Biological roles of neural J proteins.

J proteins are chief regulators of the Hsp70 family, a highly conserved family of ATPases that mediate conformational changes in a broad range of proteins. The J protein family has been the central focus of numerous prokaryote and eukaryote biologists. Common questions that arise include: How does the J protein/Hsp70 machinery support protein folding? What role do J proteins play in protein misfolding and neurodegenerative disorders? Can the J protein/ Hsp70 machinery be harnessed to provide a rational basis for recombinant protein production? The current progress that has resulted from the convergence of biochemistry with Escherichia coli and Saccharomyces cerevisiae genetics has accelerated the pace at which these questions are being elucidated. We are beginning to gain some insights into the neuronal network of J proteins. Here, we highlight recent advances in our understanding of how select J proteins harness Hsp70 s for fundamentally important conformational work in neurons.

Specific and nonspecific interactions in a molecule with flexible side chain: 2-phenylethanol and its 1:1 complex with argon studied by high-resolution UV spectroscopy.

Using high-resolution resonance-enhanced two-photon ionization spectroscopy in combination with genetic-algorithm-based computer-aided rotational fit analysis and ab initio quantum chemistry calculations we determined the conformational structure and transition moment orientation in 2-phenylethanol and its 1:1 clusters with argon. The results clearly demonstrate that the gauche structure of 2-phenylethanol, which is stabilized by the intramolecular pi-hydrogen bond between the folded side chain and the benzene ring, is the most abundant in the cold molecular beam. In this conformer the transition moment is rotated by 18 degrees from the short axis of the aromatic ring. Two distinct 1:1 complexes of 2-phenylethanol with argon in a cis- and trans-configuration with respect to the side chain have been found. Employing the Kraitchman [Am. J. Phys. 21, 17 (1953)] analysis we have found that the structure of the 2-phenylethanol moiety and the orientation of the transition moment do not change after the complexation with argon within the experimental accuracy. From the measured band intensities we conclude that in addition to the dispersion interaction of the argon atom with the aromatic ring a hydrogen-bond-type interaction with the terminal -OH group of the side chain stabilizes the cis-structure of the 1:1 complex of 2-phenylethanol with argon.

High-resolution ultraviolet spectroscopy of p-fluorostyrene-water: evidence for a sigma-type hydrogen-bonded dimer.

Ab initio calculations predict four stable conformational structures of the singly hydrated cluster of p-fluorostyrene: two out of plane with pi- and two in plane with sigma-type intermolecular hydrogen bonding between p-fluorostyrene and water. We employed mass-selective resonance-enhanced two-photon ionization high-resolution (70-MHz FWHM laser bandwidth) spectroscopy to partially resolve the rotational structure of the 0(0) (0) origin band of the S(1) <--S(0) electronic transition. A computer-aided fit based on genetic algorithms was used to analyze the experimental high-resolution spectrum and to determine the observed conformational structure. The good agreement between the experimental and the simulated spectra of the 0(0) (0) band and the assignment of the other prominent bands as inter- and intramolecular vibrational progressions clearly demonstrates that the anti in-plane conformer is the most abundant one in the molecular beam. The existence of the sigma-type hydrogen bond between p-fluorostyrene and water manifests that the electron attracting effect of fluorine dominates over the releasing mesomeric effect of the vinyl group and thus a pi-type hydrogen bonding with the aromatic ring is not favored in this case.

Fragmentation and conformation study of ephedrine by low- and high-resolution mass selective UV spectroscopy.

The neurotransmitter molecule, ephedrine, has been studied by mass-selective low- and high-resolution UV resonance enhanced two-photon ionization spectroscopy. Under all experimental conditions we observed an efficient fragmentation upon ionization. The detected vibronic peaks in the spectrum are classified according to the efficiency of the fragmentation, which leads to the conclusion that there exist three different species in the molecular beam: ephedrine-water cluster and two distinct conformers. The two-color two-photon ionization experiment with a decreased energy of the second photon leads to an upper limit of 8.3 eV for the ionization energy of ephedrine. The high-resolution (70 MHz) spectrum of the strongest vibronic peak in the spectrum measured at the fragment (m/z=58) mass channel displays a pronounced and rich rotational structure. Its analysis by the use of a specially designed computer-aided rotational fit process yields accurate rotational constants for the S(0) and S(1) states and the transition moment ratio, providing information on the respective conformational structure.

