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PubMed1991

Characterization of the gene for the bovine seminal vesicle secretory protein SVSP109.

As part of an attempt to understand androgen-regulation of SVSP109, a bovine seminal vesicle secretory protein of 109 amino acids, we have characterized the bovine SVSP109 gene. The 6.1 kb gene is organized in five exons and four introns. Regulatory sequences involved in regulation of transcription could not be identified by simple sequence homologies. The SVSP109 gene may provide an excellent example for functional properties of exons: exon 1 encodes the entire signal peptide and exon 4 the complete fibronectin type II-domain, responsible for protein-protein interactions.

Amino Acid Sequence↗

PubMed1995

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.

Amino Acid Sequence↗

PubMed1997

Docked secretory vesicles undergo Ca2+-activated exocytosis in a cell-free system.

The Ca2+-activated fusion of secretory vesicles with the plasma membrane responsible for regulated neurotransmitter and hormone secretion has previously been studied in permeable neuroendocrine cells, where requirements for ATP and cytosolic proteins were identified. As reported here, Ca2+-activated fusion mechanisms are also preserved following cell homogenization. The release of norepinephrine (NE) and other vesicle constituents from a PC12 cell membrane fraction was activated by micromolar Ca2+ (EC50 approximately 3 microM) and exhibited a dependence upon MgATP and cytosol. Ca2+-dependent NE release was inhibited by botulinum neurotoxins and by CAPS (Ca2+-dependent activator protein for secretion) antibody implying that syntaxin, synaptobrevin, SNAP-25 (synaptosomal-associated protein of 25 kDa), and CAPS are required for regulated exocytosis in this system. The exocytosis-competent membrane fraction consisted of rapidly sedimenting dense core vesicles associated with plasma membrane fragments. Free vesicles did not release NE either in the absence or presence of plasma membranes, indicating that only docked vesicles were competent for exocytosis under the reconstitution conditions used. A cell-free system for Ca2+-activated fusion will facilitate studies on the roles of essential proteins such as syntaxin, synaptobrevin, SNAP-25, and CAPS that act at post-docking steps in the regulated exocytotic pathway.

Animals↗

PubMed1993

Cloning and sequence analysis of a cDNA encoding an androgen-dependent mouse seminal vesicle secretory protein.

We report the cloning and sequencing of a new cDNA sequence encoding a protein from the mouse seminal vesicle. An open reading frame of 297 nucleotides encoded a protein of 99 amino acids with a calculated molecular mass of 11.454 kDa. The first 21 amino acids constituted a signal peptide followed by 78 amino acids encoding the secreted protein. The cDNA sequence comprised a 3' untranslated region of 226 bp and the polyadenylation signal AATAAA, 19 bp upstream from the poly(A)+ tail. A high degree of homology was found between this protein and members of the family of seminal vesicle secretory (SVS) proteins, especially rat SVS VI. Northern blot analysis indicated the presence of a 0.7 kb mRNA species in the mRNAs of seminal vesicle tissue. Castration resulted in a marked decrease in the level of the 0.7 kb mRNA encoding the protein, whereas administration of testosterone to castrated males restored the 0.7 kb mRNA.

Amino Acid Sequence↗

PubMed2005

A methionine synthase homolog is associated with secretory vesicles in tobacco pollen tubes.

Seven isoforms of 85 kDa polypeptides (p85) were identified as methionine synthase (MetE) homologs by partial aminoacid sequencing in tobacco pollen tube extracts. Immunocytochemistry data showed a localization of the antigen on the surface of tip-focussed post-Golgi secretory vesicles (SVs), that appear to be partially associated with microtubules (Mts). The chemical dissection of pollen tube high speed supernatant (HSS) showed that two distinct pools of MetE are present in pollen tubes, one being the more acidic isoforms sedimenting at 15S and the remaining at 4S after zonal centrifugation through a sucrose density gradient. The identification of the MetE within the pollen tube and its possible participation as methyl donor in a wide range of metabolic reactions, makes it a good subject for studies on pollen tube growth regulation.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed1982

Effect of castration on the synthesis of seminal vesicle secretory protein IV in the rat.

