Biomedical subjects

T C Südhof

Publications and source records attributed to T C Südhof.

At least 19 recordsLinked to original sources

PubMed1995

Distinct Ca2+ and Sr2+ binding properties of synaptotagmins. Definition of candidate Ca2+ sensors for the fast and slow components of neurotransmitter release.

Ca(2+)-dependent neurotransmitter release consists of at least two components: a major fast component that is insensitive to Sr2+ and a minor slow component that is potentiated by Sr2+ (Goda, Y., and Stevens, C. F. (1994) Proc. Natl. Acad. U. S. A. 91, 12942-12946). These results suggest that at least two Ca2+ sensors act in synaptic vesicle fusion with distinct Ca2+ and Sr2+ binding properties. We have now investigated the relative Ca2+ and Sr2+ binding activities of synaptotagmins to evaluate their potential roles as Ca2+ sensors for the fast and slow components. Our results demonstrate that the first C2 domains of synaptotagmins I, II, III, V, and VII have very similar Ca2+ requirements for phospholipid binding (range of EC50 = 2.6 microM to 5.0 microM), but distinct Sr2+ requirements (EC50 range = 23 microM to 133 microM); synaptotagmins I and II had the lowest Sr2+ affinity, and synaptotagmin III the highest Sr2+ affinity. Purified synaptotagmin I from bovine brain exhibited similar properties as its recombinant first C2 domain, suggesting that the first C2 domain fully accounts for its Ca(2+)-dependent phospholipid binding properties. Sr2+ was unable to trigger syntaxin binding by synaptotagmin I at all concentrations tested, whereas it was effective for synaptotagmin III. These results suggest that different C2 domains have distinct Sr2+ binding properties. They support the hypothesis that synaptotagmins localized on the same vesicle perform distinct functions, with synaptotagmins I and II serving as candidate Ca2+ sensors for the fast component in release and synaptotagmin III for the slow component.

Animals↗

PubMed1995

Mammalian homologues of Caenorhabditis elegans unc-13 gene define novel family of C2-domain proteins.

The unc-13 gene in Caenorhabditis elegans is essential for normal presynaptic function and encodes a large protein with C1- and C2-domains. In protein kinase C and synaptotagmin, C1- and/or C2-domains are regulatory domains for Ca2+, phospholipids, and diacylglycerol, suggesting a role for unc-13 in regulating neurotransmitter release. To determine if a similar protein is a component of the presynaptic machinery for neurotransmitter release in vertebrates, we studied unc-13 homologues in rat. Molecular cloning revealed that three homologues of unc-13 called Munc13-1, -13-2, and -13-3 are expressed in rat brain. Munc13s are large, brain-specific proteins with divergent N termini but conserved C termini containing C1- and C2-domains. Specific antibodies demonstrated that Munc13-1 is a peripheral membrane protein that is enriched in synaptosomes and localized to plasma membranes but absent from synaptic vesicles. Our data suggest that the function of unc-13 in C. elegans is conserved in mammals and that Munc13s act as plasma membrane proteins in nerve terminals. The presence of C1- and C2-domains in these proteins and the phenotype of the C. elegans mutants raise the possibility that Munc13s may have an essential signaling role during neurotransmitter release.

Amino Acid Sequence↗

PubMed1995

High affinity binding of alpha-latrotoxin to recombinant neurexin I alpha.

alpha-Latrotoxin is a potent neurotoxin from black widow spider venom that stimulates neurotransmitter release. alpha-Latrotoxin is thought to act by binding to a high affinity receptor on presynaptic nerve terminals. In previous studies, high affinity alpha-latrotoxin binding proteins were isolated and demonstrated to contain neurexin I alpha as a major component. Neurexin I alpha is a cell surface protein that exists in multiple differentially spliced isoforms and belongs to a large family of neuron-specific proteins. Using a series of neurexin I-IgG fusion proteins, we now show that recombinant neurexin I alpha binds alpha-latrotoxin directly with high affinity (Kd approximately 4 nM). Binding of alpha-latrotoxin to recombinant neurexin I alpha is dependent on Ca2+ (EC50 approximately 30 microM). Our data suggest that neurexin I alpha is a Ca(2+)-dependent high affinity receptor for alpha-latrotoxin.

Alternative Splicing↗

PubMed1995

Complexins: cytosolic proteins that regulate SNAP receptor function.