Human follicular stem cells: their presence in plucked hair and follicular cell culture.

BACKGROUND AND OBJECTIVES: A considerable portion of the hair follicle remains attached to plucked hair and can be used for follicle cell culture. In this study we have phenotyped these cells in an attempt to identify the stem cell fraction. Reports in the literature have indicated that this cell population may be positive for cytokeratin (CK) 19. Because stem cells in general need to be protected from apoptosis, the presence of the apoptosis-suppressing Bcl-2 protein, together with the absence of the apoptosis-promoting Bax and the CK profile may be used as an indicator of the stem cell population in the hair follicle, and in cultures of hair follicle cells. METHODS: Hair follicles from skin biopsies and plucked hair were derived from the scalps of healthy volunteers. Follicular cells were cultured from the plucked hairs. These hair follicles, plucked hairs and cultured cells were examined for their CK profiles, which are indicative of the type of cell (basal/stem cells) and for their status with respect to the proliferation marker Ki-67, Bax and Bcl-2. RESULTS: We found coexpression for CK19 and Bcl-2, but not Bax in two distinct areas, localized in the upper and lower third of the follicle from both skin biopsies and plucked hairs, while proliferation markers were negative in these areas. CK19 and Bcl-2 were also coexpressed in combination in a fraction of the follicular cell culture. The skin basal cell marker CK14 could be found throughout the outer root sheath of the hair follicle from both skin biopsies and plucked hairs, as well as in the follicular cell culture. CONCLUSIONS: Thus, CK19/Bcl-2-positive and Bax-negative cells can be obtained from cells derived from plucked hair and are retained in cultures made from these cells. If this phenotype represents follicular stem cells, our finding endorses the assumption that stem cells are located in the bulge area of the hair follicle, as we did not find them in or near the dermal papilla.

The expression and role of Fas ligand in intestinal inflammation.

Fas ligand (FasL) is involved in the pathogenesis of inflammatory diseases and immune privilege. We examined the expression of FasL in the enteric nervous system (ENS) in murine colitis and guinea-pig ileitis. We studied FasL immunoreactivity, functional integrity of the ENS, severity of colitis, and distribution of neutrophils in wild type and B6/gld mice that lack functional FasL. In ileitis, the distribution of FasL, CD4+ and CD8+ T cells was examined. FasL expression was increased in the ENS of wild type mice with colitis, but decreased labelling of nerve fibres was noted in B6/gld mice. Neutrophils were more abundant and widely distributed in B6/gld mice. Colitis was more severe and persistent in B6/gld mice 7 days after induction. Functional parameters of intestinal secretion and motility in B6/gld mice were the same as controls. In ileitis, FasL expression was increased in the guinea-pig ENS and returned to control levels following the resolution of inflammation. While T cells were not present in the ENS of controls, they were observed during inflammation, but were excluded from ganglia. The number of enteric neurons was unchanged over the course of inflammation. The expression of FasL is altered in intestinal inflammation and contributes to its resolution in experimental colitis.

Modulation of the SNARE core complex by dopamine.

