Pantophysin is a phosphoprotein component of adipocyte transport vesicles and associates with GLUT4-containing vesicles

Pantophysin, a protein related to the neuroendocrine-specific synaptophysin, recently has been identified in non-neuronal tissues. In the present study, Northern blots showed that pantophysin mRNA was abundant in adipose tissue and increased during adipogenesis of 3T3-L1 cells. Immunoblot analysis of subcellular fractions showed pantophysin present exclusively in membrane fractions and relatively evenly distributed in the plasma membrane and internal membrane fractions. Sucrose gradient ultracentrifugation demonstrated that pantophysin and GLUT4 exhibited overlapping distribution profiles. Furthermore, immunopurified GLUT4 vesicles contained pantophysin, and both GLUT4 and pantophysin were depleted from this vesicle population following treatment with insulin. Additionally, a subpopulation of immunopurified pantophysin vesicles contained insulin-responsive GLUT4. Consistent with the interaction of synaptophysin with vesicle-associated membrane protein 2 in neuroendocrine tissues, pantophysin associated with vesicle-associated membrane protein 2 in adipocytes. Furthermore, in [(32)P]orthophosphate-labeled cells, pantophysin was phosphorylated in the basal state. This phosphorylation was unchanged in response to insulin; however, insulin stimulated the phosphorylation of a 77-kDa protein associated with alpha-pantophysin immunoprecipitates. Although the functional role of pantophysin in vesicle trafficking is unclear, its presence on GLUT4 vesicles is consistent with the emerging role of soluble N-ethylmaleimide-sensitive protein receptor (SNARE) factor complex and related proteins in regulated vesicle transport in adipocytes. In addition, pantophysin may provide a marker for the analysis of other vesicles in adipocytes.     

Biogenesis of synaptic vesicles in vitro

Synaptic vesicles are synthesized at a rapid rate in nerve terminals to compensate for their rapid loss during neurotransmitter release. Their biogenesis involves endocytosis of synaptic vesicle membrane proteins from the plasma membrane and requires two steps, the segregation of synaptic vesicle membrane proteins from other cellular proteins, and the packaging of those unique proteins into vesicles of the correct size. By labeling an epitope-tagged variant of a synaptic vesicle protein, VAMP (synaptobrevin), at the cell surface of the neuroendocrine cell line PC12, synaptic vesicle biogenesis could be followed with considerable precision, quantitatively and kinetically. Epitope-tagged VAMP was recovered in synaptic vesicles within a few minutes of leaving the cell surface. More efficient targeting was obtained by using the VAMP mutant, del 61-70. Synaptic vesicles did not form at 15 degrees C although endocytosis still occurred. Synaptic vesicles could be generated in vitro from a homogenate of cells labeled at 15 degrees C. The newly formed vesicles are identical to those formed in vivo in their sedimentation characteristics, the presence of the synaptic vesicle protein synaptophysin, and the absence of detectable transferrin receptor. Brain, but not fibroblast cytosol, allows vesicles of the correct size to form. Vesicle formation is time and temperature-dependent, requires ATP, is calcium independent, and is inhibited by GTP-gamma S. Thus, two key steps in synaptic vesicle biogenesis have been reconstituted in vitro, allowing direct analysis of the proteins involved.     

Pantophysin is a ubiquitously expressed synaptophysin homologue and defines constitutive transport vesicles

