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R Jahn

Publications and source records attributed to R Jahn.

At least 55 records · Page 3Linked to original sources
PubMed1999

Reconstitution of regulated exocytosis in cell-free systems: a critical appraisal.

Regulated exocytosis involves the tightly controlled fusion of a transport vesicle with the plasma membrane. It includes processes as diverse as the release of neurotransmitters from presynaptic nerve endings and the sperm-triggered deposition of a barrier preventing polyspermy in oocytes. Cell-free model systems have been developed for studying the biochemical events underlying exocytosis. They range from semi-intact permeabilized cells to the reconstitution of membrane fusion from isolated secretory vesicles and their target plasma membranes. Interest in such cell-free systems has recently been reinvigorated by new evidence suggesting that membrane fusion is mediated by a basic mechanism common to all intracellular fusion events. In this chapter, we review some of the literature in the light of these new developments and attempt to provide a critical discussion of the strengths and limitations of the various cell-free systems.

Animals↗
PubMed1998

Conserved structural features of the synaptic fusion complex: SNARE proteins reclassified as Q- and R-SNAREs.

SNARE [soluble NSF (N-ethylmaleimide-sensitive fusion protein) attachment protein receptor] proteins are essential for membrane fusion and are conserved from yeast to humans. Sequence alignments of the most conserved regions were mapped onto the recently solved crystal structure of the heterotrimeric synaptic fusion complex. The association of the four alpha-helices in the synaptic fusion complex structure produces highly conserved layers of interacting amino acid side chains in the center of the four-helix bundle. Mutations in these layers reduce complex stability and cause defects in membrane traffic even in distantly related SNAREs. When syntaxin-4 is modeled into the synaptic fusion complex as a replacement of syntaxin-1A, no major steric clashes arise and the most variable amino acids localize to the outer surface of the complex. We conclude that the main structural features of the neuronal complex are highly conserved during evolution. On the basis of these features we have reclassified SNARE proteins into Q-SNAREs and R-SNAREs, and we propose that fusion-competent SNARE complexes generally consist of four-helix bundles composed of three Q-SNAREs and one R-SNARE.

Amino Acid Sequence↗
PubMed1998

Synaptic transmission: two players team up for a new tune.

An unexpected link has been found between two molecules that were thought to perform seemingly unrelated functions. The AMPA-subclass glutamate receptor GluR2 interacts with the membrane-fusion protein NSF in a manner that appears important for receptor-mediated intracellular signalling.

Carrier Proteins↗
PubMed1998

Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 A resolution.

The evolutionarily conserved SNARE proteins and their complexes are involved in the fusion of vesicles with their target membranes; however, the overall organization and structural details of these complexes are unknown. Here we report the X-ray crystal structure at 2.4 A resolution of a core synaptic fusion complex containing syntaxin-1 A, synaptobrevin-II and SNAP-25B. The structure reveals a highly twisted and parallel four-helix bundle that differs from the bundles described for the haemagglutinin and HIV/SIV gp41 membrane-fusion proteins. Conserved leucine-zipper-like layers are found at the centre of the synaptic fusion complex. Embedded within these leucine-zipper layers is an ionic layer consisting of an arginine and three glutamine residues contributed from each of the four alpha-helices. These residues are highly conserved across the entire SNARE family. The regions flanking the leucine-zipper-like layers contain a hydrophobic core similar to that of more general four-helix-bundle proteins. The surface of the synaptic fusion complex is highly grooved and possesses distinct hydrophilic, hydrophobic and charged regions. These characteristics may be important for membrane fusion and for the binding of regulatory factors affecting neurotransmission.

Antigens, Surface↗
PubMed1998

Identification of a minimal core of the synaptic SNARE complex sufficient for reversible assembly and disassembly.