A family of proteins called complexins was discovered that compete with alpha-SNAP, but not synaptotagmin, for SNAP receptor binding. Complexins I and II are highly homologous hydrophilic proteins that are tightly conserved, with 100% identity among mouse, rat, and human complexin II. They are enriched in neurons where they colocalize with syntaxin and SNAP-25; in addition, complexin II is expressed ubiquitously at low levels. Complexins bind weakly to syntaxin alone and not at all to synaptobrevin and SNAP-25, but strongly to the SNAP receptor-core complex composed of these three molecules. They compete with alpha-SNAP for binding to the core complex but not with other interacting molecules, including synaptotagmin I, suggesting that the complexins regulate the sequential interactions of alpha-SNAP and synaptotagmins with the SNAP receptor during exocytosis.

Adaptor Proteins, Vesicular Transport↗

PubMed1995

The synaptic vesicle cycle: a cascade of protein-protein interactions.

The synaptic vesicle cycle at the nerve terminal consists of vesicle exocytosis with neurotransmitter release, endocytosis of empty vesicles, and regeneration of fresh vesicles. Of all cellular transport pathways, the synaptic vesicle cycle is the fastest and the most tightly regulated. A convergence of results now allows formulation of molecular models for key steps of the cycle. These developments may form the basis for a mechanistic understanding of higher neural function.

Animals↗

PubMed1995

Ca(2+)-dependent and -independent activities of neural and non-neural synaptotagmins.

Synaptotagmins (Syts) are brain-specific Ca2+/phospholipid-binding proteins. In hippocampal synapses, Syt I is essential for fast Ca(2+)-dependent synaptic vesicle exocytosis but not for Ca(2+)-independent exocytosis. In vertebrates and invertebrates, Syt may therefore participate in Ca(2+)-dependent synaptic membrane fusion, either by serving as the Ca2+ sensor in the last step of fast Ca(2+)-triggered neurotransmitter release, or by collaborating with an additional Ca2+ sensor. While Syt I binds Ca2+ (refs 10, 11), its phospholipid binding is triggered at lower calcium concentrations (EC50 = 3-6 microM) than those required for exocytosis. Furthermore, Syts bind clathrin-AP2 with high affinity, indicating that they may play a general role in endocytosis rather than being confined to a specialized function in regulated exocytosis. Here we resolve this apparent contradiction by describing four Syts, three of which (Syt VI, VII and VIII) are widely expressed in non-neural tissues. All Syts tested share a common domain structure, with a cytoplasmic region composed of two C2 domains that interacts with clathrin-AP2 (Kd = 0.1-1.0 nM) and with neural and non-neural syntaxins. The first C2 domains of Syt I, II, III, V and VII, but not of IV, VI or VIII, bind phospholipids with a similar Ca(2+)-concentration dependence (EC50 = 3-6 microM). The same C2 domains also bind syntaxin as a function of Ca2+ but the Ca(2+)-concentration dependence of Syt I, II and V (> 200 microM) differs from that of Syt III and VII (< 10 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1995

Essential functions of synapsins I and II in synaptic vesicle regulation.

Synaptic vesicles are coated by synapsins, phosphoproteins that account for 9% of the vesicle protein. To analyse the functions of these proteins, we have studied knockout mice lacking either synapsin I, synapsin II, or both. Mice lacking synapsins are viable and fertile with no gross anatomical abnormalities, but experience seizures with a frequency proportional to the number of mutant alleles. Synapsin-II and double knockouts, but not synapsin-I knockouts, exhibit decreased post-tetanic potentiation and severe synaptic depression upon repetitive stimulation. Intrinsic synaptic-vesicle membrane proteins, but not peripheral membrane proteins or other synaptic proteins, are slightly decreased in individual knockouts and more severely reduced in double knockouts, as is the number of synaptic vesicles. Thus synapsins are not required for neurite outgrowth, synaptogenesis or the basic mechanics of synaptic vesicle traffic, but are essential for accelerating this traffic during repetitive stimulation. The phenotype of the synapsin knockouts could be explained either by deficient recruitment of synaptic vesicles to the active zone, or by impaired maturation of vesicles at the active zone, both of which could lead to a secondary destabilization of synaptic vesicles.