Certain properties of the highly specialized synaptic transmitter vesicles are shared by constitutively occurring vesicles. We and others have thus identified a cDNA in various nonneuroendocrine cell types of rat and human that is related to synaptophysin, one of the major synaptic vesicle membrane proteins, which we termed pantophysin. Here we characterize the gene structure, mRNA and protein expression, and intracellular distribution of pantophysin. Its mRNA is detected in murine cell types of nonneuroendocrine as well as of neuroendocrine origin. The intron/exon structure of the murine pantophysin gene is identical to that of synaptophysin except for the last intron that is absent in pantophysin. The encoded polypeptide of calculated mol wt 28,926 shares many sequence features with synaptophysin, most notably the four hydrophobic putative transmembrane domains, although the cytoplasmic end domains are completely different. Using antibodies against the unique carboxy terminus pantophysin can be detected by immunofluorescence microscopy in both exocrine and endocrine cells of human pancreas, and in cultured cells, colocalizing with constitutive secretory and endocytotic vesicle markers in nonneuroendocrine cells and with synaptophysin in cDNA-transfected epithelial cells. By immunoelectron microscopy, the majority of pantophysin reactivity is detected at vesicles with a diameter of < 100 nm that have a smooth surface and an electron-translucent interior. Using cell fractionation in combination with immunoisolation, these vesicles are enriched in a light fraction and shown to contain the cellular vSNARE cellubrevin and the ubiquitous SCAMPs in epithelial cells and synaptophysin in neuroendocrine or cDNA-transfected nonneuroendocrine cells and neuroendocrine tissues. Pantophysin is therefore a broadly distributed marker of small cytoplasmic transport vesicles independent of their content.     

v-SNARE composition distinguishes synaptic vesicle pools

Synaptic vesicles belong to two distinct pools, a recycling pool responsible for the evoked release of neurotransmitter and a resting pool unresponsive to stimulation. The uniform appearance of synaptic vesicles has suggested that differences in location or cytoskeletal association account for these differences in function. We now find that the v-SNARE tetanus toxin-insensitive vesicle-associated membrane protein (VAMP7) differs from other synaptic vesicle proteins in its distribution to the two pools, providing evidence that they differ in molecular composition. We also find that both resting and recycling pools undergo spontaneous release, and when activated by deletion of the longin domain, VAMP7 influences the properties of release. Further, the endocytosis that follows evoked and spontaneous release differs in mechanism, and specific sequences confer targeting to the different vesicle pools. The results suggest that different endocytic mechanisms generate synaptic vesicles with different proteins that can endow the vesicles with distinct properties.     

Mice lacking synaptophysin reproduce and form typical synaptic vesicles

Synaptophysin is one of the major integral membrane proteins of the small (30–50 nm diameter) electron-translucent transmitter-containing vesicles in neurons and of similar vesicles in neuroendocrine cells. Since its expression is tightly linked to the occurrence of these vesicle types, we mutated the X-chromosomally located synaptophysin gene in embryonic stem cells for the generation of synaptophysin-deficient mice in order to study the consequence of synaptophysin ablation for the formation and function of such vesicles in vivo. the behavior and appearance of mice lacking synaptophysin was indistinguishable from that of their litter mates and reproductive capacity was comparable to normal mice. Furthermore, no drastic compensatory changes were noted in the expression of several other neuronal polypeptides or in the mRNA levels of synaptophysin isoforms, the closely related neuronal synaptoporin/synaptophysinII, and the ubiquitous pantophysin. Immunofluorescence microscopy of several neuronal and neuroendocrine tissues showed that overall tissue architecture was maintained in the absence of synaptophysin, and that the distribution of other synaptic vesicle components was not visibly affected. In electron-microscopic preparations, large numbers of vesicles with a diameter of 39.9 nm and an electron-translucent interior were seen in synaptic regions of synaptophysin-deficient mice; these vesicles could be labeled by antibodies against synaptic vesicle proteins, such as synaptobrevin 2.This research was supported by the DFG-SFB 317     

VAMP2, but not VAMP3/cellubrevin, mediates insulin-dependent incorporation of GLUT4 into the plasma membrane of L6 myoblasts