The effects of castration on the synthesis (accumulation) of a major seminal vesicle secretory protein (SVS IV) were examined in young adult rats. In vitro incorporation of labeled amino acids into SVS IV by minced tissue was monitored by immunological methods. Castration resulted in a large decrease in the differential synthesis of SVS IV. A significant decrease in the relative incorporation of isotope into SVS IV was evident within 3 days of castration, and by 4 weeks relative incorporation dropped some 30-fold. These changes took place in the presence of a large generalized decline in protein synthesis so that incorporation into SVS IV on an organ basis decreased by over 200-fold. SVS IV messenger RNA levels were estimated by RNA excess solution hybridization using a cloned cDNA probe. Relative message levels declined after castration in harmony with the declines in SVS IV synthesis. SVS IV mRNA was decreased by a relative factor of approximately 20 and an absolute factor of approximately 200 in long-term (40-day) castrates. Accordingly, the seminal vesicle conforms to the general pattern of steroid regulated systems in which hormone withdrawal leads to differential decreases in the steady-state pool size for specific mRNAs. The seminal vesicle is unusual, however, in that a prolonged period is required for maximum differential effects to occur.

Animals↗

PubMed1989

Purification and characterization of a cortical secretory vesicle membrane fraction.

A membrane fraction has been prepared by sucrose density gradient fractionation of purified cortical secretory vesicles from the eggs of the sea urchin Strongylocentrotus purpuratus. The purified cortical vesicle membrane fraction has a phospholipid to protein ratio of 1.76 and exhibits a morphology typical of biological membranes as seen by electron microscopy. The protein composition of the purified membranes was analyzed by SDS-polyacrylamide gel electrophoresis and shown to be distinct from that of eggs, cell surface complex, cortical vesicles, fertilization product, and yolk platelets. Alkaline extraction (pH 11.0) of peripheral membrane proteins increased the phospholipid to protein ratio to 2.55 and removed several polypeptides. Immunoblot analysis of the isolated cortical vesicle membrane fraction revealed low levels of contamination with two major cortical vesicle content proteins. Fractions enriched in egg plasma membranes and yolk platelet membranes also have been isolated and compared with the cortical vesicle membranes by SDS-polyacrylamide gel electrophoresis. The protein compositions of the three membrane fractions were found to contain very little overlap, indicating that the cortical vesicle membrane preparation is relatively free of contamination from these likely noncortical vesicle sources of membrane. Both the plasma membrane and cortical vesicle membrane samples were found by immunoblotting to contain actin.

Actins↗

PubMed2003

Autosomal dominant growth hormone deficiency disrupts secretory vesicles in vitro and in vivo in transgenic mice.

Autosomal dominant GH deficiency type II (IGHDII) is often associated with mutations in the human GH gene (GH1) that give rise to products lacking exon-3 ((Deltaexon3)hGH). In the heterozygous state, these act as dominant negative mutations that prevent the release of human pituitary GH (hGH). To determine the mechanisms of these dominant negative effects, we used a combination of transgenic and morphological approaches in both in vitro and in vivo models. Rat GC cell lines were generated expressing either wild-type GH1 (WT-hGH-GC) or a genomic GH1 sequence containing a G->A transition at the donor splice site of IVS3 ((Deltaexon3)hGH-GC). WT-hGH-GC cells grew normally and produced equivalent amounts of human and rGH packaged in dense-cored secretory vesicles (SVs). In contrast, (Deltaexon3)hGH-GC cells showed few SVs but accumulated secretory product in amorphous cytoplasmic aggregates. They produced much less rGH and grew more slowly than WT-hGH-GC cells. When cotransfected with an enhanced green fluorescent protein construct (GH-eGFP), which copackages with GH in SVs, WT-hGH-GC cells showed normal electron microscopy morphology and SV movements, tracked with total internal reflectance fluorescence microscopy. In contrast, coexpression of (Deltaexon3)hGH with GH-eGFP abolished the vesicular targeting of GH-eGFP, which instead accumulated in static aggregates. Transgenic mice expressing (Deltaexon3)hGH in somatotrophs showed an IGHD-II phenotype with mild to severe pituitary hypoplasia and dwarfism, evident at weaning in the most severely affected lines. Hypothalamic GHRH expression was up-regulated and somatostatin expression reduced in (Deltaexon3)hGH transgenic mice, consistent with their profound GHD. Few SVs were detectable in the residual pituitary somatotrophs in (Deltaexon3)hGH transgenic mice, and these cells showed grossly abnormal morphology. A low copy number transgenic line showed a mild effect relatively specific for GH, whereas two severely affected lines with higher transgene copy numbers showed early onset, widespread pituitary damage, macrophage invasion, and multiple hormone deficiencies. These new in vitro and in vivo models shed new light on the cellular mechanisms involved in IGHDII, as well as its phenotypic consequences in vivo.

Animals↗

PubMedDate not supplied

Clathrin light chains and secretory vesicle binding proteins are distinct.

