Browsing by Author "Jahn, R."

We have recently solubilized and enriched a chloride- and calcium-dependent glutamate-binding protein from rat brain (Brose, N., Halpain, S., Suchanek, C., and Jahn, R. (1989) J. Biol. Chem. 264, 9619-9625). The partially purified protein fraction, containing two major protein components of 51,000 Da and 105,000 Da, was used to generate a rabbit antiserum. This serum quantitatively precipitated the binding activity from membrane extracts. Small amounts of the antiserum inhibited glutamate binding when chloride was absent from the incubation medium. Three protein bands were labeled by the serum on immunoblots. From the affinity purified antibody fractions contained in the serum, only the antibodies directed against a 51,000-Da protein were able to immunoprecipitate the binding activity, indicating that this protein is an essential component of the binding site. A survey of a variety of rat tissues by immunoblot analysis revealed a ubiquitous distribution of the protein. After subcellular fractionation of liver and brain, the 51,000-Da protein copurified with mitochondrial markers. Furthermore, exclusive labeling of mitochondria was observed by light and electron microscopy immunocytochemistry. Subfractionation of purified liver mitochondria resulted in a selective association of the protein with inner mitochondrial membranes. Pharmacological characterization of glutamate binding to liver mitochondrial membranes revealed a pattern almost identical to that of the chloride- and calcium-dependent glutamate-binding site in rat brain.

Sets of SNARE proteins mediate membrane fusion by assembling into core complexes. Multiple SNAREs are thought to function in different intracellular trafficking steps but it is often unclear which of the SNAREs cooperate in individual fusion reactions. We report that syntaxin 7, syntasin 8, vti1b and endobrevin/ VAMP-8 form a complex that functions in the fusion of late endosomes. Antibodies specific for each protein coprecipitate the complex, inhibit homotypic fusion of late endosomes in vitro and retard delivery of endocytosed epidermal growth factor to lysosomes. The purified proteins form core complexes with biochemical and biophysical properties remarkably similar to the neuronal core complex, although each of the four proteins carries a transmembrane domain and three have independently folded N-terminal domains. Substitution experiments, sequence and structural comparisons revealed that each protein occupies a unique position in the complex, with syntaxin 7 corresponding to syntasin 1, and vti1b and syntaxin 8 corresponding to the N- and C-terminal domains of SNAP-25, respectively. We conclude that the structure of core complexes and their molecular mechanism in membrane fusion is highly conserved between distant SNAREs.

SNARE proteins are crucial for intracellular membrane fusion in all eukaryotes. These proteins assemble into tight complexes that connect membranes and may induce fusion. The crystal structure of the neuronal core complex is represented by an unusually long bundle of four alpha-helices connected by 16 layers of mostly hydrophobic amino acids. Here we report the 1.9 Angstrom resolution crystal structure of an endosomal SNARE core complex containing four SNAREs: syntaxin 7, syntaxin 8, vti1b and endobrevin/VAMP-8. Despite limited sequence homology, the helix alignment and the layer structure of the endosomal complex are remarkably similar to those of the neuronal complex. However, subtle variations are evident that characterize different SNARE subfamilies. We conclude that the structure of the SNARE core complex is an evolutionarily conserved hallmark of all SNARE complexes and is intimately associated with the general role of SNAREs in membrane fusion.

Synaptotagmin (p65) is an abundant and evolutionarily conserved protein of synaptic vesicles that contains two copies of an internal repeat homologous to the regulatory region of protein kinase C. In the current study, we have investigated the biochemical properties of synaptotagmin, demonstrating that it contains five protein domains: an intravesicular amino-terminal domain that is glycosylated but lacks a cleavable signal sequence; a single transmembrane region; a sequence separating the transmembrane region from the two repeats homologous to protein kinase C; the two protein kinase C-homologous repeats; and a conserved carboxyl-terminal sequence following the two repeats homologous to protein kinase C. Sucrose density gradient centrifugations and gel electrophoresis indicate that synaptotagmin monomers associate into dimers and are part of a larger molecular weight complex. A sequence predicted to form an amphipathic alpha-helix that may cause the stable dimerization of synaptotagmin is found in its third domain between the transmembrane region and the protein kinase C-homologous repeats. Synaptotagmin contains a single hypersensitive proteolytic site that is located immediately amino-terminal to the amphipathic alpha-helix, suggesting that synaptotagmin contains a particularly exposed region as the peptide backbone emerges from the dimer. Finally, subcellular fractionation and antibody bead purification demonstrate that synaptotagmin co-purifies with synaptophysin and other synaptic vesicle markers in brain. However, in the adrenal medulla, synaptotagmin was found in both synaptophysin-containing microvesicles and in chromaffin granules that are devoid of synaptophysin, suggesting a shared role for synaptotagmin in the exocytosis of small synaptic vesicles and large dense core catecholaminergic vesicles.

Concept and design of an independent scientific evaluation of different pathways of care for schizophrenia patients in Germany with respect to effectiveness and efficiency are presented. In this prospective, observational study, schizophrenia patients receiving an integrated care treatment, the intervention group (IG), are compared with patients under routine care conditions treated by the same physician (first control group, CG 1). A second control group (CG 2) of patients treated by office-based psychiatrists not participating in the integrated care program will be recruited and their data compared with the two other groups. The total amount of psychiatric hospital days after 12 months is defined as primary outcome parameter. Secondary outcome parameters comprise the frequency of psychiatric inpatient readmissions, severity of schizophrenia symptoms, remission rates and quality of life. Patients undergo assessments at baseline, month 6 and 12 using standardized and experimental questionnaires. Routine data of a regional German social health insurance fund complement information on included patients. Additionally, a cost-effectiveness and cost-utility analysis will be performed. Until now, 137 psychiatrists included 980 patients in the integrated care project in Lower Saxony, Germany, and 47 psychiatrists (IG and both CGs) are willing to participate in the independent evaluation. For the first time, a prospective observational controlled evaluation study of a countrywide integrated care project planning to recruit 500 schizophrenia patients has started using comprehensive assessments as well as routine data of a social health insurance fund.

Endobrevin/VAMP-8 is an R-SNARE localized to endosomes, but it is unknown in which intracellular fusion step it operates. Using subcellular fractionation and quantitative immunogold electron microscopy, we found that endobrevin/VAMP-8 is present on all membranes known to communicate with early endosomes, including the plasma membrane, clathrin-coated pits, late endosomes, and membranes of the trans-Golgi network. Affinity-purified antibodies that block the ability of endobrevin/VAMP-8 to form SNARE core complexes potently inhibit homotypic fusion of both early and late endosomes in vitro. Fab fragments were as active as intact immunoglobulin Gs. Recombinant endobrevin/VAMP-8 inhibited both fusion reactions with similar potency. We conclude that endobrevin/VAMP-8 operates as an R-SNARE in the homotypic fusion of early and late endosomes.