Communication between nerve cells in the brain occurs primarily through specialized junctions called synapses. Recently, many details of synaptic transmission have emerged. The identities of specific proteins important for synaptic vesicle release have now been established. We have investigated three synaptic proteins, VAMP (vesicle associated membrane protein; also called synaptobrevin), syntaxin, and SNAP25 (synaptosomal associated protein of 25kDa) as possible targets in the dopamine-mediated modulation of synaptic function in rat striatal slices. These three proteins form a SNARE (soluble N-ethylmalemide-sensitive factor attachment protein receptors) core complex that is known to be essential for synaptic transmission. Although it is envisioned that the SNAREs undergo dynamic and cyclic interactions to elicit synaptic vesicle release, their precise functions in neurotransmission remains unknown. We have examined SNARE complexes in intact rat striatal slices. Cellular proteins were solubilized, separated electrophoretically by SDS-PAGE, and then identified immunologically. Application of dopamine to striatal slices results in SNAREs favoring the SNARE core complex, a complex which forms spontaneously in the absence of crosslinking agents, rather than the monomer form. In addition, rapid crosslinking of dopamine-treated striatal slices demonstrates that the SNARE complex is increased 4 fold in dopamine treated striatal slices compared with control slices. Haloperidol blocked the dopamine-induced change in the core complex. These results suggest that changes in the activities of SNAREs may be involved in the underlying cellular mechanisms(s) of dopamine-regulated synaptic plasticity of the striatum.

Cysteine string protein regulates G protein modulation of N-type calcium channels.

Cysteine string proteins (CSPs) are secretory vesicle proteins bearing a "J domain" and a palmitoylated cysteine-rich "string" region that are critical for neurotransmitter release. The precise role of CSP in neurotransmission is controversial. Here, we demonstrate a novel interaction between CSP, receptor-coupled trimeric GTP binding proteins (G proteins), and N-type Ca2+ channels. G. subunits interact with the J domain of CSP in an ATP-dependent manner; in contrast, Gbetagamma subunits interact with the C terminus of CSP in both the presence and absence of ATP. The interaction of CSP with both G proteins and N-type Ca2+ channels results in a tonic G protein inhibition of the channels. In view of the crucial importance of N-type Ca2+ channels in presynaptic vesicle release, our data attribute a key role to CSP in the fine tuning of neurotransmission.

A novel SNAP25-caveolin complex correlates with the onset of persistent synaptic potentiation.

We have identified synaptic protein complexes in intact rat hippocampal slices using the rapid chemical cross-linking reagent paraformaldehyde. Cellular proteins were rapidly cross-linked, solubilized, separated electrophoretically by SDS-PAGE, and then identified immunologically. Multiple complexes containing syntaxin, the synaptosomal-associated protein of 25 kDa (SNAP25), and vesicle-associated membrane protein (VAMP) were observed to coexist in a single hippocampal slice including a 100 kDa cross-linked protein complex that exhibited the same electrophoretic migration as a member of the previously identified SDS-resistant soluble N-ethylmaleimide-sensitive fusion attachment protein receptor "core" of the 20 S complex. A VAMP-synaptophysin complex, reported previously in vitro, was also observed in the hippocampal slices. This study links biochemical and physiological studies involving presynaptic proteins implicated in secretion and confirms that these proteins that have been studied extensively previously in the presence of detergent do form "bona fide" cellular complexes. Importantly, we have also detected additional novel protein complexes that do not correspond to complexes identified previously in vitro. After the induction of persistent synaptic potentiation, an abundant 40 kDa SNAP25-caveolin1 complex was observed. The SNAP25-caveolin1 complex was not abundant in control slices and, therefore, represents the first demonstration of a reorganization of protein complexes in intact hippocampal slices during the induction of synaptic potentiation. The interaction between caveolin1 and SNAP25 was confirmed biochemically by demonstration of the association of caveolin with recombinant-immobilized SNAP25 and by the coimmunoprecipitation of SNAP25 using caveolin-specific antisera. Caveolin1, like SNAP25, was observed to be abundant in isolated hippocampal nerve terminals (synaptosomes). Immunofluorescent studies demonstrated that both SNAP25 and caveolin1 are present in neurons and colocalize in axonal varicosities. These results suggest that a short-lasting SNAP25-caveolin interaction may be involved in the early phase of synaptic potentiation.

Prevalence of hepatitis C virus seropositivity among hospitalized US veterans.