Animals↗

PubMed1995

A novel ubiquitous form of Munc-18 interacts with multiple syntaxins. Use of the yeast two-hybrid system to study interactions between proteins involved in membrane traffic.

Munc-18-1 is a 67-kDa neuronal protein that binds tightly to syntaxin 1 and functions in synaptic vesicle exocytosis (Hata, Y., Slaughter, C.A., and Südhof, T.C. (1993a) Nature 366, 347-351). We have now characterized a new Munc-18 isoform, Munc-18-2, that exhibits 63% amino acid sequence identity with Munc-18-1. Munc-18-2 is expressed in most tissues, whereas Munc-18-1 is primarily expressed in brain. Using recombinant Munc-18-1 and Munc-18-2 produced in COS cells, we show that both forms of Munc-18 bind tightly to syntaxins 1A, 2, and 3 but not to syntaxin 4. In an independent approach to study the binding specificities of Munc-18-1 and Munc-18-2, we used the yeast two-hybrid system. This assay system depends on protein-protein interactions in the cell nucleus. We validated its utility for studying membrane trafficking proteins by testing well characterized interactions between cytosolic proteins that are known to be physiologically important in exocytosis. Strong interactions, such as the binding of syntaxins 1-4 with SNAP-25, were effectively detected by the yeast two-hybrid assay, but weak binding, such as the binding of syntaxins to synaptotagmin or of synaptotagmin to neurexins, was not. Studies on full-length and truncated forms of Munc-18s by the yeast two-hybrid system confirmed their interactions with syntaxins. Both the N and the C terminus of Munc-18 were essential for binding. Munc-18-1 and Munc-18-2 bind only to syntaxins 1A, 2, and 3 but not 4 and 5 by yeast-two hybrid system assays. Our studies demonstrate that neural and non-neural tissues have distinct forms of Munc-18, which may function in different types of exocytosis. The lack of specificity of the interactions between syntaxins and Munc-18s indicates that specificity of membrane trafficking reactions is not dependent on this interaction.

Amino Acid Sequence↗

PubMed1995

Neuroligin 1: a splice site-specific ligand for beta-neurexins.

Neurexins are neuronal cell surface proteins with hundreds of isoforms generated by alternative splicing. Here we describe neuroligin 1, a neuronal cell surface protein that is enriched in synaptic plasma membranes and acts as a splice site-specific ligand for beta-neurexins. Neuroligin 1 binds to beta-neurexins only if they lack an insert in the alternatively spliced sequence of the G domain, but not if they contain an insert. The extracellular sequence of neuroligin 1 is composed of a catalytically inactive esterase domain homologous to acetylcholinesterase. In situ hybridization reveals that alternative splicing of neurexins at the site recognized by neuroligin 1 is highly regulated. These findings support a model whereby alternative splicing of neurexins creates a family of cell surface receptors that confers interactive specificity onto their resident neurons.

Alternative Splicing↗

PubMed1995

The appendage domain of alpha-adaptin is a high affinity binding site for dynamin.

Dynamin is a GTPase that appears to be required for endocytosis. Even though this molecule is known to be in surface-coated pits, the identity of the resident coat proteins that account for this localization is not known. Here we show that dynamin is one of three synaptic terminal proteins that bind with specificity to the appendage domain of alpha-adaptin. Binding is sensitive to both salt and pH levels but is not affected by nucleotides. Using recombinant dynamin expressed in SF9 cells, we estimate that the binding affinity is approximately 200 nM. Binding does not require GTP, and the GTPase activity of dynamin is not stimulated by this interaction. These results suggest that the COOH terminus of alpha-adaptin may be a domain within AP2 that mediates the initial interactions between dynamin and surface-coated pits. This may be an essential step in the regulation of coated pit budding.

Adaptor Protein Complex 2↗

PubMed1995

Structure of the first C2 domain of synaptotagmin I: a novel Ca2+/phospholipid-binding fold.

C2 domains are regulatory sequence motifs that occur widely in nature. Synaptotagmin I, a synaptic vesicle protein involved in the Ca2+ regulation of exocytosis, contains two C2 domains, the first of which acts as a Ca2+ sensor. We now describe the three-dimensional structure of this C2 domain at 1.9 A resolution in both the Ca(2+)-bound and Ca(2+)-free forms. The C2 polypeptide forms an eight-stranded beta sandwich constructed around a conserved four-stranded motif designated as a C2 key. Ca2+ binds in a cup-shaped depression between two polypeptide loops located at the N- and C-termini of the C2-key motif.

