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E R Chapman

Publications and source records attributed to E R Chapman.

34 records · Page 2Linked to original sources
PubMed1995

Poisoning by botulinum neurotoxin A does not inhibit formation or disassembly of the synaptosomal fusion complex.

We investigated the effect of poisoning rat brain synaptosomes with botulinum neurotoxin A on the NSF-mediated disassembly of a complex consisting of syntaxin, SNAP-25 and synaptobrevin (fusion complex). Botulinum neurotoxin A specifically removes 9 amino acids from the C-terminus of SNAP-25 and efficiently blocks KCl-evoked glutamate release from synaptosomes. We report that truncated SNAP-25 is incorporated into the fusion complex of poisoned synaptosomes. The presence of truncated SNAP-25 does not interfere with the NSF-induced disassembly of the fusion complex. Also, the release of truncated SNAP-25 from the fusion complex is similar to that of the native SNAP-25. Since neither the formation of the complex nor its disassembly seems to be affected by the SNAP-25 fragment, this fragment is likely to block exocytosis by disrupting events between disassembly of the synaptosomal fusion complex and membrane fusion itself.

Animals↗
PubMed1995

Synaptobrevin binding to synaptophysin: a potential mechanism for controlling the exocytotic fusion machine.

The synaptic vesicle protein synaptobrevin (VAMP) has recently been implicated as one of the key proteins involved in exocytotic membrane fusion. It interacts with the synaptic membrane proteins syntaxin I and synaptosome-associated protein (SNAP)-25 to form a complex which precedes exocytosis [Söllner et al. (1993b) Cell, 75, 409-418]. Here we demonstrate that the majority of synaptobrevin is bound to the vesicle protein synaptophysin in detergent extracts. No syntaxin I was found in this complex when synaptophysin-specific antibodies were used for immunoprecipitation. Conversely, no synaptophysin was associated with the synaptobrevin-syntaxin I complex when syntaxin-specific antibodies were used for immunoprecipitation. Thus, the synaptobrevin pool bound to synaptophysin is not available for binding to syntaxin I and SNAP-25, and vice versa. Synaptobrevin-synaptophysin binding was also demonstrated by chemical cross-linking in isolated nerve terminals. Furthermore, recombinant synaptobrevin II efficiently bound synaptophysin and its isoform synaptoporin, but not the more distantly related synaptic vesicle protein p29. Recombinant synaptobrevin I bound with similar efficiency, whereas the non-neuronal isoform cellubrevin displayed a lower affinity towards synaptophysin. Treatment with high NaCl concentrations resulted in a dissociation of the synaptobrevin-synaptophysin complex. In addition, the interaction of synaptobrevin with synaptophysin was irreversibly abolished by low amounts of SDS, while the interaction with syntaxin I was enhanced. We conclude that synaptophysin selectively interacts with synaptobrevin in a complex which excludes the t-SNAP receptors syntaxin I and SNAP-25, suggesting a role for synaptophysin in the control of exocytosis.

Amino Acid Sequence↗
PubMed1995

The t-SNAREs syntaxin 1 and SNAP-25 are present on organelles that participate in synaptic vesicle recycling.

Syntaxin 1 and synaptosome-associated protein of 25 kD (SNAP-25) are neuronal plasmalemma proteins that appear to be essential for exocytosis of synaptic vesicles (SVs). Both proteins form a complex with synaptobrevin, an intrinsic membrane protein of SVs. This binding is thought to be responsible for vesicle docking and apparently precedes membrane fusion. According to the current concept, syntaxin 1 and SNAP-25 are members of larger protein families, collectively designated as target-SNAP receptors (t-SNAREs), whose specific localization to subcellular membranes define where transport vesicles bind and fuse. Here we demonstrate that major pools of syntaxin 1 and SNAP-25 recycle with SVs. Both proteins cofractionate with SVs and clathrin-coated vesicles upon subcellular fractionation. Using recombinant proteins as standards for quantitation, we found that syntaxin 1 and SNAP-25 each comprise approximately 3% of the total protein in highly purified SVs. Thus, both proteins are significant components of SVs although less abundant than synaptobrevin (8.7% of the total protein). Immunoisolation of vesicles using synaptophysin and syntaxin specific antibodies revealed that most SVs contain syntaxin 1. The widespread distribution of both syntaxin 1 and SNAP-25 on SVs was further confirmed by immunogold electron microscopy. Botulinum neurotoxin C1, a toxin that blocks exocytosis by proteolyzing syntaxin 1, preferentially cleaves vesicular syntaxin 1. We conclude that t-SNAREs participate in SV recycling in what may be functionally distinct forms.