BACKGROUND: Hepatitis C virus (HCV) is a major cause of acute and chronic hepatitis in the United States and abroad. HCV antibody prevalences ranging from 10 to 90% have been reported in intravenous drug abusers, hemodialysis patients, and persons suffering from other liver diseases, whereas HCV seropositivity rates for volunteer-blood donor populations are generally under 1%. However no information has been available concerning the prevalence of HCV in general hospital populations in the United States. METHODS: We examined the rate of HCV seropositivity in 530 patients admitted to the Atlanta VA Medical Center between November 1993 and November 1994. The test population consisted of 400 random hospital admissions, 100 successive admissions to the surgical service, and 30 random admissions to the gastrointestinal service. Serum samples were assayed for HCV antibodies by a second generation EIA, and all repeat reactives were re-examined using a supplemental research assay to confirm the presence of HCV antibodies. Complete chart reviews were carried out on all HCV seropositive patients and on 100 HCV seronegative patients. RESULTS: Sixty-two of the 530 patients tested (11.7%) were repeatedly positive for HCV antibodies. Of these 62 repeat reactives, 56 (90.3%) were positive and 3 others (4.8%) indeterminate by the supplemental assay. The HCV seropositivity rate after supplemental testing was 11.8% for random admissions, 5.0% for surgical admissions, and 13.3% for patients admitted to the gastroenterology service. HCV-associated risk factors in HCV seropositive patients included a history of intravenous drug abuse, current or previous alcohol abuse, previous or concurrent liver disease, previous blood transfusions, hemodialysis, and multiple sex partners or unsafe sex. CONCLUSIONS: HCV infection may be more prevalent among hospitalized VA patients (and among other US hospital populations) than previously expected.

G protein modulation of N-type calcium channels is facilitated by physical interactions between syntaxin 1A and Gbetagamma.

The direct modulation of N-type calcium channels by G protein betagamma subunits is considered a key factor in the regulation of neurotransmission. Some of the molecular determinants that govern the binding interaction of N-type channels and Gbetagamma have recently been identified (see, i.e., Zamponi, G. W., Bourinet, E., Nelson, D., Nargeot, J., and Snutch, T. P. (1997) Nature 385, 442-446); however, little is known about cellular mechanisms that modulate this interaction. Here we report that a protein of the presynaptic vesicle release complex, syntaxin 1A, mediates a crucial role in the tonic inhibition of N-type channels by Gbetagamma. When syntaxin 1A was coexpressed with (N-type) alpha(1B) + alpha(2)-delta + beta(1b) channels in tsA-201 cells, the channels underwent a 18 mV negative shift in half-inactivation potential, as well as a pronounced tonic G protein inhibition as assessed by its reversal by strong membrane depolarizations. This tonic inhibition was dramatically attenuated following incubation with botulinum toxin C, indicating that syntaxin 1A expression was indeed responsible for the enhanced G protein modulation. However, when G protein betagamma subunits were concomitantly coexpressed, the toxin became ineffective in removing G protein inhibition, suggesting that syntaxin 1A optimizes, rather than being required for G protein modulation of N-type channels. We also demonstrate that Gbetagamma physically binds to syntaxin 1A, and that syntaxin 1A can simultaneously interact with Gbetagamma and the synprint motif of the N-type channel II-III linker. Taken together, our experiments suggest a mechanism by which syntaxin 1A mediates a colocalization of G protein betagamma subunits and N-type calcium channels, thus resulting in more effective G protein coupling to, and regulation of, the channel. Thus, the interactions between syntaxin, G proteins, and N-type calcium channels are part of the structural specialization of the presynaptic terminal.

Cysteine string protein (CSP) is an insulin secretory granule-associated protein regulating beta-cell exocytosis.