Amino Acid Sequence↗

PubMed1995

Synaptic core complex of synaptobrevin, syntaxin, and SNAP25 forms high affinity alpha-SNAP binding site.

SNAPs (soluble NSF attachment proteins) are cytoplasmic proteins that bind to specific membrane receptors and mediate the membrane binding of NSF (N-ethylmaleimide-sensitive factor), a protein that is required for membrane fusion reactions. Three synaptic proteins in brain (SNAP25 (synaptosomal-associated protein of 25 kDa; no relation to the SNAPs for NSF), synaptobrevin/VAMP, and syntaxin) were identified as SNAP receptors by affinity chromatography on immobilized alpha-SNAP complexed to NSF (Söllner, T., Whiteheart, S. W., Brunner, M., Erdjument-Bromage, H., Geromanos, S., Tempst, P. and Rothman, J. E. (1993) Nature 362, 318-324). However, the nature of the alpha-SNAP binding site is unclear. We now show that alpha-SNAP binds tightly to the complex of syntaxin with synaptobrevin. SNAP25 is not required for tight binding of alpha-SNAP to this complex but stabilizes the syntaxin-synaptobrevin complex by forming a trimeric core complex with it. alpha-SNAP does not bind to synaptobrevin individually and binds only weakly to syntaxin and SNAP25 in the absence of synaptobrevin. These data suggest that the complex of the vesicular protein synaptobrevin with the plasma membrane protein syntaxin is required for physiological alpha-SNAP binding. Thus, alpha-SNAP probably functions in a late step of the membrane fusion reaction after the formation of the synaptobrevin-syntaxin-SNAP25 core complex.

Carrier Proteins↗

PubMed1995

Cartography of neurexins: more than 1000 isoforms generated by alternative splicing and expressed in distinct subsets of neurons.

Neurexins, a family of cell surface proteins specific to brain, are transcribed from two promoters in three genes, resulting in three alpha- and three beta-neurexins. In situ hybridization revealed differential but overlapping distributions of neurexin isoforms in different classes of neurons. PCRs demonstrated that alpha-neurexins are alternatively spliced at five canonical positions, and beta-neurexins at two. Characterization of many independent bovine neurexin I alpha cDNAs suggests that different splice sites are used independently. This creates the potential to express more than 1000 distinct neurexin proteins in brain. The splicing pattern is conserved in rat and cow. Thus, in addition to somatic gene rearrangement (immunoglobulins and T cell receptors) and large gene families (odorant receptors), alternative splicing potentially represents a third mechanism for creating a large number of cell surface receptors that are expressed by specific subsets of cells.

Alternative Splicing↗

PubMed1995

Mapping of synapsin II (SYN2) genes to human chromosome 3p and mouse chromosome 6 band F.

Synapsins are neuron-specific phosphoproteins of small synaptic vesicles encoded by two different genes. While the gene for synapsin I (SYN1) is on the X chromosome, we have now assigned the human and mouse synapsin II (SYN2) genes to autosomes. By using PCR primers derived from rat synapsin II cDNA sequences we were able to amplify homologous sequences of the 3'-untranslated regions and to localize the human SYN2 gene to 3p and the mouse Syn2 gene to mouse chromosome 6 by single strand conformation analysis of PCR products from panels of somatic hybrid cell lines. The mouse gene was further mapped by FISH to chromosome 6 band F in a region of known conserved synteny with human 3p. Genotyping of a M. musculus x M. spretus backcross panel placed Syn2 close to a cluster of previously mapped loci on chromosome 6 in an interval between interleukin 5 receptor alpha (Il5ra) and hematopoietic cell phosphatase 1C (Hcph). Both physical and genetic mapping data indicate that Syn2 is near two mutant loci defined by neuromuscular disorders, opisthotonus (opt) and deaf waddler (dfw).

Animals↗

PubMed1995

Phosphorylation of rabphilin-3A by Ca2+/calmodulin- and cAMP-dependent protein kinases in vitro.