Animals↗
PubMed1994

SNAP-25, a t-SNARE which binds to both syntaxin and synaptobrevin via domains that may form coiled coils.

The membrane proteins SNAP-25, syntaxin, and synaptobrevin (vesicle-associated membrane protein) have recently been implicated as central elements of an exocytotic membrane fusion complex in neurons. Here we report that SNAP-25 binds directly to both syntaxin and synaptobrevin. The SNAP-25-binding domain of syntaxin lies between residues 199 and 243, within the region previously shown to mediate synaptobrevin binding (Calakos, N., Bennett, M. K., Peterson, K. E., and Scheller, R. H. (1994) Science 263, 1146-1149). The syntaxin-binding domain of SNAP-25 encompasses most of the amino-terminal half of SNAP-25, including its putative palmitoylation sites. Truncation of the carboxyl-terminal 9 residues of SNAP-25, which yields a fragment corresponding to that generated by botulinum neurotoxin A, diminishes the interaction of SNAP-25 with synaptobrevin, but not with syntaxin. Sequence analysis revealed that the regions that mediate the interaction between SNAP-25 and syntaxin contain heptad repeats characteristic of certain classes of alpha-helices. Similar repeats are also present at the carboxyl terminus of SNAP-25 and in synaptobrevin. These domains have a moderate to high probability of forming coiled coils. We conclude that SNAP-25 can interact with both syntaxin and synaptobrevin and that binding may be mediated by alpha-helical domains that form intermolecular coiled-coil structures.

Animals↗
PubMed1994

Calcium-dependent interaction of the cytoplasmic region of synaptotagmin with membranes. Autonomous function of a single C2-homologous domain.

The synaptic vesicle protein synaptotagmin has been implicated in the docking and subsequent calcium-regulated exocytosis of synaptic vesicles. We demonstrate that synaptotagmin is a major constituent of synaptic vesicle membranes, comprising 7-8% of the total vesicle protein. A proteolytic fragment of synaptotagmin, containing two repeats homologous to the C2-domain of protein kinase C, bound to a variety of natural membranes in a calcium-dependent manner (EC50 approximately 30 microM calcium). Binding was insensitive to proteolysis of the acceptor membranes suggesting an interaction with the lipid constituents. This interaction was confirmed using a recombinant fusion protein, containing both C2-like domains of synaptotagmin, that bound to artificial liposomes in a calcium-dependent manner. Phospholipid binding properties were preserved in a 114-amino acid domain corresponding to the first C2-like repeat of the protein and represents the shortest functional cassette yet reported. Furthermore, deletion of a highly conserved 9-amino acid motif, within this region, was sufficient to abolish the calcium-dependent phospholipid binding properties of this domain. This mutation may provide a means to selectively disrupt individual C2-domains in order to assess their relative contributions to function.

Amino Acid Sequence↗
PubMed1994

Synaptic targeting of rabphilin-3A, a synaptic vesicle Ca2+/phospholipid-binding protein, depends on rab3A/3C.

rab3A, a low molecular weight GTP-binding protein of synaptic vesicles with a putative function in synaptic vesicle docking, interacts in a GTP-dependent manner with rabphilin-3A, a peripheral membrane protein that binds Ca2+ and phospholipids. We now show that rabphilin-3A is an evolutionarily conserved synaptic vesicle protein that is attached to synaptic vesicle membranes via its N terminus and exhibits a heterogeneous distribution among synapses. In rab3A-deficient mice, rabphilin-3A is decreased in synapses belonging to neurons that primarily express rab3A and accumulates in the perikarya of these neurons. In contrast, neurons expressing significant levels of rab3C still contain normal levels of rabphilin-3A in a synaptic pattern, and rabphilin-3A binds rab3C in vitro. These results suggest that analogous to the membrane recruitment of raf by ras, rab3A and rab3C may function in recruiting rabphilin-3A to the synaptic vesicle membrane in a GTP-dependent manner.

Adaptor Proteins, Signal Transducing↗
PubMed1993

Botulinum neurotoxin A selectively cleaves the synaptic protein SNAP-25.

Neurotransmitter release is potently blocked by a group of structurally related toxin proteins produced by Clostridium botulinum. Botulinum neurotoxin type B (BoNT/B) and tetanus toxin (TeTx) are zinc-dependent proteases that specifically cleave synaptobrevin (VAMP), a membrane protein of synaptic vesicles. Here we report that inhibition of transmitter release from synaptosomes caused by botulinum neurotoxin A (BoNT/A) is associated with the selective proteolysis of the synaptic protein SNAP-25. Furthermore, isolated or recombinant L chain of BoNT/A cleaves SNAP-25 in vitro. Cleavage occurred near the carboxyterminus and was sensitive to divalent cation chelators. In addition, a glutamate residue in the BoNT/A L chain, presumably required to stabilize a water molecule in the zinc-containing catalytic centre, was required for proteolytic activity. These findings demonstrate that BoNT/A acts as a zinc-dependent protease that selectively cleaves SNAP-25. Thus, a second component of the putative fusion complex mediating synaptic vesicle exocytosis is targeted by a clostridial neurotoxin.