Recently, several groups have initiated studies on cytosolic proteins that bind to isolated secretory vesicle membranes in the presence of Ca2+ in order to identify proteins that may regulate exocytosis. Two major chromaffin granule binding proteins, of molecular weights 32,000 (32K) and 34,000 (34K), were reported to have the same mobility on one-dimensional SDS gels as clathrin-associated light chains from the adrenal medulla, and the 34K granule binding protein the same one-dimensional peptide map as the 34K clathrin light chain. These observations support the hypothesis that Ca2+-dependent recruitment of soluble light chains to the vesicle membrane may nucleate the assembly of a clathrin coat and initiate endocytosis. Here we report that two-dimensional peptide maps of the clathrin light chains and of all chromaffin granule membrane binding proteins in the 30K range are distinct, and therefore fail to support this hypothesis. It has also been suggested that some or all of the vesicle binding proteins require calmodulin for their interaction with the membrane. However, we find that antagonism of calmodulin by trifluoperazine does not prevent the association of the other cytosolic proteins with the chromaffin granule membrane.

Adrenal Medulla↗

PubMed1979

Isolation of a protein from the plasma membrane of adrenal medulla which binds to secretory vesicles.

Solubilized proteins of the plasma membrane of bovine adrenal medulla were fractionated on the basis of their affinity for secretory vesicles. The isolation procedure included preparation of a highly purified fraction of plasma membranes, its solubilization in detergent, and application to a column prepared from glutaraldehyde-fixed chromaffin granules. Using this technique, one major polypeptide (80% of the material bound) was isolated. This protein has been shown to originate from the plasma membrane and has no affinity for fixed bovine adrenal medullary mitochondria or lysosomes. It is eluted most effectively by low pH (3.0) and can be rebound and re-eluted from fixed secretory granules. In sodium dodecyl sulfate and beta-mercaptoethanol it has an apparent molecular weight of 51,000. In addition, two minor components, comprising about 20% of the material bound were detected having apparent molecular weights in sodium dodecyl sulfate of 14,000 and 62,000. It is suggested that such a molecule could function as a plasma membrane-located receptor for chromaffin granules during the secretory process.

Adrenal Medulla↗

PubMed2002

Enlargement of secretory vesicles by protein tyrosine phosphatase PTP-MEG2 in rat basophilic leukemia mast cells and Jurkat T cells.

Stimulus-induced secretion of bioactive polypeptides is a fundamental aspect of the immune system. Secretory proteins are synthesized in the endoplasmic reticulum and are transported through the Golgi apparatus to the trans-Golgi network, where they are sorted into transport vesicles that bud off and fuse into condensing vacuoles, which subsequently undergo an editing and concentration process to become mature secretory vesicles. In this study, we report that the PTP-MEG2 protein tyrosine phosphatase is located on these vesicles in mast cells. Expression of PTP-MEG2 caused a striking enlargement of these vesicles in both rat basophilic leukemia mast cells and Jurkat T leukemia cells into giant vesicles with diameters of up to several micrometers. The fused vesicles did not acquire markers for other compartments and were adjacent to the trans-Golgi network, contained carboxypeptidase E, chromogranin C, and IL-2, and had an electron-dense core typical of secretory vesicles. Expression of PTP-MEG2 also caused a reduction in the secretion of IL-2 from stimulated Jurkat cells. The effects of PTP-MEG2 on secretory vesicles required the catalytic activity of PTP-MEG2 and was rapidly reversed by pervanadate. We propose that PTP-MEG2 represents a novel connection between tyrosine dephosphorylation and the regulation of secretory vesicles in hematopoietic cells.

Animals↗

PubMed1987

Comparison of seminal vesicle secretory proteins of rodents using antibody and nucleotide probes.

The copulatory vaginal plug is a conspicuous feature of rodent reproduction. The five major seminal vesicle secretory proteins of Rattus norvegicus (proteins I-V), which form the copulatory plug, constitute a closely related androgen-regulated family that appears to share a common evolutionary origin. The relationships between these rat proteins and the major seminal vesicle proteins of other rodents were explored using antibodies specific for the individual rat proteins. Immunoblotting of proteins separated by SDS-PAGE showed that the vesicular proteins of R. rattus are identical to those of R. norvegicus except for an additional protein related to protein III. No differences were seen in inbred and outbred strains of R. norvegicus. Of the major proteins of Mus musculus, one showed strong homology with rat protein II and three others were weakly homologous to proteins I, IV (or S) and V (or F); none showed homology to rat protein III. The only homology between the vesicular proteins of Mesocricetus auratus (Syrian hamster) and Meriones ungulatus (Mongolian gerbil) was with rat protein II while those of Cavia porcellus (guinea pig) showed no homology at all with the rat proteins. In addition, cDNA probes for rat genes IV and V both detected weak homologues in seminal vesicle RNA from mice but not guinea pigs.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1999

Evidence for the proenkephalin processing enzyme prohormone thiol protease (PTP) as a multicatalytic cysteine protease complex: activation by glutathione localized to secretory vesicles.

