SNC1, a yeast homolog of the synaptic vesicle-associated membrane protein/synaptobrevin gene family: genetic interactions with the RAS and CAP genes.

SNC1, a gene from the yeast Saccharomyces cerevisiae, encodes a homolog of vertebrate synaptic vesicle-associated membrane proteins (VAMPs) or synaptobrevins. SNC1 was isolated by its ability to suppress the loss of CAP function in S. cerevisiae strains possessing an activated allele of RAS2. CAP is a component of the RAS-responsive S. cerevisiae adenylyl cyclase complex. The N-terminal domain of CAP is required for full cellular responsiveness to activated RAS proteins. The C-terminal domain of CAP is required for normal cellular morphology and responsiveness to nutrient extremes. Multicopy plasmids expressing SNC1 suppress only the loss of the C-terminal functions of CAP and only in the presence of activated RAS2.     

Synaptobrevin/vesicle-associated membrane protein (VAMP) of Aplysia californica: structure and proteolysis by tetanus toxin and botulinal neurotoxins type D and F.

Synaptobrevin/vesicle-associated membrane protein (VAMP) and syntaxin are potential vesicle donor and target membrane receptors of a docking complex that requires N-ethylmaleimide-sensitive factor (NSF) and soluble NSF-attachment proteins as soluble factors for vesicle fusion with target membranes. Members of this docking complex are the target of clostridial neurotoxins that act as zinc-dependent proteases. Molecular cloning of the Aplysia californica synaptobrevin cDNA revealed a 180-residue polypeptide (M(r), 19,745) with a central transmembrane region and an atypically large C-terminal intravesicular domain. This polypeptide integrates into membranes at both the co- and posttranslational level, as shown by modification of an artificially introduced N-glycosylation site. The soluble and membrane-anchored forms of synaptobrevin are cleaved by the light chains of the botulinal toxins type D and F and by tetanus toxin involving the peptide bonds Lys49-Ile50, Gln48-Lys49, and Gln66-Phe67, respectively. The active center of teh tetanus toxin light chain was identified by site-specific mutagenesis. His233, His237, Glu234, and Glu270/271 are essential to this proteolytic activity. Modification of histidine residues resulted in loss of zinc binding, whereas a replacement of Glu234 only slightly reduced the zinc content.     

The vesicle-associated membrane protein family of proteins in rat pancreatic and parotid acinar cells

BACKGROUND & AIMS: The vesicle-associated membrane protein (VAMP) family of proteins may play an important role in regulating enzyme secretion from pancreatic and parotid acini. The purpose of this study was to characterize the isoforms produced in pancreatic and parotid acini and determine their subcellular locations. METHODS: Using a battery of specific antisera and recombinant tetanus toxin light chain (which cleaves VAMP-2 and cellubrevin), the presence of each VAMP molecule in the acini was determined by immunoblotting of subcellular membrane fractions; their localization was determined by confocal immunofluorescence microscopy and immunogold electron microscopy. RESULTS: Both VAMP-2 and cellubrevin were present on both the zymogen granule membrane and plasma membrane. VAMP-1 was not present in the acinar cell but was found in the nerve endings innervating the acini. As expected, pancreatic acinar VAMP-2 and cellubrevin were sensitive to cleavage by recombinant tetanus toxin. CONCLUSIONS: VAMP-2 and cellubrevin may play integral roles in exocytosis of the pancreatic and parotid acinar cells, whereas VAMP-1 is restricted to nerves that innervate the acini and may function to modulate exocrine activity. (Gastroenterology 1996 Dec;111(6):1661-9)     

Calcium dependence of neurotransmitter release and rate of spontaneous vesicle fusions are altered in Drosophila synaptotagmin mutants.