Assembly of the three neuronal membrane proteins synaptobrevin, syntaxin, and SNAP-25 is thought to be one of the key steps in mediating exocytosis of synaptic vesicles. In vivo and in vitro, these proteins form a tight complex. Assembly is associated with a large increase in alpha-helical content, suggesting that major structural and conformational changes are associated with the assembly reaction. Limited proteolysis by trypsin, chymotrypsin, and proteinase K of the ternary complex formed from recombinant proteins lacking their membrane anchors revealed a SDS-resistant minimal core. The components of this core complex were purified and characterized by N-terminal sequencing and mass spectrometry. They include a slightly shortened synaptobrevin fragment, C- and N-terminal fragments of SNAP-25, and a C-terminal fragment of syntaxin that is slightly larger than the previously characterized H3 domain. Recombinant proteins corresponding to these fragments are sufficient for assembly and disassembly. In addition, each of the two SNAP-25 fragments can individually form complexes with syntaxin and synaptobrevin, suggesting that they both contribute to the assembly of the SNARE complex. Upon complex assembly, a large increase in alpha-helical content is observed along with a significantly increased melting temperature (Tm). Like the full-length complex, the minimal complex tends to form an oligomeric species; global analysis of equilibrium ultracentrifugation data suggests a monomer-trimer equilibrium exists. These conserved biophysical properties may thus be of fundamental importance in the mechanism of membrane fusion.

Amino Acid Sequence↗
PubMed1998

Vesicle transport: klarsicht clears up the matter.

Lipid droplets can be seen to move around on microtubule tracks in a characteristic manner within a developing Drosophila embryo. This phenomenon has allowed genetic studies to be combined with biophysical measurements of single moving droplets, providing a clear view of motor protein control in vivo.

Animals↗
PubMed1998

The heterotrimeric G protein Go2 regulates catecholamine uptake by secretory vesicles.

Secretory vesicles store neurotransmitters that are released by exocytosis. Their membrane contains transporters responsible for transmitter loading that are driven by an electrochemical proton gradient across the vesicle membrane. We have now examined whether uptake of noradrenaline is regulated by heterotrimeric G proteins. In streptolysin O-permeabilized PC 12 cells, GTP-analogues and AlF4- inhibited noradrenaline uptake, an effect that was sensitive to treatment with pertussis toxin. Inhibition of uptake was prevented by Galphao-specific antibodies and mimicked by purified activated Galphao2. No effect was seen when Galphao2 in its inactive GDP-bound form or purified activated Galphao1, Galphai1 and Galphai2 were tested. Down-regulation of uptake remained unchanged when exocytosis was inhibited by the light chain of tetanus toxin. Vesicular acidification was not affected whereas binding of [3H]reserpine was reduced by GTPgammaS and Galphao2. These data suggest that the monoamine transporter rather than the vacuolar ATPase is affected. We conclude that catecholamine uptake is controlled by Galphao2, suggesting a novel function for heterotrimeric G proteins in the control of neurotransmitter storage.

Animals↗
PubMed1998

Structure of the ATP-dependent oligomerization domain of N-ethylmaleimide sensitive factor complexed with ATP.

N-ethylmaleimide-sensitive factor (NSF) is a hexameric ATPase which primes and/or dissociates SNARE complexes involved in intracellular fusion events. Each NSF protomer contains three domains: an N-terminal domain required for SNARE binding and two ATPase domains, termed D1 and D2, with D2 being required for oligomerization. We have determined the 1.9 A crystal structure of the D2 domain of NSF complexed with ATP using multi-wavelength anomalous dispersion phasing. D2 consists of a nucleotide binding subdomain with a Rossmann fold and a C-terminal subdomain, which is structurally unique among nucleotide binding proteins. There are interactions between the ATP moiety and both the neighboring D2 protomer and the C-terminal subdomain that may be important for ATP-dependent oligomerization. Of particular importance are three well-ordered and conserved lysine residues that form ionic interactions with the beta- and gamma-phosphates, one of which likely contributes to the low hydrolytic activity of D2.

Adenosine Triphosphatases↗
PubMed1998

[Case report of segmental neurofibromatosis of the 3rd finger ray].

We report on a 36-year-old woman suffering from a segmental neurofibromatosis solely confined to the third finger. Microscopically, the tumor showed mesenchymal tissue containing myxoid material with features of the nerve sheath--myxoid neurofibroma. The tumor was characterized by the peculiar tendency to grow in a multifocal pattern involving the distribution of every nerve of one finger, by an extreme proneness to local recurrences as well as by a concomitant bone defect.