The cysteine protease known as "prohormone thiol protease" (PTP) has been identified as a major proenkephalin processing enzyme in secretory vesicles of adrenal medulla (known as chromaffin granules). This study provides the first demonstration that PTP exists as a multicatalytic cysteine protease complex that can be activated by endogenous glutathione present in chromaffin granules. The high molecular mass nature of PTP, of approximately 185 kDa, was demonstrated by elution of a single peak of 35S-enkephalin precursor cleaving activity by Sephacryl S200 gel filtration chromatography and by a single band of 35S-enkephalin precursor cleaving activity detected on radiozymogram gels under native buffer conditions. Importantly, when 0.1% SDS was included in radiozymogram gels, PTP activity was resolved into three bands of proteolytic activity with apparent molecular masses of 88, 81, and 61 kDa. These activities were all cysteine proteases, since they were inhibited by the cysteine protease inhibitor E-64c but not by pepstatin A or EDTA that inhibit aspartyl protease and metalloprotease, respectively. Purification of native PTP by preparative gel electrophoresis indicated that PTP was composed of four polypeptides of 66, 60, 33, and 29 kDa detected on SDS-PAGE gels. These four protein subunits accounted for the three catalytic activities of PTP, as demonstrated on 35S-enkephalin precursor radiozymogram gels. Results also indicated that the electrophoretic mobilities of the four subunits differed under reducing compared to nonreducing conditions. The multicatalytic activities of the PTP complex all require reducing conditions for activity, which can be provided by endogenous reduced glutathione in chromaffin granules. These novel findings provide the first evidence for a role of a multicatalytic cysteine protease complex, PTP, in chromaffin granules that may be involved in the proteolytic processing of proenkephalin and perhaps other precursors into active neuropeptides.

Amino Acid Sequence↗

PubMedDate not supplied

Currents through the fusion pore that forms during exocytosis of a secretory vesicle.

Exocytosis, or the fusion of cytoplasmic vesicles with the cell membrane, occurs in nearly all eukaryotic cells, but its mechanism is not understood. Morphological and electrophysiological studies have suggested that membrane fusion begins with the formation of a 'fusion pore', a narrow channel across the closely adjacent membranes of vesicle and cell that forms the first connection of the vesicle lumen with the cell exterior and later dilates to allow release of vesicle contents. We used the patch clamp technique to study exocytosis of single giant secretory vesicles in mast cells of beige mice. The first opening of the fusion pore was found to generate a brief current transient, whose size and direction indicated an initial pore conductance of about 230 pS and a lumen-positive vesicle membrane potential. In time-resolved a.c. admittance measurements, the pore conductance was found to increase to much larger values within milliseconds, as if the pore dilated soon after opening. We conclude that the earliest fusion event may be the formation of a structure similar to an ion channel. Its conductance is of the same order of magnitude as that of a single gap junction channel, the only other known channel that spans two membranes.

Animals↗

PubMed1994

Functional expression of P-glycoproteins in secretory vesicles.

We expressed P-glycoproteins (P-gps) encoded by the three mouse mdr genes in the membranes of secretory vesicles (SV) accumulating in the yeast mutant strain sec 6-4. Expression of the Mdr1 and Mdr3 isoforms in SV membranes caused a significant increased accumulation of the drug vinblastine (VBL) over background levels measured in control SV. The Mdr1/Mdr3-mediated increased drug accumulation could be completely abolished by the P-gp modulator verapamil. By contrast, overexpression of Mdr2 in these vesicles failed to increase intravesicular VBL accumulation over background levels. Mdr3-mediated VBL transport was not affected by changes in the membrane potential, since identical rates of VBL uptake were measured in the presence or absence of the endogenous proton-translocating PMA1 H(+)-ATPase responsible for the strong electrochemical membrane potential across SV membranes. Moreover, in the presence of a delta micro-H+ across the SV membranes (inside positive) of almost 90 mV, we detected in Mdr3-expressing SV an enhanced accumulation of the lipophilic cation and P-gp substrate tetraphenylphosphonium, suggesting that P-gp-mediated uptake of this cation occurs against an intravesicular depolarized membrane. Likewise, VBL transport in Mdr3-expressing SV was not affected by the presence or absence of a steep proton gradient (inside acid) and was independent of any proton movements, excluding a proton synport or antiport mechanism for P-gp-mediated drug transport. Finally, we could demonstrate that colchicine accumulation in Mdr3-expressing SV occurred against a significant substrate concentration gradient, reaching a 7-fold increase in intravesicular colchicine concentration above the extravesicular medium drug concentration. Our studies show that SV isolated from the temperature-sensitive yeast sec 6-4 mutants are an ideal tool to express and to functionally characterize heterologous membrane proteins, in general and P-gps, in particular.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2006