Since the demonstration that Ca2+ influx into the presynaptic terminal is essential for neurotransmitter release, there has been much speculation about the Ca2+ receptor responsible for initiating exocytosis. Numerous experiments have shown that the protein, or protein complex, binds multiple Ca2+ ions, resides near the site of Ca2+ influx, and has a relatively low affinity for Ca2+. Synaptotagmin is an integral membrane protein of synaptic vesicles that contains two copies of a domain known to be involved in Ca(2+)-dependent membrane interactions. Synaptotagmin has been shown to bind Ca2+ in vitro with a relatively low affinity. In addition, synaptotagmin has been shown to bind indirectly to Ca2+ channels, positioning the protein close to the site of Ca2+ influx. Recently, a negative regulatory role for synaptotagmin has been proposed, in which it functions as a clamp to prevent fusion of synaptic vesicles with the presynaptic membrane. Release of the clamp would allow exocytosis. Here we present genetic and electrophysiological evidence that synaptotagmin forms a multimeric complex that can function as a clamp in vivo. However, upon nerve stimulation and Ca2+ influx, all synaptotagmin mutations dramatically decrease the ability of Ca2+ to promote release, suggesting that synaptotagmin probably plays a key role in activation of synaptic vesicle fusion. This activity cannot simply be attributed to the removal of a barrier to secretion, as we can electrophysiologically separate the increase in rate of spontaneous vesicle fusion from the decrease in evoked response. We also find that some syt mutations, including those that lack the second Ca(2+)-binding domain, decrease the fourth-order dependence of release on Ca2+ by approximately half, consistent with the hypothesis that a synaptotagmin complex functions as a Ca2+ receptor for initiating exocytosis.     

Role of vesicle-associated membrane protein 2 in exocytosis of glucagon-like peptide-1 from the murine intestinal L cell

Aims/hypothesis

Glucagon-like peptide-1 (GLP-1), secreted by the enteroendocrine L cell, is an incretin hormone that potently stimulates insulin secretion. Although signalling pathways promoting GLP-1 release are well characterised, the mechanisms by which GLP-1-containing granules fuse to the L cell membrane are unknown. As soluble NSF attachment proteins (SNAREs) are known to mediate granule–membrane fusion, the role of vesicle-associated membrane proteins (VAMPs) in GLP-1 exocytosis was examined.

Methods

SNARE expression was determined in murine GLUTag L cells by RT-PCR and immunoblot and in primary murine L cells by immunofluorescence. Co-immunoprecipitation was used to examine SNARE interactions, while tetanus toxin (TetX)-mediated cleavage of VAMP was used with a GLP-1 secretion assay and total internal reflection fluorescence microscopy to determine the role of VAMP2 in exocytosis.

Results

VAMP2 was expressed in murine L cells and localised to secretory granules in GLUTag cells. VAMP1/3 and the core membrane proteins syntaxin1a and synaptosomal-associated protein 25 kDa (SNAP25) were also detected. TetX cleaved VAMPs in GLUTag cells. However, only VAMP2 interacted with syntaxin1a, as did SNAP25 and Munc18-1. TetX treatment of GLUTag cells prevented glucose-dependent insulinotrophic peptide- and oleic-acid-stimulated GLP-1 secretion (p?<?0.05–0.01), as well as K⁺-stimulated single-cell exocytosis (p?<?0.05–0.001), while TetX-resistant VAMP2 expression rescued GLP-1 secretion (p?<?0.01–0.001).

Conclusions/interpretation

Together, these findings indicate an essential role for VAMP2 in GLP-1 exocytosis from the GLUTag L cell in response to a variety of established secretagogues. An improved understanding of the mechanisms governing the release of GLP-1 may lead to new therapeutic approaches to enhance the levels of this incretin hormone in patients with type 2 diabetes.     

Glycosylation site binding protein and protein disulfide isomerase are identical and essential for cell viability in yeast.

Glycosylation site binding protein (GSBP) has been shown to be identical to protein disulfide isomerase (PDI; EC 5.3.4.1) in a variety of multicellular organisms. We have utilized immunological and biochemical techniques to determine if GSBP and PDI are identical in yeast. Antiserum prepared against yeast GSBP identified in microsomes by its ability to be labeled with a peptide photoaffinity probe was found to recognize PDI purified from yeast. Moreover, this purified yeast PDI was found to be specifically labeled by the photoaffinity probe originally used to identify GSBP in a variety of eukaryotes. On the basis of these observations, we conclude that yeast GSBP and PDI are the same protein. The structure of the yeast PDI gene revealed a product with sequence similarity to higher eukaryotic PDI/GSBP. Disruption of this gene in yeast resulted in a recessive lethal mutation, indicating that PDI/GSBP is required for cell viability.     