Cysteine string proteins (CSPs) are novel synaptic vesicle-associated protein components characterized by an N-terminal J-domain and a central palmitoylated string of cysteine residues. The cellular localization and functional role of CSP was studied in pancreatic endocrine cells. In situ hybridization and RT-PCR analysis demonstrated CSP mRNA expression in insulin-producing cells. CSP1 mRNA was present in pancreatic islets; both CSP1 and CSP2 mRNAs were seen in insulin-secreting cell lines. Punctate CSP-like immunoreactivity (CSP-LI) was demonstrated in most islets of Langerhans cells, acinar cells and nerve fibers of the rat pancreas. Ultrastructural analysis showed CSP-LI in close association with membranes of secretory granules of cells in the endo- and exocrine pancreas. Subcellular fractionation of insulinoma cells showed CSP1 (34/36 kDa) in granular fractions; the membrane and cytosol fractions contained predominantly CSP2 (27 kDa). The fractions also contained proteins of 72 and 70 kDa, presumably CSP dimers. CSP1 overexpression in INS-1 cells or intracellular administration of CSP antibodies into mouse ob/ob beta-cells did not affect voltage-dependent Ca2+-channel activity. Amperometric measurements showed a significant decrease in insulin exocytosis in individual INS-1 cells after CSP1 overexpression. We conclude that CSP is associated with insulin secretory granules and that CSP participates in the molecular regulation of insulin exocytosis by mechanisms not involving changes in the activity of voltage-gated Ca2+-channels.

Influence of nucleotide excision repair of Escherichia coli on radiation-induced mutagenesis of double-stranded M13 DNA.

To investigate a possible role of nucleotide excision repair (NER) of E. coli in the removal of gamma-radiation-induced DNA lesions, double-stranded M13mp10 DNA, which contains a part of the lac operon, including the promoter/operator region, the lacZ alpha gene and a 144 basepair (bp) inframe insert in the lacZ alpha gene, as mutational target was gamma-irradiated in a phosphate buffer under N2. Subsequently, the radiation-exposed DNA was transfected to wild-type or NER-deficient (uvrA-) E. coli, mutants in the mutational target selected, followed by characterization of the mutants by sequence analysis. Both the mutations obtained from wild-type and uvrA- E. coli appeared to consist mainly of bp substitutions. However, in contrast to wild-type cells, a relatively large proportion of the mutations obtained from the NER-deficient cells (about 25%) is represented by -1 bp deletions, indicating that NER may be responsible for the removal of lesions which cause this particular type of frameshift. Comparison of the bp substitutions between both E. coli strains showed considerable differences. Thirty per cent of all bp substitutions in the NER-deficient host are T/A-->C/G transitions which are virtually absent in wild-type E. coli. This indicates that NER is involved in the elimination of lesions responsible for these transitions. This may also be true for a part of the lesions which cause C/G-->T/A transitions, which make up 52% of the bp substitutions in uvrA- cells versus 17% in wild-type cells. Strikingly, C/G-->G/C transversions appeared to be only formed in wild-type, where they make up 22% of all bp substitutions, and not in the NER-deficient E. coli. This result suggests, that due to the action of NER, a particular type of mutation may be introduced. A similar indication holds for C/G-->A/T transversions, which are predominant in wild-type (58%) and in the minority in uvrA- cells (15%).

Effect of the sulfhydryl compound cysteamine on gamma-radiation-induced mutations in double-stranded M13 DNA.

Sulfhydryl compounds can protect DNA against free-radical-induced DNA damages not only by scavenging of radicals, but also by chemical non-enzymatic repair or modification of such damages by hydrogen-donation. To investigate the influence of chemical repair and modification on mutations, induced by gamma-radiation-generated free radicals (.OH, .H), phosphate-buffered aqueous solutions of double-stranded (ds) M13 DNA were exposed to gamma-rays under N2 in the presence of 5 mM cysteamine. The exposed DNA was subsequently transfected to wild-type E. coli and mutations in the mutational target were characterized. This target in fact contains three different target sequences, i.e., the lac promoter/operator, the lacZ alpha gene and a 144 bp inframe insert. The mutation spectrum obtained was compared with those in the absence of cysteamine under N2 and N2O. In the latter case, the ratio of .OH and .H available for reacting with DNA is about the same as under N2 + cysteamine. The results show that chemical repair and/or modification by cysteamine of potentially lethal lesions takes place, leading to a much higher survival of ds M13 DNA in the presence of cysteamine than could be expected on basis of scavenging of .OH and .H alone. This higher survival appeared to be accompanied with a higher mutation induction. However, the N2 + cysteamine mutation spectrum shows a remarkable resemblance with the N2O-spectrum. This holds for the total mutation target, as well as each of the three targets, although the mutations obtained in each of the three targets under the same irradiation conditions are quite different. Thus, it can be concluded that cysteamine is mainly effective on radiation-induced potentially lethal DNA lesions, and not so much on (pre)mutagenic damages. Moreover, the type of mutation appeared to be strongly dependent on the mutational target sequence.