Loss of P4 ATPases Drs2p and Dnf3p disrupts aminophospholipid transport and asymmetry in yeast post-Golgi secretory vesicles.

Eukaryotic plasma membranes generally display asymmetric lipid distributions with the aminophospholipids concentrated in the cytosolic leaflet. This arrangement is maintained by aminophospholipid translocases (APLTs) that use ATP hydrolysis to flip phosphatidylserine (PS) and phosphatidylethanolamine (PE) from the external to the cytosolic leaflet. The identity of APLTs has not been established, but prime candidates are members of the P4 subfamily of P-type ATPases. Removal of P4 ATPases Dnf1p and Dnf2p from budding yeast abolishes inward translocation of 6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminocaproyl] (NBD)-labeled PS, PE, and phosphatidylcholine (PC) across the plasma membrane and causes cell surface exposure of endogenous PE. Here, we show that yeast post-Golgi secretory vesicles (SVs) contain a translocase activity that flips NBD-PS, NBD-PE, and NBD-PC to the cytosolic leaflet. This activity is independent of Dnf1p and Dnf2p but requires two other P4 ATPases, Drs2p and Dnf3p, that reside primarily in the trans-Golgi network. Moreover, SVs have an asymmetric PE arrangement that is lost upon removal of Drs2p and Dnf3p. Our results indicate that aminophospholipid asymmetry is created when membrane flows through the Golgi and that P4-ATPases are essential for this process.

Biological Transport↗

PubMed1994

Secretogogue-induced gating of chloride channels in the secretory vesicles of parafollicular cells.

Thyroid parafollicular (PF) cells are neural crest-derived endocrine cells that secrete serotonin and calcitonin. The secretory vesicles of PF cells acidify when secretion is induced by increased extracellular Ca2+ or TSH. We tested the hypothesis that acidification is regulated by secretogogue-gated Cl- channels in vesicular membranes. Cl- channel (p64) immunoreactivity was enriched in purified PF vesicles. X-Ray microanalysis showed a change in chlorine level in PF vesicles in response to secretogogue-stimulation of isolated cells. Secretogogue stimulation also altered the degree of p64 channel phosphorylation. Protein kinase and phosphatase inhibitors antagonized secretogogue-induced vesicle acidification and secretion; however, secretion could occur even when acidification was blocked. We conclude that acidification of PF vesicles is regulated by a gatable Cl- channel in vesicle membranes and that protein phosphorylation and dephosphorylation are involved in channel activation. Acidification of vesicles is not required for exocytosis.

Acridine Orange↗

PubMed2003

Axonal transport of British and Danish amyloid peptides via secretory vesicles.

The ABri and ADan amyloid peptides deposited in familial British and Danish neurodegenerative disorders are generated by processing mutant forms of the precursor protein BRI2. Although the pathogenic process that leads to deposition of amyloid in the brains of patients has been studied extensively, the cellular processes and normal function of the precursor protein did not receive much attention. We observed in a variety of transfected cell lines the presence of two independent proteolytic processing events. In addition to the previously described cleavage, which results in the production of carboxyl-terminal approximately 3 kDa wild-type peptide or approximately 4 kDa ABri or ADan peptides, we describe a novel amino-terminal cleavage site within BRI2. Both cleavages occur within the cis- or medial-Golgi. Following cleavage, the BRI2-derived carboxyl-terminal peptides are transported via a regulated secretory pathway into secretory vesicles in neuronal cells. Worth noting is that expression of wild-type British or Danish mutants of BRI2, in mouse neuroblastoma N2a cells that do not express endogenous BRI2, induces elongation of neurites, which suggests a role for this protein in differentiation of neuronal cells.

Adaptor Proteins, Signal Transducing↗