Botulinum Toxins↗
PubMed1993

Botulinum neurotoxin C1 blocks neurotransmitter release by means of cleaving HPC-1/syntaxin.

The anaerobic bacterium Clostridium botulinum produces several related neurotoxins that block exocytosis of synaptic vesicles in nerve terminals and that are responsible for the clinical manifestations of botulism. Recently, it was reported that botulinum neurotoxin type B as well as tetanus toxin act as zinc-dependent proteases that specifically cleave synaptobrevin, a membrane protein of synaptic vesicles (Link et al., Biochem. Biophys. Res. Commun., 189, 1017-1023; Schiavo et al., Nature, 359, 832-835). Here we report that inhibition of neurotransmitter release by botulinum neurotoxin type C1 was associated with the proteolysis of HPC-1 (= syntaxin), a membrane protein present in axonal and synaptic membranes. Breakdown of HPC-1/syntaxin was selective since no other protein degradation was detectable. In vitro studies showed that the breakdown was due to a direct interaction between HPC-1/syntaxin and the toxin light chain which acts as a metallo-endoprotease. Toxin-induced cleavage resulted in the generation of a soluble fragment of HPC-1/syntaxin that is 2-4 kDa smaller than the native protein. When HPC-1/syntaxin was translated in vitro, cleavage occurred only when translation was performed in the presence of microsomes, although a full-length product was obtained in the absence of membranes. However, susceptibility to toxin cleavage was restored when the product of membrane-free translation was subsequently incorporated into artificial proteoliposomes. In addition, a translated form of HPC-1/syntaxin, which lacked the putative transmembrane domain at the C-terminus, was soluble and resistant to toxin action. We conclude that HPC-1/syntaxin is involved in exocytotic membrane fusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Fluorescence energy transfer analysis of calmodulin-peptide complexes.

The interactions between calmodulin and the tryptophan residues of synthetic peptides corresponding to the calmodulin binding domains of skeletal muscle myosin light-chain kinase and the plasma membrane calcium pump were examined. The single tryptophan residue contained in each peptide became relatively immobilized and inaccessible to iodide ion upon binding to calmodulin, indicating that the indole side chain was inserted into a hydrophobic cleft in the surface of calmodulin. Fluorescence energy transfer from peptidyl tryptophan residues to an AEDANS moiety attached to cysteine-26 of spinach calmodulin was measured. Included in these analyses was a tryptophan-containing peptide analog of the calmodulin binding domain of neuromodulin. These data indicated that the indole ring of each peptide inserted 32-35 A away from cysteine-26 and may therefore interact with the carboxyl-terminal lobe of CaM in its "bent" conformation [Persechini & Kretsinger (1988a) J. Cardiovasc. Pharmacol. 12 (Suppl 5), S1-S12; Ikura et al. (1992) Science 256, 632-638; Vorherr et al. (1992) Eur. J. Biochem. 204, 931-937]. The interchange of tryptophan-3 and phenylalanine-21 of the calcium pump peptide increased the efficiency of energy transfer to the AEDANS-moiety approximately 12-fold, reducing the calculated distance to 20 A. These data suggest that phenylalanine-21 of the calcium pump peptide interacts with the hydrophobic cleft in the amino-terminal lobe of CaM.

Amino Acid Sequence↗
PubMed1992

Palmitylation of neuromodulin (GAP-43) is not required for phosphorylation by protein kinase C.

Neuromodulin (also designated GAP-43, B-50, and F-1) is a prominent protein kinase C substrate attached to the membranes of neuronal growth cones during development and to presynaptic membranes in discrete subsets of adult synapses. In this study, we have examined the relationship between the attachment of neuromodulin to membranes and its phosphorylation by protein kinase C. To address this issue, we have compared wild-type and mutant neuromodulins expressed in cells that normally lack the protein. Wild-type neuromodulin expressed in Chinese hamster ovary cells was associated with membranes, incorporated [3H]palmitic acid, and was phosphorylated in response to phorbol ester treatment. Substitution of serine 41, the in vitro protein kinase C site, abolished the phorbol ester response, indicating that serine 41 serves as the sole protein kinase C phosphorylation site in vivo. Substitution of the putative fatty acylation sites, cysteines 3 and 4, abolished membrane association as well as [3H]palmitic acid labeling of neuromodulin. Fatty acylation therefore appears to serve as the mechanism for anchoring neuromodulin to membranes. Surprisingly, the soluble cysteine substitution mutant was phosphorylated by protein kinase C at a rate indistinguishable from that of the wild-type protein. Therefore, membrane association may not be required for the phosphorylation of neuromodulin by protein kinase C.