Like neuronal synaptic vesicles, intracellular GLUT4-containing vesicles must dock and fuse with the plasma membrane, thereby facilitating insulin-regulated glucose uptake into muscle and fat cells. GLUT4 colocalizes in part with the vesicle SNAREs VAMP2 and VAMP3. In this study, we used a single-cell fluorescence-based assay to compare the functional involvement of VAMP2 and VAMP3 in GLUT4 translocation. Transient transfection of proteolytically active tetanus toxin light chain cleaved both VAMP2 and VAMP3 proteins in L6 myoblasts stably expressing exofacially myc-tagged GLUT4 protein and inhibited insulin-stimulated GLUT4 translocation. Tetanus toxin also caused accumulation of the remaining C-terminal VAMP2 and VAMP3 portions in Golgi elements. This behavior was exclusive to these proteins, because the localization of intracellular myc-tagged GLUT4 protein was not affected by the toxin. Upon cotransfection of tetanus toxin with individual vesicle SNARE constructs, only toxin-resistant VAMP2 rescued the inhibition of insulin-dependent GLUT4 translocation by tetanus toxin. Moreover, insulin caused a cortical actin filament reorganization in which GLUT4 and VAMP2, but not VAMP3, were clustered. We propose that VAMP2 is a resident protein of the insulin-sensitive GLUT4 compartment and that the integrity of this protein is required for GLUT4 vesicle incorporation into the cell surface in response to insulin.     

VAMP/synaptobrevin isoforms 1 and 2 are widely and differentially expressed in nonneuronal tissues

VAMP/synaptobrevin is part of the synaptic vesicle docking and fusion complex and plays a central role in neuroexocytosis. Two VAMP (vesicle- associated membrane protein) isoforms are expressed in the nervous system and are differently distributed among the specialized parts of the tissue. Here, VAMP-1 and -2 are shown to be present in all rat tissues tested, including kidney, adrenal gland, liver, pancreas, thyroid, heart, and smooth muscle. The two isoforms are differentially expressed in various tissues and their level may depend on differentiation. VAMP-1 is restricted to exocrine pancreas and to kidney tubular cells, whereas VAMP-2 is the predominant isoform present in Langerhans islets and in glomerular cells. Both isoforms show a patchy vesicular intracellular distribution in confocal microscopy. The present results provide evidence for the importance of neuronal VAMP proteins in the physiology of all cells.     

Differential Regulation of the Serotonin Transporter by Vesicle-Associated Membrane Protein 2 in Cells of Neuronal versus Non-Neuronal Origin

The serotonin transporter (SERT) is a key regulator of serotonergic signalling as it mediates the re-uptake of synaptic serotonin into nerve terminals, thereby terminating or modulating its signal. It is well-known that SERT regulation is a dynamic process orchestrated by a wide array of proteins and mechanisms. However, molecular details on possible coordinated regulation of SERT activity and 5-HT release are incomplete. Here, we report that vesicle-associated membrane protein 2 (VAMP2), a SNARE protein that mediates vesicle fusion with the plasma membrane, interacts with SERT. This was documented in vitro, through GST pull-down assays, by co-immunoprecipitation experiments on heterologous cells and rat hippocampal synaptosomes, and with FRET analysis in live transfected HEK-293 MSR cells. The related isoforms VAMP1 and VAMP3 also physically interact with SERT. However, comparison of the three VAMP isoforms shows that only VAMP2 possesses a functionally distinct role in relation to SERT. VAMP2 influences 5-HT uptake, cell surface expression and the delivery rate of SERT to the plasma membrane differentially in HEK-293 MSR and PC12 cells. Moreover, siRNA-mediated knock-down of endogenous VAMP2 reduces 5-HT uptake in CAD cells stably expressing low levels of heterologous SERT. Deletion and mutant analysis suggest a role for the isoform specific C-terminal domain of VAMP2 in regulating SERT function. Our data identify a novel interaction between SERT and a synaptic vesicle protein and support a link between 5-HT release and re-uptake.     