Mutations induced by gamma-irradiation of M13 bacteriophages containing single-stranded DNA.

Oxygenated suspensions of M13 bacteriophages, containing single-stranded M13mp10 DNA, were gamma-irradiated followed by infection of E. coli cells. Mutants in the mutational target sequence, which consists of the lac promoter /operator region, the lacZ alpha gene, and a 144 bp inframe insert in the lacZ alpha gene, were selected and characterized. Except for three one-base deletions, all of the 51 mutations characterized were base substitutions. All base substitutions appeared to involve guanines and cytosines and none affect adenines and thymines. Since most of the known repair systems do not act on single-stranded DNA, the conclusion can be drawn that radiation induces under these conditions only mutagenic damages on guanine and cytosine. Although all possible G- and C-transversions and transitions were found, there is a strong preference for G-->C and G-->T transversions (21 and 25% of all base substitutions, respectively) and C-->T transitions (48% of all base substitutions). These results indicate, that the G/C-->C/G and G/C-->T/A transversions, found after irradiation of double-stranded M13 DNA, are mainly due to radiation guanine products, whereas cytosine damage is mainly responsible for G/C-->A/T transitions.

The cysteine string secretory vesicle protein activates Hsc70 ATPase.

Cysteine string protein (CSP) is a 34 kDa secretory vesicle protein bearing a "J-domain" as well as a palmitoylated cysteine-rich "string" region used for membrane attachment. Mutation of the CSP gene causes impaired presynaptic neuromuscular transmission in Drosophila melanogaster, implicating CSP as part of the exocytotic protein machinery. The J-domain of CSP shares homology with the universally conserved DnaJ family, a group of proteins that act as co-chaperones with Hsc70 and its homologs. Hsc70 is an abundant neural protein with coupled protein binding and ATPase activities. We have investigated the CSP modulation of Hsc70 ATPase activity. Here we demonstrated that CSP enhances Hsc70 ATPase activity in a dose-dependent manner. CSP activation of Hsc70 was maximal ( approximately 12 times) at 1:1 stoichiometry and above. We show that a J-domain-containing fragment (amino acids 1-82) of CSP is sufficient for the activation of Hsc70. Neither CSP nor the amino-terminal fragment stimulate the activity of the isolated Hsc70 ATPase domain (amino acids 1-386). CSP does not significantly increase the activity of N-ethylmaleimide-sensitive fusion protein, another ATPase required for transport vesicle function. Our results suggest that CSP, a DnaJ family member associated with the secretory vesicle cycle regulates Hsc70 functions. Hsc70 may function within the biochemical pathways of exo- and endocytosis to promote the formation or dissociation of multimeric complexes or to regulate conformational changes.

Cis-elements in the promoter region of the human myeloperoxidase (MPO) gene.

We recently reported the identification and initial characterization of the human myeloperoxidase (MPO) promoter. The minimal or basic MPO promoter lies within the proximal 128 bp of the 5'-flanking region of the MPO gene. Plasmids containing progressively larger segments of the 5'-flanking region show correspondingly greater MPO promoter activity and increased tissue specificity compared with smaller promoter fragments. These findings suggested the presence of a multiple important regulatory cis-elements in the 5'-flanking region of the MPO gene. We now report results of studies which reveal the presence of seven discrete nuclear protein binding sites (DP1-DP7) within the proximal 600 bp of 5'-flanking MPO DNA. DNase I footprinting and gel shift analyses indicate tissue-specific and/or maturation-specific differences in nuclear protein binding to most of these sites, suggesting that they play a role in transcriptional regulation. Mutation of site DP7 stimulates the activity of a 594-bp MPO promoter construct in transfection studies, whereas mutation of any of the six other sites (DP1-DP6) reduces promoter activity. These results indicate that oligonucleotides DP1-DP7 constitute cis-elements which contribute to the activity of the human MPO promoter.