Amino Acid Sequence↗
PubMed1991

Characterization of the calmodulin binding domain of neuromodulin. Functional significance of serine 41 and phenylalanine 42.

Neuromodulin (also designated P-57, GAP-43, B-50) is a major presynaptic substrate for protein kinase C. Phosphorylation of neuromodulin decreases its affinity for calmodulin, suggesting that neuromodulin may function to bind and concentrate calmodulin at specific sites within neurons, releasing calmodulin locally in response to phosphorylation by protein kinase C (Alexander, K. A., Cimler, B. M., Meier, K. E., and Storm, D. R. (1987) J. Biol. Chem. 262, 6108-6113). In the present study, we have constructed and characterized several mutant neuromodulins to demonstrate that the amino acid sequence 39-56 is required for calmodulin binding, and that this domain contains the sole in vitro protein kinase C phosphorylation site at serine 41. We also demonstrate that the adjacent phenylalanine 42, interacts hydrophobically with calmodulin. These hydrophobic interactions may be disrupted by the introduction of negative charge at serine 41, and thereby regulate the neuromodulin/calmodulin binding interactions. The sensitivity of the neuromodulin/calmodulin binding interaction to negative charge at serine 41 was determined by substitution of serine 41 with an aspartate or an asparagine residue. The asparagine mutant retained its affinity for calmodulin-Sepharose while the aspartate mutant did not adsorb to calmodulin-Sepharose. We conclude that protein kinase C phosphorylation of neuromodulin abolishes calmodulin binding by introducing negative charges within the calmodulin binding domain at a position adjacent to the phenylalanine.

Amino Acid Sequence↗
PubMed1991

Targeting of neuromodulin (GAP-43) fusion proteins to growth cones in cultured rat embryonic neurons.

Neuromodulin (GAP-43) is a membrane protein that is transported to neuronal growth cones. Zuber and co-workers have proposed that the N-terminal 10 amino acid sequence of neuromodulin is sufficient to target proteins to growth cones. We demonstrate that a neuromodulin-beta-galactosidase fusion protein is transported to growth cones of cultured rat neurons, whereas a fusion protein containing the N-terminal 10 amino acids of neuromodulin and beta-galactosidase is not. A mutant neuromodulin lacking cysteines 3 and 4, the palmitylation sites required for membrane attachment, does not target beta-galactosidase to growth cones. We conclude that membrane attachment is required for growth cone accumulation and that structural elements, in addition to the first 10 amino acids of neuromodulin, may be required for growth cone targeting.

Animals↗
PubMed1989

Expression of cDNAs encoding wild-type and mutant neuromodulins in Escherichia coli: comparison with the native protein from bovine brain.

Murine cDNA that encodes neuromodulin, a neurospecific calmodulin binding protein, was inserted into the plasmid pKK223-3 for expression in Escherichia coli. After being transformed into E. coli strain SG20252 (lon-), the expression vector directed the synthesis of a protein that was recognized by polyclonal antibodies raised against bovine neuromodulin. The recombinant protein expressed in E. coli was found to be tightly associated with insoluble cell material and was extractable only with guanidine hydrochloride or sodium dodecyl sulfate. Following solubilization with guanidine hydrochloride, the protein was purified to apparent homogeneity by a single CaM-Sepharose affinity column step with a yield of 0.2 mg of protein/L of E. coli culture. The availability of the purified recombinant neuromodulin made it possible to answer several specific questions concerning the structure and function of the protein. Despite the fact that murine neuromodulin is 12 amino acid residues shorter than the bovine protein and the recombinant protein expressed in E. coli may lack any posttranslational modifications, the two proteins displayed similar biochemical properties in almost all respects examined. They both had higher affinity for CaM-Sepharose in the absence of Ca2+ than in its presence; they were both phosphorylated in vitro by protein kinase C in a Ca2+- and phospholipid-dependent manner; neither form of the proteins was autophosphorylated, and the phosphorylated form of the proteins did not bind calmodulin. The recombinant neuromodulin and neuromodulin purified from bovine brain had similar, but not identical, affinities of calmodulin, indicating that the palmitylation of the protein that occurs in animal cells is not crucial for calmodulin interactions.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