Adult↗
PubMed1997

Export of cellubrevin from the endoplasmic reticulum is controlled by BAP31.

Cellubrevin is a ubiquitously expressed membrane protein that is localized to endosomes throughout the endocytotic pathway and functions in constitutive exocytosis. We report that cellubrevin binds with high specificity to BAP31, a representative of a highly conserved family of integral membrane proteins that has recently been discovered to be binding proteins of membrane immunoglobulins. The interaction between BAP31 and cellubrevin is sensitive to high ionic strength and appears to require the transmembrane regions of both proteins. No other proteins of liver membrane extracts copurified with BAP31 on immobilized recombinant cellubrevin, demonstrating that the interaction is specific. Synaptobrevin I bound to BAP31 with comparable affinity, whereas only weak binding was detectable with synaptobrevin II. Furthermore, a fraction of BAP31 and cellubrevin was complexed when each of them was quantitatively immunoprecipitated from detergent extracts of fibroblasts (BHK 21 cells). During purification of clathrin-coated vesicles or early endosomes, BAP31 did not cofractionate with cellubrevin. Rather, the protein was enriched in ER-containing fractions. When BHK cells were analyzed by immunocytochemistry, BAP31 did not overlap with cellubrevin, but rather colocalized with resident proteins of the ER. In addition, immunoreactive vesicles were clustered in a paranuclear region close to the microtubule organizing center, but different from the Golgi apparatus. When microtubules were depolymerized with nocodazole, this accumulation disappeared and BAP31 was confined to the ER. Truncation of the cytoplasmic tail of BAP31 prevented export of cellubrevin, but not of the transferrin receptor from the ER. We conclude that BAP31 represents a novel class of sorting proteins that controls anterograde transport of certain membrane proteins from the ER to the Golgi complex.

Animals↗
PubMed1997

Structural changes are associated with soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor complex formation.

SNAP-25, syntaxin, and synaptobrevin play a key role in the regulated exocytosis of synaptic vesicles, but their mechanism of action is not understood. In vitro, the proteins spontaneously assemble into a ternary complex that can be dissociated by the ATPase N-ethylmaleimide-sensitive fusion protein and the cofactors alpha-, beta-, and gamma-SNAP. Since the structural changes associated with these reactions probably form the basis of membrane fusion, we have embarked on biophysical studies aimed at elucidating such changes in vitro using recombinant proteins. All proteins were purified in a monomeric form. Syntaxin showed significant alpha-helicity, whereas SNAP-25 and synaptobrevin exhibited characteristics of largely unstructured proteins. Formation of the ternary complex induced dramatic increases in alpha-helicity and in thermal stability. This suggests that structure is induced in SNAP-25 and synaptobrevin upon complex formation. In addition, the stoichiometry changed from 2:1 in the syntaxin-SNAP-25 complex to 1:1:1 in the ternary complex. We propose that the transition from largely unstructured monomers to a tightly packed, energetically favored ternary complex connecting two membranes is a key step in overcoming energy barriers for membrane fusion.

Carrier Proteins↗
PubMed1997

Structure and conformational changes in NSF and its membrane receptor complexes visualized by quick-freeze/deep-etch electron microscopy.

Using quick-freeze/deep-etch electron microscopy of recombinant proteins adsorbed to mica, we show that NSF, the oligomeric ATPase involved in membrane fusion, is a hollow 10 x 16 nm cylinder whose conformation depends upon nucleotide binding. Depleted of nucleotide, NSF converts to a "splayed" protease-sensitive conformation that reveals its subunit composition. NSF's synaptic membrane substrate, the ternary SNARE complex containing syntaxin, SNAP-25, and synaptobrevin, is a 4 x 14 nm rod with a "tail" at one end, corresponding to the N-terminus of syntaxin. Using epitope tags, antibodies, and maltose-binding protein markers, we find that syntaxin and synaptobrevin are aligned in parallel in the complex, with their membrane anchors located at the same end of the rod. This SNARE rod binds with alpha-SNAP to one end of the NSF cylinder to form an asymmetric "20S" complex. Together, these images suggest how NSF could dissociate the SNARE complex and how association and dissociation of the complex could be related to membrane fusion.

