. C2-domains are unique among these modules because phospholipid binding to many C2-domains is regulated by Ca . For this reason, C2-domains are sometimes referred to as Ca -dependent lipid binding domains. However, C2-domains are not obligatory Ca and phospholipid-binding modules. C2-domains have diverged evolutionarily into Ca-dependent and Ca-independent forms that interact with multiple targets. Thus, although most C2-domains are probably Ca-binding domains, they represent a family of versatile protein modules with diverse functions. C2-domains comprise approximately 130 residues and were first identified in protein kinase C (1). Close to 100 C2-domain sequences are listed in the current data banks. Although reviews of several C2-domain proteins have been published (2–11), recent results on the structure and interactions of C2-domains by x-ray crystallography and NMR spectroscopy offer a new opportunity to rationalize the properties of C2-domains in structural terms. In this minireview, we will attempt to use this opportunity and correlate the functional properties of C2-domains with their structures.

Jasmonate perception by inositol-phosphate-potentiated COI1–JAZ co-receptor

Jasmonates are a family of plant hormones that regulate plant growth, development and responses to stress. The F-box protein CORONATINE INSENSITIVE 1 (COI1) mediates jasmonate signalling by promoting hormone-dependent ubiquitylation and degradation of transcriptional repressor JAZ proteins. Despite its importance, the mechanism of jasmonate perception remains unclear. Here we present structural and pharmacological data to show that the true Arabidopsis jasmonate receptor is a complex of both COI1 and JAZ. COI1 contains an open pocket that recognizes the bioactive hormone (3R,7S)-jasmonoyl-l-isoleucine (JA-Ile) with high specificity. High-affinity hormone binding requires a bipartite JAZ degron sequence consisting of a conserved α-helix for COI1 docking and a loop region to trap the hormone in its binding pocket. In addition, we identify a third critical component of the jasmonate co-receptor complex, inositol pentakisphosphate, which interacts with both COI1 and JAZ adjacent to the ligand. Our results unravel the mechanism of jasmonate perception and highlight the ability of F-box proteins to evolve as multi-component signalling hubs.

A conformational switch in syntaxin during exocytosis: role of munc18

Syntaxin 1, an essential protein in synaptic membrane fusion, contains a helical autonomously folded N‐terminal domain, a C‐terminal SNARE motif and a transmembrane region. The SNARE motif binds to synaptobrevin and SNAP‐25 to assemble the core complex, whereas almost the entire cytoplasmic sequence participates in a complex with munc18‐1, a neuronal Sec1 homolog. We now demonstrate by NMR spectroscopy that, in isolation, syntaxin adopts a ‘closed’ conformation. This default conformation of syntaxin is incompatible with core complex assembly which requires an ‘open’ syntaxin conformation. Using site‐directed mutagenesis, we find that disruption of the closed conformation abolishes the ability of syntaxin to bind to munc18‐1 and to inhibit secretion in PC12 cells. These results indicate that syntaxin binds to munc18‐1 in a closed conformation and suggest that this conformation represents an essential intermediate in exocytosis. Our data suggest a model whereby, during exocytosis, syntaxin undergoes a large conformational switch that mediates the transition between the syntaxin–munc18‐1 complex and the core complex.

Snares and munc18 in synaptic vesicle fusion

The release of neurotransmitters by Ca2+-triggered synaptic vesicle exocytosis is an exquisitely regulated process that is fundamental for interneuronal communication. This process involves several steps and is controlled by a protein machinery that must prevent release before Ca2+ entry into presynaptic terminals, and yet must rapidly induce release on Ca2+ influx. Extensive studies of the components of this machinery have indicated that SNAREs and Munc18-1 are central proteins for membrane fusion during exocytosis. An increasing amount of information derived from a convergence of structural, physiological and genetic studies is providing important insights into the mechanism of neurotransmitter release.