Synaptophysin I selectively specifies the exocytic pathway of synaptobrevin 2/VAMP2

Biogenesis and recycling of synaptic vesicles are accompanied by sorting processes that preserve the molecular composition of the compartments involved. In the present study, we have addressed the targeting of synaptobrevin 2/VAMP2 (vesicle-associated membrane protein 2), a critical component of the synaptic vesicle--fusion machinery, in a heterotypic context where its sorting is not confounded by the presence of other neuron-specific molecules. Ectopically expressed synaptophysin I interacts with VAMP2 and alters its default surface targeting to a prominent vesicular distribution, with no effect on the targeting of other membrane proteins. Protein-protein interaction is not sufficient for the control of VAMP2 sorting, which is mediated by the C-terminal domain of synaptophysin I. Synaptophysin I directs the sorting of VAMP2 to vesicles before surface delivery, without influencing VAMP2 endocytosis. Consistent with this, dynamin and alpha-SNAP (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein) mutants which block trafficking at the plasma membrane do not abrogate the effect of synaptophysin I on VAMP2 sorting. These results indicate that the sorting determinants of synaptic vesicle proteins can operate independently of a neuronal context and implicate the association of VAMP2 with synaptophysin I in the specification of the pathway of synaptic vesicle biogenesis.     

Vesicle-associated membrane protein 2 mediates trafficking of α5β1 integrin to the plasma membrane

Integrins are major receptors for cell adhesion to the extracellular matrix (ECM). As transmembrane proteins, the levels of integrins at the plasma membrane or the cell surface are ultimately determined by the balance between two vesicle trafficking events: endocytosis of integrins at the plasma membrane and exocytosis of the vesicles that transport integrins. Here, we report that vesicle-associated membrane protein 2 (VAMP2), a SNARE protein that mediates vesicle fusion with the plasma membrane, is involved in the trafficking of α5β1 integrin. VAMP2 was present on vesicles containing endocytosed β1 integrin. Small interfering RNA (siRNA) silencing of VAMP2 markedly reduced cell surface α5β1 and inhibited cell adhesion and chemotactic migration to fibronectin, the ECM ligand of α5β1, without altering cell surface expression of α2β1 integrin or α3β1 integrin. By contrast, silencing of VAMP8, another SNARE protein, had no effect on cell surface expression of the integrins or cell adhesion to fibronectin. In addition, VAMP2-mediated trafficking is involved in cell adhesion to collagen but not to laminin. Consistent with disruption of integrin functions in cell proliferation and survival, VAMP2 silencing diminished proliferation and triggered apoptosis. Collectively, these data indicate that VAMP2 mediates the trafficking of α5β1 integrin to the plasma membrane and VAMP2-dependent integrin trafficking is critical in cell adhesion, migration and survival.     

Southern rice black-streaked dwarf virus hijacks SNARE complex of its insect vector for its effective transmission to rice

Vesicular trafficking is an important dynamic process that facilitates intracellular transport of biological macromolecules and their release into the extracellular environment. However, little is known about whether or how plant viruses utilize intracellular vesicles to their advantage. Here, we report that southern rice black-streaked dwarf virus (SRBSDV) enters intracellular vesicles in epithelial cells of its insect vector by engaging VAMP7 and Vti1a proteins in the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. The major outer capsid protein P10 of SRBSDV was shown to interact with VAMP7 and Vti1a of the white-backed planthopper and promote the fusion of vesicles into a large vesicle, which finally fused with the plasma membrane to release virions from midgut epithelial cells. Downregulation of the expression of either VAMP7 or Vti1a did not affect viral entry and accumulation in the gut, but significantly reduced viral accumulation in the haemolymph. It also did not affect virus acquisition, but significantly reduced the virus transmission efficiency to rice. Our data reveal a critical mechanism by which a plant reovirus hijacks the vesicle transport system to overcome the midgut escape barrier in vector insects and provide new insights into the role of the SNARE complex in viral transmission and the potential for developing novel strategies of viral disease control.     

Members of the VAMP family of synaptic vesicle proteins are components of glucose transporter-containing vesicles from rat adipocytes.

