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PubMed1991

[The dependence of electrical reactions on the exocytosis of secretory vesicles in mouse mammary alveoli].

The effect of acetylcholine inducing the exocytosis of secretory vesicles, upon the development of changes in electrical resistance in mammal gland's alveoli, was studied in mice. A decrease in the ACh concentration and in potassium ions concentration gradient was followed by a deceleration of the reduction of transepithelial electrical resistance. The data obtained suggest that the exocytosis of secretory vesicles is not reflected in changes of transepithelial electrical resistance in mammal gland's alveoli.

Acetylcholine↗

PubMed1992

Processing of adrenocorticotropin by two proteases in bovine intermediate lobe secretory vesicle membranes. A distinct acidic, tetrabasic residue-specific calcium-activated serine protease and a PC2-like enzyme.

Adrenocorticotropin (ACTH) is cleaved at the tetrabasic residue site, in pituitary intermediate lobe secretory vesicles, to yield ACTH1-17 and corticotropin-like intermediate lobe peptide (CLIP). ACTH1-17 is then converted to alpha-melanocyte-stimulating hormone (N-AcACTH1-13NH2) by first removing the Lys15-Lys16-Arg17 residues, followed by amidation of the COOH terminus and acetylation of the NH2 terminus. Bovine intermediate lobe secretory vesicle membranes were screened for proteolytic enzyme activity that will cleave the tetrabasic residues of ACTH. Two activities with pH optima of 5.0-6.0 and 7.5-8.0 were detected. The acidic, ACTH-converting enzyme cleaved ACTH1-39 at the tetrabasic residues between the Arg17-Arg18 bond to yield ACTH1-17 and CLIP, but did not cleave paired basic residues of pro-opiomelanocortin. This enzyme activity was characterized as a Ca(2+)-activated serine protease with unique specificity for the tetrabasic residues of ACTH1-39. The neutral activity preferentially generated ACTH1-17 and to a small extent ACTH1-16 from ACTH1-39 and ACTH1-24. This enzyme activity was Ca(2+)-dependent but was not inhibited by serine or aspartic protease inhibitors. The neutral activity was significantly immunodepleted by antiserum raised against bovine PC2/PC3, and together with specificity studies, suggests that the enzyme is a PC2-like serine protease. The pH optimum, distinct specificity for tetrabasic residues, and subcellular localization of the acidic ACTH-converting enzyme indicate a function of this enzyme in the in vivo conversion of ACTH1-39 to alpha-melanocyte-stimulating hormone in intermediate lobe secretory vesicles which have an acidic internal pH.

Adrenocorticotropic Hormone↗

PubMed2002

Physical mobilization of secretory vesicles facilitates neuropeptide release by nerve growth factor-differentiated PC12 cells.

It has been speculated that neurosecretion can be enhanced by increasing the motion, and hence, the availability of cytoplasmic secretory vesicles. However, facilitator-induced physical mobilization of secretory vesicles has not been observed directly in living cells, and recent experimental results call this hypothesis into question. Here, high resolution green fluorescent protein (GFP)-based measurements in nerve growth factor-differentiated PC12 cells are used to test whether altering dense core vesicle (DCV) motion affects neuropeptide release. Experiments with mycalolide B and jasplakinolide demonstrate that neuropeptidergic DCV motion at the ends of processes is proportional to F-actin. Furthermore, Ba2+ increases DCV mobility without detectably modifying F-actin. Finally, we show that altering DCV motion by changing F-actin or stimulating with Ba2+ proportionally changes sustained neuropeptide release. Therefore, increasing DCV mobility facilitates prolonged neuropeptide release.

Actins↗

PubMed1991

Newly synthesized synaptophysin is transported to synaptic-like microvesicles via constitutive secretory vesicles and the plasma membrane.

The biogenesis of synaptic-like microvesicles (SLMVs) in neuroendocrine cells was investigated by studying the traffic of newly synthesized synaptophysin to SLMVs in PC12 cells. Synaptophysin was found to be sulfated, which facilitated the determination of its exit route from the trans-Golgi network (TGN). Virtually all [35S]sulfate-labeled synaptophysin was found to leave the TGN in vesicles which were indistinguishable from constitutive secretory vesicles but distinct from immature secretory granules and SLMVs. [35S]sulfate-labeled synaptophysin was rapidly transported from the TGN to the cell surface, with a t1/2 of approximately 10 min in resting cells. After arrival at the cell surface, [35S]sulfate-labeled synaptophysin cycled for at least 1 h between the plasma membrane and an intracellular compartment likely to be the early endosome. Up to approximately 40% of the [35S]sulfate-labeled synaptophysin eventually (after 3 h and later) reached SLMVs, which could be distinguished from the other post-TGN compartments by their lower buoyant density in a sucrose gradient and their selective inclusion upon permeation chromatography using a controlled-pore glass column. Our results suggest that newly synthesized membrane proteins of SLMVs in neuroendocrine cells, and possibly of small synaptic vesicles in neurons, reach these organelles via the TGN----plasma membrane----early endosome.