Requirement for distinct vesicle-associated membrane proteins in insulin- and AMP-activated protein kinase (AMPK)-induced translocation of GLUT4 and CD36 in cultured cardiomyocytes

Aims/hypothesis  

Upon stimulation of insulin signalling or contraction-induced AMP-activated protein kinase (AMPK) activation, the glucose transporter GLUT4 and the long-chain fatty acid (LCFA) transporter CD36 similarly translocate from intracellular compartments to the plasma membrane of cardiomyocytes to increase uptake of glucose and LCFA, respectively. This similarity in regulation of GLUT4 traffic and CD36 traffic suggests that the same families of trafficking proteins, including vesicle-associated membrane proteins (VAMPs), are involved in both processes. While several VAMPs have been implicated in GLUT4 traffic, nothing is known about the putative function of VAMPs in CD36 traffic. Therefore, we compared the involvement of the myocardially produced VAMP isoforms in insulin- or contraction-induced GLUT4 and CD36 translocation.     

Characterization of the role of the Synaptotagmin family as calcium sensors in facilitation and asynchronous neurotransmitter release

Ca(2+) influx into presynaptic nerve terminals activates synaptic vesicle exocytosis by triggering fast synchronous fusion and a slower asynchronous release pathway. In addition, a brief rise in Ca(2+) after consecutive action potentials has been correlated with a form of short-term synaptic plasticity with enhanced vesicle fusion termed facilitation. Although the synaptic vesicle protein Synaptotagmin 1 (Syt1) has been implicated as the Ca(2+) sensor for synchronous fusion, the molecular identity of the Ca(2+) sensors that mediate facilitation and asynchronous release is unknown. To test whether the synchronous Ca(2+) sensor, Syt1, or the asynchronous Ca(2+) sensor is involved in facilitation, we analyzed whether genetic elimination of Syt1 in Drosophila results in a concomitant impairment in facilitation. Our results indicate that Syt1 acts as a redundant Ca(2+) sensor for facilitation, with the asynchronous Ca(2+) sensor contributing significantly to this form of short-term plasticity. We next examined whether other members of the Drosophila Syt family functioned in Ca(2+)-dependent asynchronous release or facilitation in vivo. Genetic elimination of other panneuronally expressed Syt proteins did not alter these forms of exocytosis, indicating a non-Syt Ca(2+) sensor functions for both facilitation and asynchronous release. In light of these findings, the presence of two presynaptic Ca(2+) sensors can be placed in a biological context, a Syt1-based Ca(2+) sensor devoted primarily to baseline synaptic transmission and a second non-Syt Ca(2+) sensor for short-term synaptic plasticity and asynchronous release.     

Vesicle-associated membrane protein 3 (VAMP-3) and VAMP-8 are present in human platelets and are required for granule secretion

Secretion of platelet granules is necessary for normal hemostasis. Platelet secretion requires soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) complex formation between different members of the syntaxin, SNAP-25, and vesicle-associated membrane protein (VAMP) gene families. Using microcapillary reverse-phase high-performance liquid chromatography-nano-electrospray tandem mass spectrometry, we identified VAMP-3 and VAMP-8 as VAMP isoforms coimmunoprecipitated from platelets with syntaxin 4. Immunoblotting experiments confirmed the presence of VAMP-3 and VAMP-8 but not VAMP-1 or VAMP-2 in platelets. To examine the effect of VAMP proteins on platelet secretion, soluble recombinant (r) VAMP-2, rVAMP-3, and rVAMP-8 were incubated with streptolysin O-permeabilized platelets. Secretion of alpha granules (monitored by flow cytometric measurement of P-selectin) was blocked, and dense-granule secretion (assessed by release of carbon 14-serotonin) was almost completely inhibited by rVAMP-3, whereas rVAMP-8 inhibited secretion of dense granules but not alpha granules. In contrast, rVAMP-2, which formed SNARE complexes in vitro, had no effect on platelet exocytosis. We conclude that VAMP-3 and VAMP-8 form SNARE complexes with platelet syntaxin 4 and are required for platelet granule secretion.     