Mixed Lineage Kinase Domain-like Protein MLKL Causes Necrotic Membrane Disruption upon Phosphorylation by RIP3

Programmed necrotic cell death induced by the tumor necrosis factor alpha (TNF-α) family of cytokines is dependent on a kinase cascade consisting of receptor-interacting kinases RIP1 and RIP3. How these kinase activities cause cells to die by necrosis is not known. The mixed lineage kinase domain-like protein MLKL is a functional RIP3 substrate that binds to RIP3 through its kinase-like domain but lacks kinase activity of its own. RIP3 phosphorylates MLKL at the T357 and S358 sites. Reported here is the development of a monoclonal antibody that specifically recognizes phosphorylated MLKL in cells dying of this pathway and in human liver biopsy samples from patients suffering from drug-induced liver injury. The phosphorylated MLKL forms an oligomer that binds to phosphatidylinositol lipids and cardiolipin. This property allows MLKL to move from the cytosol to the plasma and intracellular membranes, where it directly disrupts membrane integrity, resulting in necrotic death.

Synaptotagmins: C2-domain proteins that regulate membrane traffic.

We wish to thank Drs. M. S. Brown, J. L. Goldstein, Y. Goda, R. Jahn, E. Kandel, E. Liebe, and C. F. Stevens for invaluable advice and discussions. The work described in this review was partially supported by the National Institutes of Health, Human Frontier Science Project, and W. M. Keck Foundation.

Synaptic vesicle fusion.

The core of the neurotransmitter release machinery is formed by SNARE complexes, which bring the vesicle and plasma membranes together and are key for fusion, and by Munc18-1, which controls SNARE-complex formation and may also have a direct role in fusion. In addition, SNARE complex assembly is likely orchestrated by Munc13s and RIMs, active-zone proteins that function in vesicle priming and diverse forms of presynaptic plasticity. Synaptotagmin-1 mediates triggering of release by Ca2+, probably through interactions with SNAREs and both membranes, as well as through a tight interplay with complexins. Elucidation of the release mechanism will require a full understanding of the network of interactions among all these proteins and the membranes.

A Complexin/Synaptotagmin 1 Switch Controls Fast Synaptic Vesicle Exocytosis

Ca(2+) binding to synaptotagmin 1 triggers fast exocytosis of synaptic vesicles that have been primed for release by SNARE-complex assembly. Besides synaptotagmin 1, fast Ca(2+)-triggered exocytosis requires complexins. Synaptotagmin 1 and complexins both bind to assembled SNARE complexes, but it is unclear how their functions are coupled. Here we propose that complexin binding activates SNARE complexes into a metastable state and that Ca(2+) binding to synaptotagmin 1 triggers fast exocytosis by displacing complexin from metastable SNARE complexes. Specifically, we demonstrate that, biochemically, synaptotagmin 1 competes with complexin for SNARE-complex binding, thereby dislodging complexin from SNARE complexes in a Ca(2+)-dependent manner. Physiologically, increasing the local concentration of complexin selectively impairs fast Ca(2+)-triggered exocytosis but retains other forms of SNARE-dependent fusion. The hypothesis that Ca(2+)-induced displacement of complexins from SNARE complexes triggers fast exocytosis accounts for the loss-of-function and gain-of-function phenotypes of complexins and provides a molecular explanation for the high speed and synchronicity of fast Ca(2+)-triggered neurotransmitter release.

Three-dimensional structure of the complexin/SNARE complex.

During neurotransmitter release, the neuronal SNARE proteins synaptobrevin/VAMP, syntaxin, and SNAP-25 form a four-helix bundle, the SNARE complex, that pulls the synaptic vesicle and plasma membranes together possibly causing membrane fusion. Complexin binds tightly to the SNARE complex and is essential for efficient Ca(2+)-evoked neurotransmitter release. A combined X-ray and TROSY-based NMR study now reveals the atomic structure of the complexin/SNARE complex. Complexin binds in an antiparallel alpha-helical conformation to the groove between the synaptobrevin and syntaxin helices. This interaction stabilizes the interface between these two helices, which bears the repulsive forces between the apposed membranes. These results suggest that complexin stabilizes the fully assembled SNARE complex as a key step that enables the exquisitely high speed of Ca(2+)-evoked neurotransmitter release.