Regulation of neurotransmitter release is thought to involve modulation of the release probability by protein phosphorylation. In order to identify novel targets for such regulatory processes, we have studied the phosphorylation of rabphilin-3A in vitro. Rabphilin-3A is a synaptic vesicle protein that interacts with rab3A in a GTP-dependent manner and binds Ca2+ in a phospholipid-dependent manner. Here we show that rabphilin-3A is an efficient substrate for Ca2+/calmodulin-dependent protein kinase II, which phosphorylates rat rabphilin-3A at residue 234 and 274, and for cAMP-dependent protein kinase, which phosphorylates rat rabphilin-3A at residue 234. This identifies the middle region of rabphilin-3A situated between the N-terminal rab3A-binding sequences and the C-terminal C2-domains involved in Ca2+/phospholipid binding as a regulatory domain. Thus, rabphilin-3A is a second phosphoprotein on synaptic vesicles that, similar to synapsin I, may integrate phosphorylation signals from multiple protein kinase signaling pathways in the cell.

Adaptor Proteins, Signal Transducing↗

PubMed1994

Synaptotagmin I: a major Ca2+ sensor for transmitter release at a central synapse.

Mice carrying a mutation in the synaptotagmin I gene were generated by homologous recombination. Mutant mice are phenotypically normal as heterozygotes, but die within 48 hr after birth as homozygotes. Studies of hippocampal neurons cultured from homozygous mutant mice reveal that synaptic transmission is severely impaired. The synchronous, fast component of Ca(2+)-dependent neurotransmitter release is decreased, whereas asynchronous release processes, including spontaneous synaptic activity (miniature excitatory postsynaptic current frequency) and release triggered by hypertonic solution or alpha-latrotoxin, are unaffected. Our findings demonstrate that synaptotagmin I function is required for Ca2+ triggering of synchronous neurotransmitter release, but is not essential for asynchronous or Ca(2+)-independent release. We propose that synaptotagmin I is the major low affinity Ca2+ sensor mediating Ca2+ regulation of synchronous neurotransmitter release in hippocampal neurons.

Animals↗

PubMed1994

Ca(2+)-dependent conformational change in synaptotagmin I.

Synaptotagmin I is a Ca2+/phospholipid binding protein of synaptic vesicles with a proposed function as a Ca2+ sensor in synaptic vesicle exocytosis. Using controlled partial proteolysis as an assay, we now show that synaptotagmin I undergoes a conformational change as a function of Ca2+ binding. As observed for phospholipid binding, Ba2+ and Sr2+ but not Mg2+, substitute for Ca2+ in effecting this conformational change. The first C2 domain from synaptotagmin I that represents the Ca(2+)-dependent phospholipid binding domain of synaptotagmin also undergoes a Ca(2+)-dependent change in controlled partial proteolysis. In contrast, no effect of Ca2+ was observed with mutant C2 domains containing point mutations that abolish Ca2+ binding. The Ca2+ concentration dependence of the effect of Ca2+ on proteolysis mirrors the Ca2+ dependence of phospholipid binding. The conformational shift in synaptotagmin I caused by Ca2+/phospholipid binding could be the basis for its Ca(2+)-regulated function in triggering neurotransmitter release.

Animals↗

PubMed1994

Co-expression in vertebrate tissues and cell lines of multiple inositol 1,4,5-trisphosphate (InsP3) receptors with distinct affinities for InsP3.

Inositol 1,4,5-trisphosphate (InsP3) is a ubiquitous second messenger in eukaryotic cells that triggers Ca2+ release from intracellular stores. Three types of InsP3 receptors have been identified in mammals. The three receptor types are encoded by homologous genes and are structurally similar, suggesting two alternative hypotheses about the biological significance of multiple InsP3 receptors: (a) the different InsP3 receptors could have similar functions as InsP3-gated Ca2+ channels, and the presence of multiple genes could then serve as a mechanism to allow tissue-specific differential expression of receptors; or (b) the different receptors are co-expressed in cells but have distinct biological roles in these cells. To test these hypothesis, we have investigated the similarities and differences between the expression, alternative splicing, and ligand binding of different receptors. Our results demonstrate co-expression of different InsP3 receptors in almost all tissues and cell lines tested. Although all receptor types exhibit a similar specificity for inositol phosphates, the different receptors have different affinities for InsP3, with a relative order of affinities of type II > type I > type III. These findings suggest that the presence of multiple InsP3-sensitive Ca2+ pools with differential responsiveness to InsP3 may be a general property of all cells mediated by the presence of multiple types of InsP3 receptors.

Animals↗