Wiskott Aldrich syndrome protein (WASP) and N-WASP are critical for T cell development

Although T cell dysfunction and lymphopenia are key features of immunodeficient patients with the Wiskott-Aldrich syndrome and Wiskott-Aldrich syndrome protein (WASP)-deficient mice, T cell development appears relatively normal. We hypothesized that N-WASP, a ubiquitously expressed homologue of WASP, may serve a redundant function with WASP. To examine the unique and redundant activities of WASP and N-WASP, we generated ES cells devoid of WASP and N-WASP [double knockout (DKO)] and used the RAG-2-deficient blastocyst complementation system to generate DKO lymphocytes. Moreover, we mated WASP KO mice with mice containing a conditionally targeted N-WASP allele and used the Cre-loxP system to generate mice lacking WASP and N-WASP in T cells [conditional DKO (cDKO)]. In both systems, N-WASP-deficient cells were indistinguishable from WT cells. In contrast, T cell development in DKO and cDKO mice was markedly altered, as shown by thymic hypocellularity and reduced numbers of peripheral T cells. We found that the combined activity of WASP and N-WASP was important for CD4(-)CD8(-) double-negative (DN)-to-CD4(+)CD8(+) double-positive (DP) cell transition, and this may be partly explained by reduced cycling DN3 cells. In addition, decreased migratory responses of CD4(+)CD8(-) and CD4(-)CD8(+) single-positive (SP) cells and increased percentage of CD69(low)CD24(low) and CD62L(low) SP cells in cDKO cells imply retention of SP cells in the thymus. In summary, this study suggests that, although WASP serves a unique role for peripheral T cell function, T cell development depends on the combined activity of WASP and N-WASP.     

Effects of synapsin I and calcium/calmodulin-dependent protein kinase II on spontaneous neurotransmitter release in the squid giant synapse.

The molecular events that control synaptic vesicle availability in chemical synaptic junctions have not been fully clarified. Among the protein molecules specifically located in presynaptic terminals, synapsin I and calcium/calmodulin-dependent protein kinase II (CaM kinase II) have been shown to modulate evoked transmitter release in the squid giant synapse. In the present study, analysis of synaptic noise in this chemical junction was used to determine whether these proteins also play a role in the control of spontaneous and enhanced spontaneous transmitter release. Injections of dephosphorylated synapsin I into the presynaptic terminal reduced the rate of spontaneous and enhanced quantal release, whereas injection of phosphorylated synapsin I did not modify such release. By contrast CaM kinase II injection increased enhanced miniature release without affecting spontaneous miniature frequency. These results support the view that dephosphorylated synapsin I "cages" synaptic vesicles while CaM kinase II, by phosphorylating synapsin I, "decages" these organelles and increases their availability for release without affecting the release mechanism itself.     

Physiologically important secondary modifications of red cell membrane in hereditary spherocytosis-evidence for in vivo oxidation and lipid rafts protein variations

Hereditary spherocytosis (HS) is a heterogeneous group of disorders. The abnormal red cell morphology (resulting in shortened cell survival) is due to a primary deficiency in spectrin, ankyrin-1, band 3 or protein 4.2. Secondary protein deficiencies are often observed and may be involved in the outcome of the disease. In the present study, we searched for secondary erythrocyte membrane protein alterations in HS, including the lipid raft associated proteins and the oxidative index. For this purpose, 12 patients with clinical and laboratory diagnosis of mild to typical HS were examined. Erythrocyte membrane ghosts and skeletons were subjected to SDS-PAGE and immunoblotting analysis using antibodies against red cell membrane proteins and DNP moiety, after 2,4-dinitrophenylhydrazine derivatization. Protein deficiencies, degradation, aggregation and enhanced binding of cytoplasmic components, band 8, hemoglobin and immunoglobulins G to the membrane as well as increased oxidative index, were found in the majority of the HS patients. Proportion of the membrane- and skeleton-bound globin was oxidized/denatured Hb or hemichromes and crosslinkings. Some HS membranes are deficient in lipid rafts proteins and contain sorcin. A context of these distortions is more pronounced in typical HS cases compared to the mild ones. Similar defects in thalassemia and senescent RBCs are dictated by increased oxidative stress and are positively correlated with perturbations in membrane properties. These data add some new insight in the field of HS pathophysiology and clinical variability.     