Existing data support the hypothesis that insulin triggers the exocytosis of small vesicles containing the GluT4 isoform of the glucose transporter. The data also suggest that these vesicles reform through endocytosis of GluT4. These processes resemble those described for synaptic vesicles after depolarization of nerve cells. To determine whether GluT4 vesicles are related to synaptic vesicles, rat adipocyte low density microsomes (LDM), which are rich in GluT4 vesicles, were screened for the synaptic vesicle proteins synaptotagmin, synaptophysin, SV2, p29, rab3, and VAMP (synaptobrevin) by immunoblotting. Two polypeptides that reacted with antibodies against the VAMPs were identified, one with the same apparent size as the two isoforms of VAMP in the brain (18 kDa) and one that was slightly smaller (17 kDa). These members of the VAMP family were highly enriched in GluT4 vesicles isolated by immunoadsorption and translocated from the LDM to the plasma membrane in response to insulin. With the exception of rab3, which was observed in the LDM but was not localized in the GluT4 vesicles, the other synaptic vesicle proteins were not detected. The presence of the VAMPs in both GluT4 and synaptic vesicles suggests that the genesis and/or exocytosis of these two types of vesicles involve shared processes.     

The t-SNAREs syntaxin4 and SNAP23 but not v-SNARE VAMP2 are indispensable to tether GLUT4 vesicles at the plasma membrane in adipocyte

SNARE proteins (VAMP2, syntaxin4, and SNAP23) have been thought to play a key role in GLUT4 trafficking by mediating the tethering, docking and subsequent fusion of GLUT4-containing vesicles with the plasma membrane. The precise functions of these proteins have remained elusive, however. We have now shown that depletion of the vesicle SNARE (v-SNARE) VAMP2 by RNA interference in 3T3-L1 adipocytes inhibited the fusion of GLUT4 vesicles with the plasma membrane but did not affect tethering of the vesicles to the membrane. In contrast, depletion of the target SNAREs (t-SNAREs) syntaxin4 or SNAP23 resulted in impairment of GLUT4 vesicle tethering to the plasma membrane. Our results indicate that the t-SNAREs syntaxin4 and SNAP23 are indispensable for the tethering of GLUT4 vesicles to the plasma membrane, whereas the v-SNARE VAMP2 is not required for this step but is essential for the subsequent fusion event.     

Protein kinase D 3 is localized in vesicular structures and interacts with vesicle-associated membrane protein 2

Protein kinase D localizes in the Golgi and regulates protein transport from the Golgi to the plasma membrane. In the present study, we found that PKD3, a novel member of the PKD family, and its fluorescent protein fusions localized in the Golgi and in the vesicular structures that are in part marked by endosome markers. Fluorescent recovery after photobleaching (FRAP) showed that the PKD3-associated vesicular structures were constantly forming and dissolving, reflecting active subcellular structures. FRAP on plasma membrane-located PKD3 indicated a slower recovery of PKD3 fluorescent signal compared to those of PKC isoforms, implying a different targeting mechanism at the plasma membrane. VAMP2, the vesicle-localized v-SNARE, was later identified as a novel binding partner of PKD3 through yeast two-hybrid screening. PKD3 directly interacted with VAMP2 in vitro and in vivo, and colocalized in part with VAMP2 vesicles in cells. PKD3 did not phosphorylate VAMP-GFP and the purified GST-VAMP2 protein in in vitro phosphorylation assays. Rather, PKD3 was found to promote the recruitment of VAMP2 vesicles to the plasma membrane in response to PMA, while the kinase dead PKD3 abolished this effect. Thus, the kinase activity of PKD3 was required for PMA-induced plasma membrane trafficking of VAMP2. In summary, our findings suggest that PKD3 localizes to vesicular structures that are part of the endocytic compartment. The vesicular distribution may be attributed in part to the direct interaction between PKD3 and vesicle-associated membrane protein VAMP2, through which PKD3 may regulate VAMP2 vesicle trafficking by facilitating its recruitment to the target membrane.     