Cysteine string protein, a DnaJ family member, is present on diverse secretory vesicles.

Synaptic vesicles are the secretory organelles responsible for the regulated secretion of neurotransmission. Proteins associated with or integral components of the lipid bilayer likely represent important components in regulated secretion. CSP (cysteine string protein) is a 34 kDa protein which copurifies with cholinergic synaptic vesicles from the marine ray Torpedo californica. In Drosophila melanogaster deletion of the CSP gene causes impaired presynaptic neuromuscular transmission. A rat brain complementary DNA (cDNA) encoding CSP was isolated and sequence analysis predicts a protein with 86% identity to Torpedo CSP. Rat CSP contains a "J domain" as well as a cysteine rich "string" region. The J domain "fingerprints" the CSP family as members of the universally conserved DnaJ/hsp40 (heat shock protein) chaperone family. Polyclonal antisera raised against a seventeen amino acid peptide representing the carboxy terminus of rat CSP detected a 35 kDa immunoreactive protein in a rat brain synaptic vesicle enriched preparation. A 35 kDa immunoreactive protein that comigrates electrophoretically with rat brain CSP was also detected in zymogen granule membranes. These results establish the presence of a CSP in rat brain and in the zymogen granule of the rat pancreas and suggest that CSPs have a role in exocrine and neural secretion.

Specificity and regulation of a synaptic vesicle docking complex.

Synaptic vesicles are proposed to dock at the presynaptic plasma membrane through the interaction of two integral membrane proteins of synaptic vesicles, VAMP and synaptotagmin, and two plasma membrane proteins, syntaxin and SNAP-25. We have characterized the binding properties of these proteins and observed SNAP-25 potentiation of VAMP 2 binding to syntaxins 1a and 4 but not syntaxins 2 or 3. n-sec1, a neuron-specific syntaxin-binding protein, bound syntaxin with nanomolar affinity, forming a complex that is distinct from the previously identified 7S and 20S syntaxin-containing complexes. This suggests that syntaxin exists in at least three states: bound to n-sec1, in a 7S particle, and in a 20S particle. Recombinant n-sec1 inhibited VAMP or SNAP-25 binding to syntaxin. We propose that the specific associations of VAMP, SNAP-25, and syntaxin mediate vesicle docking and that a syntaxin/n-sec1 complex precedes and/or regulates formation of these complexes.

Identification of a vesicle-associated membrane protein (VAMP)-like membrane protein in zymogen granules of the rat exocrine pancreas.

Zymogen granules of the exocrine pancreas are the secretory organelles responsible for the regulated secretion of digestive enzymes. Several proteins are associated with or are integral components of the lipid bilayer that forms the zymogen granule membrane. These proteins likely represent important components in the regulated secretion of digestive enzymes. VAMPs (vesicle-associated membrane proteins)/synaptobrevins are a family of 18-kDa integral membrane proteins originally characterized in synaptic vesicles. Polyclonal antisera raised against either a VAMP/glutathione S-transferase (GST) fusion protein or rat brain synaptic vesicles, detected an 18-kDa immunoreactive protein in zymogen granule membranes that co-migrates electrophorectically with rat brain synaptic vesicle VAMP. Rat brain synaptic vesicle VAMP was detected by both antisera. Botulinum-B toxin treatment of zymogen granule membranes did not result in cleavage of zymogen granule membrane VAMP, indicating that exocrine pancreatic VAMP is either VAMP1 or a novel VAMP-isoform. Immunofluorescent studies demonstrated that exocrine pancreatic VAMP localized with GP2, a zymogen granule membrane protein, to the apical region of pancreatic acinar cells. No significant labeling was observed in basolateral regions of pancreatic acinar cells. These results establish the presence of a VAMP protein in the zymogen granule of the rat pancreas and suggest that VAMPs have a role in exocrine secretion.