Age-dependent variation in the cytosol/membrane distribution of red cell protein kinase-C

An age-dependent increase in membrane association of protein kinase-c and a decrease in the cytosolic enzyme, especially in the densest fraction rich in senescent red cells was observed in Stractan-gradient-separated normal erythrocytes.     

Ribozyme knockdown functionally links a 1,25(OH)2D3 membrane binding protein (1,25D3-MARRS) and phosphate uptake in intestinal cells

We used a ribozyme loss-of-function approach to demonstrate that the protein product of a cDNA encoding a multifunctional membrane-associated protein binds the seco-steroid 1,25(OH)(2)D(3) and transduces its stimulatory effects on phosphate uptake. These results are paralleled by studies in which the ability of the hormone to stimulate phosphate uptake in isolated chick intestinal epithelial cells is abolished by preincubation with Ab099 directed against the amino terminus of the protein. We now report the complete sequence of the cloned chicken cDNA for the 1,25D(3)-MARRS (membrane-associated, rapid-response steroid-binding) protein and reveal it to be identical to the multifunctional protein ERp57. Functional studies showed that active ribozyme, but not a scrambled control, decreased specific membrane-associated 1,25(OH)(2)D(3) binding, but did not affect binding to the nuclear receptor for 1,25(OH)(2)D(3). Seco-steroid-dependent stimulation of protein kinase C activity was diminished as 1,25D(3)-MARRS protein levels were reduced in the presence of the ribozyme, as judged by Western blot analyses. Phosphate uptake in isolated cells is an index of intestinal phosphate transport that occurs during growth and maturation. Whereas cells and perfused duodena robustly responded to 1,25(OH)(2)D(3) in preparations from young birds, older animals no longer responded with stimulated phosphate uptake or transport. The age-related decline was accompanied by a decrease in 1,25D(3)-MARRS mRNA that was apparent up to 1 year of age. Together, these studies functionally link phosphate transport in the chick duodenum with the 1,25D(3)-MARRS protein and point to a previously uncharacterized role for this multifunctional protein class.     

Formation and release of arrestin domain-containing protein 1-mediated microvesicles (ARMMs) at plasma membrane by recruitment of TSG101 protein

Mammalian cells are capable of delivering multiple types of membrane capsules extracellularly. The limiting membrane of late endosomes can fuse with the plasma membrane, leading to the extracellular release of multivesicular bodies (MVBs), initially contained within the endosomes, as exosomes. Budding viruses exploit the TSG101 protein and endosomal sorting complex required for transport (ESCRT) machinery used for MVB formation to mediate the egress of viral particles from host cells. Here we report the discovery of a virus-independent cellular process that generates microvesicles that are distinct from exosomes and which, like budding viruses, are produced by direct plasma membrane budding. Such budding is driven by a specific interaction of TSG101 with a tetrapeptide PSAP motif of an accessory protein, arrestin domain-containing protein 1 (ARRDC1), which we show is localized to the plasma membrane through its arrestin domain. This interaction results in relocation of TSG101 from endosomes to the plasma membrane and mediates the release of microvesicles that contain TSG101, ARRDC1, and other cellular proteins. Unlike exosomes, which are derived from MVBs, ARRDC1-mediated microvesicles (ARMMs) lack known late endosomal markers. ARMMs formation requires VPS4 ATPase and is enhanced by the E3 ligase WWP2, which interacts with and ubiquitinates ARRDC1. ARRDC1 protein discharged into ARMMs was observed in co-cultured cells, suggesting a role for ARMMs in intercellular communication. Our findings reveal an intrinsic cellular mechanism that results in direct budding of microvesicles from the plasma membrane, providing a formal paradigm for the evolutionary recruitment of ESCRT proteins in the release of budding viruses.