Synaptophysin I controls the targeting of VAMP2/synaptobrevin II to synaptic vesicles

Synaptic vesicle (SV) proteins are synthesized at the level of the cell body and transported down the axon in membrane precursors of SVs. To investigate the mechanisms underlying sorting of proteins to SVs, fluorescent chimeras of vesicle-associated membrane protein (VAMP) 2, its highly homologous isoform VAMP1 and synaptotagmin I (SytI) were expressed in hippocampal neurons in culture. Interestingly, the proteins displayed a diffuse component of distribution along the axon. In addition, VAMP2 was found to travel in vesicles that constitutively fuse with the plasma membrane. Coexpression of VAMP2 with synaptophysin I (SypI), a major resident of SVs, restored the correct sorting of VAMP2 to SVs. The effect of SypI on VAMP2 sorting was dose dependent, being reversed by increasing VAMP2 expression levels, and highly specific, because the sorting of the SV proteins VAMP1 and SytI was not affected by SypI. The cytoplasmic domain of VAMP2 was found to be necessary for both the formation of VAMP2-SypI hetero-dimers and for VAMP2 sorting to SVs. These data support a role for SypI in directing the correct sorting of VAMP2 in neurons and demonstrate that a direct interaction between the two proteins is required for SypI in order to exert its effect.     

A Highlights from MBoC Selection: Characterization of VAMP isoforms in 3T3-L1 adipocytes: implications for GLUT4 trafficking

The fusion of GLUT4-containing vesicles with the plasma membrane of adipocytes is a key facet of insulin action. This process is mediated by the formation of functional soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complexes between the plasma membrane t-SNARE complex and the vesicle v-SNARE or VAMP. The t-SNARE complex consists of Syntaxin4 and SNAP23, and whereas many studies identify VAMP2 as the v-SNARE, others suggest that either VAMP3 or VAMP8 may also fulfil this role. Here we characterized the levels of expression, distribution, and association of all the VAMPs expressed in 3T3-L1 adipocytes to provide the first systematic analysis of all members of this protein family for any cell type. Despite our finding that all VAMP isoforms form SDS-resistant SNARE complexes with Syntaxin4/SNAP23 in vitro, a combination of levels of expression (which vary by >30-fold), subcellular distribution, and coimmunoprecipitation analyses lead us to propose that VAMP2 is the major v-SNARE involved in GLUT4 trafficking to the surface of 3T3-L1 adipocytes.     

Differential Phosphorylation of Syntaxin and Synaptosome-Associated Protein of 25 kDa (SNAP-25) Isoforms

Abstract : The synaptic plasma membrane proteins syntaxin and synaptosome-associated protein of 25 kDa (SNAP-25) are central participants in synaptic vesicle trafficking and neurotransmitter release. Together with the synaptic vesicle protein synaptobrevin/vesicle-associated membrane protein (VAMP), they serve as receptors for the general membrane trafficking factors N -ethylmaleimide-sensitive factor (NSF) and soluble NSF attachment protein (α-SNAP). Consequently, syntaxin, SNAP-25, and VAMP (and their isoforms in other membrane trafficking pathways) have been termed SNAP receptors (SNAREs). Because protein phosphorylation is a common and important mechanism for regulating a variety of cellular processes, including synaptic transmission, we have investigated the ability of syntaxin and SNAP-25 isoforms to serve as substrates for a variety of serine/threonine protein kinases. Syntaxins 1A and 4 were phosphorylated by casein kinase II, whereas syntaxin 3 and SNAP-25 were phosphorylated by Ca²⁺ - and calmodulin-dependent protein kinase II and cyclic AMP-dependent protein kinase, respectively. The biochemical consequences of SNARE protein phosphorylation included a reduced interaction between SNAP-25 and phosphorylated syntaxin 4 and an enhanced interaction between phosphorylated syntaxin 1A and the synaptic vesicle protein synaptotagmin I, a potential Ca²⁺ sensor in triggering synaptic vesicle exocytosis. No other effects on the formation of SNARE complexes (comprised of syntaxin, SNAP-25, and VAMP) or interactions involving n-Sec1 or α-SNAP were observed. These findings suggest that although phosphorylation does not directly regulate the assembly of the synaptic SNARE complex, it may serve to modulate SNARE complex function through other proteins, including synaptotagmin I.     