Adenosine Triphosphatases↗
PubMed1997

The secretory granule protein syncollin binds to syntaxin in a Ca2(+)-sensitive manner.

The membrane proteins synaptobrevin, syntaxin, and SNAP-25 form the core of a ubiquitous fusion machine that interacts with the soluble proteins NSF and alpha-SNAP. During regulated exocytosis, membrane fusion is usually strictly controlled by Ca2+ ions. However, the mechanism by which Ca2+ regulates exocytosis is still unclear. Here we show that the membranes of exocrine secretory granules contain an 18-kDa protein, syncollin, that binds to syntaxin at low Ca2+ concentrations and dissociates at concentrations known to stimulate exocytosis. Syncollin has a single hydrophobic domain at its N-terminus and shows no significant homology with any known protein. Recombinant syncollin inhibits fusion in vitro between zymogen granules and pancreatic plasma membranes, and its potency falls as Ca2+ concentration rises. We suggest that syncollin acts as a Ca2(+)-sensitive regulator of exocytosis in exocrine tissues.

Amino Acid Sequence↗
PubMed1997

Assembly and disassembly of a ternary complex of synaptobrevin, syntaxin, and SNAP-25 in the membrane of synaptic vesicles.

The synaptic membrane proteins synaptobrevin, syntaxin, and SNAP-25 form a ternary complex that can be disassembled by the ATPase N-ethylmaleimide-sensitive factor (NSF) in the presence of soluble cofactors (SNAP proteins). These steps are thought to represent molecular events involved in docking and subsequent exocytosis of synaptic vesicles. Using two independent and complementary approaches, we now report that such ternary complexes form in the membrane of highly purified and monodisperse synaptic vesicles in the absence of the plasma membrane. Furthermore, the complexes are reversibly dissociated by NSF and SNAP proteins. Thus, ternary complexes can be assembled and disassembled while all three proteins are anchored as neighbors in the same membrane, suggesting that NSF is involved in priming synaptic vesicles for exocytosis.

Adenosine Triphosphatases↗
PubMed1997

Inhibition of transmitter release correlates with the proteolytic activity of tetanus toxin and botulinus toxin A in individual cultured synapses of Hirudo medicinalis.

We have studied the effects of tetanus toxin and botulinus toxin A on neurotransmitter release in the Retzius-->P-cell synapse of the leech and exploited the unique properties of this system, which allow for combined physiological and biochemical analyses in single-cell pairs. The sequences of Hirudo medicinalis synaptobrevin and synaptosomal-associated protein of 25 kDa (SNAP-25), deduced by cDNA cloning, are 61 and 55% identical, respectively, to their corresponding mammalian homologs. Whereas Hirudo synaptobrevin is proteolyzed by tetanus toxin, its SNAP-25 isoform is resistant to botulinus toxin A cleavage because of amino acid substitutions within and around the putative cleavage site. In close correlation, microinjection of tetanus toxin into the presynaptic neuron produced a block of transmitter release, whereas botulinus toxin A had no effect on synaptic transmission. Subsequent immunoblotting of single-cell pairs demonstrated directly that the tetanus toxin-mediated block of exocytosis is accompanied by cleavage of synaptobrevin in the injected neuron, resulting in the generation of a detectable C-terminal cleavage product. Immunoblotting also confirmed the resistance of SNAP-25 to botulinus toxin A cleavage in vivo. Using recombinant proteins, we show that the N-terminal fragment of synaptobrevin released by tetanus toxin, but not its C-terminal membrane-anchored cleavage product, participates with syntaxin and SNAP-25 in synaptic SNAP receptor (SNARE) ternary complex formation in Hirudo. Our data demonstrate a direct correlation between the inhibition of transmitter release and the ability of the neurotoxin to proteolyze its target protein and support the view that SNARE ternary complex formation is an important step leading to synaptic vesicle exocytosis.

Amino Acid Sequence↗