Palmitoylation of the synaptic vesicle fusion machinery

The fusion of synaptic vesicles with the pre-synaptic plasma membrane mediates the secretion of neurotransmitters at nerve terminals. This pathway is regulated by an array of protein–protein interactions. Of central importance are the soluble NSF ( N -ethylmaleimide-sensitive factor) attachment protein receptor (SNARE) proteins syntaxin 1 and SNAP25, which are associated with the pre-synaptic plasma membrane and vesicle-associated membrane protein (VAMP2), a synaptic vesicle SNARE. Syntaxin 1, SNAP25 and VAMP2 interact to form a tight complex bridging the vesicle and plasma membranes, which has been suggested to represent the minimal membrane fusion machinery. Synaptic vesicle fusion is stimulated by a rise in intraterminal Ca²⁺ levels, and a major Ca²⁺ sensor for vesicle fusion is synaptotagmin I. Synaptotagmin is likely to couple Ca²⁺ entry to vesicle fusion via Ca²⁺-dependent and independent interactions with membrane phospholipids and the SNARE proteins. Intriguingly, syntaxin 1, SNAP25, VAMP2 and synaptotagmin I have all been reported to be modified by palmitoylation in neurons. In this review, we discuss the mechanisms and dynamics of palmitoylation of these proteins and speculate on how palmitoylation might contribute to the regulation of synaptic vesicle fusion.     

Analysis of SCAMP1 function in secretory vesicle exocytosis by means of gene targeting in mice.

Secretory carrier membrane proteins (SCAMPs) comprise a family of ubiquitous membrane proteins of transport vesicles with no known function. Their universal presence in all cells suggests a fundamental role in membrane traffic. SCAMPs are particularly highly expressed in organelles that undergo regulated exocytosis, such as synaptic vesicles and mast cell granules. Of the three currently known SCAMPs, SCAMP1 is the most abundant. To investigate the possible functions of SCAMP1, we generated mice that lack SCAMP1. SCAMP1-deficient mice are viable and fertile. They exhibit no changes in the overall architecture or the protein composition of the brain or alterations in peripheral organs. Capacitance measurements in mast cells demonstrated that exocytosis could be triggered reliably by GTPgammaS in SCAMP1-deficient cells. The initial overall capacitance of mast cells was similar between wild type and mutant mice, but the final cell capacitance after completion of exocytosis, was significantly smaller in SCAMP1-deficient cells than in wild type cells. Furthermore, there was an increased proportion of reversible fusion events, which may have caused the decrease in the overall capacitance change observed after exocytosis. Our data show that SCAMP1 is not essential for exocytosis, as such, and does not determine the stability or size of secretory vesicles, but is required for the full execution of stable exocytosis in mast cells. This phenotype could be the result of a function of SCAMP1 in the formation of stable fusion pores during exocytosis or of a role of SCAMP1 in the regulation of endocytosis after formation of fusion pores.     

Secretory carrier membrane proteins 31-35 define a common protein composition among secretory carrier membranes.

A novel compositional overlap between membranes of exocrine and endocrine granules, synaptic vesicles, and a liver Golgi fraction has been identified using a monoclonal antibody (SG7C12) raised against parotid secretion granule membranes. This antibody binds secretory carrier membrane proteins with apparent Mr 31,000, 33,000 and 35,000 (designated SCAMPs 31, 33, 35). The proteins are nonglycosylated integral membrane components, and the epitope recognized by SG7C12 is on the cytoplasmic side of the granule membrane. SCAMP 33 is found in all secretory carrier membranes studied so far while SCAMP 35 is found in exocrine and certain endocrine granules and liver Golgi membranes and SCAMP31 only in exocrine granules. They are not related to other similar-sized proteins that have been studied previously in relation to vesicular transport and secretion. Immunocytochemical staining shows that these SCAMPs are highly concentrated in the apical cytoplasm of exocrine cells. Antigens are present not only on exocrine granules and synaptic vesicles but also on other smooth membrane vesicles of exocrine and neural origin as revealed by immunolocalization in subcellular fractions and immunoadsorption to antibody-coated magnetic beads. The wide tissue distribution and localization to secretory carriers and related membranes suggest that SCAMPs 31-35 may be essential components in vesicle-mediated transport/secretion.