Animals↗

PubMed1992

Sodium-dependent calcium efflux from adrenal chromaffin cells following exocytosis. Possible role of secretory vesicle membranes.

Although cytosolic Ca2+ transients are known to influence the magnitude and duration of hormone and neurotransmitter release, the processes regulating the decay of such transients after cell stimulation are not well understood. Na(+)-dependent Ca2+ efflux across the secretory vesicle membrane, following its incorporation into the plasma membrane, may play a significant role in Ca2+ efflux after stimulation of secretion. We have measured an enhanced 45Ca2+ efflux from cultured bovine adrenal chromaffin cells following cell stimulation with depolarizing medium (75 mM K+) or nicotine (10 microM). Such stimulation also causes Ca2+ uptake via voltage-gated Ca2+ channels and secretion of catecholamines. Na+ replacement with any of several substitutes (N-methyl-glucamine, Li+, choline, or sucrose) during cell stimulation inhibited the enhanced 45Ca2+ efflux, indicating and Na(+)-dependent Ca2+ efflux process. Na+ deprivation did not inhibit 45Ca2+ uptake or catecholamine secretion evoked by elevated K+. Suppression of exocytotic incorporation of secretory vesicle membranes into the plasma membrane with hypertonic medium (620 mOsm) or by lowering temperature to 12 degrees C inhibited K(+)-stimulated 45Ca2+ efflux in Na(+)-containing medium but did not inhibit the stimulated 45Ca2+ uptake. Enhancement of exocytotic secretion with pertussis toxin resulted in an enhanced 45Ca2+ efflux without affecting calcium uptake. The combined results suggest that Na(+)-dependent Ca2+ efflux across secretory vesicle membranes, following their incorporation into the plasma membrane during exocytosis, plays a significant role in regulating calcium efflux and the decay of cytosolic Ca2+ in adrenal chromaffin cells and possibly in related secretory cells.

Adrenal Medulla↗

PubMed2001

Proteomic analysis of a developmentally regulated secretory vesicle.

Secretion of spore coat proteins from the prespore secretory vesicles (PSVs) in Dictyostelium discoideum is a signal mediated event that underlies terminal cell differentiation, and represents an important case of developmentally regulated secretion. In order to study the biochemical mechanisms that govern the regulated fusion of the PSVs with the plasma membrane and the subsequent secretion of their cargo, we purified this organelle from prespore cells. Analysis of protein extracts of highly purified PSVs indicated that, in addition to the cargo of structural spore coat proteins, many more proteins are associated with the PSVs. Their identification is paramount to the understanding of the mechanism of regulated secretion in this system. In this study we have taken the first comprehensive proteomic approach to the analysis of an entire, previously uncharacterized, organelle, with the goal of identifying the major proteins associated with the PSVs. We show that in addition to the structural spore coat proteins, the PSVs contain the enzymes needed for proper spore coat assembly (thioredoxin 2 and 3), regulatory proteins which we predict receive and transduce the developmental signal for secretion (rab7 GTPase, PI-3 kinase, NDP kinase and the calcium binding proteins calfumirin-1 and calreticulin) as well as proteins that interact with the cytoskeleton to mediate movement of the PSVs to the plasma membrane (actin binding proteins coactosin and profilin 1). In addition, the results suggest that proteins can play multiple roles in the cell, and that protein function can be dictated in part by subcellular localization. The identification of the PSV proteins is allowing us to develop testable hypotheses about the roles of these proteins within the functional context of developmentally regulated secretion.

Animals↗

PubMed1995

A role for phosphatidylinositol transfer protein in secretory vesicle formation.

Vesicular traffic in eukaryotic cells is characterized by two steps of membrane rearrangement: the formation of vesicles from donor membranes and their fusion with acceptor membranes. With respect to vesicle formation, several of the cytosolic proteins implicated in budding and fission have been identified. A feature common to all these proteins is that their targets, when known, are other proteins rather than lipids. Here we report, using a previously established cell-free system derived from a neuroendocrine cell line, the purification of cytosolic factors that stimulate the formation of constitutive secretory vesicles and immature secretory granules from the trans-Golgi network. One such factor, referred to as CAST1, was identified as the alpha and beta isoforms of the mammalian phosphatidylinositol transfer protein (PtdIns-TP) (refs 3-5). The yeast PtdIns-TP, SEC14p (ref. 6), which has no sequence homology to mammalian PtdIns-TP (refs 7,8), was able to substitute for the mammalian PtdIns-TP in secretory vesicle formation. Our results suggest a highly conserved role for phosphoinositides in vesicle formation.

Animals↗

PubMed2004

The release of secretory vesicle in encysting Giardia lamblia.

Giardia is an intestinal parasite that undergoes adaptation for survival outside the host. It secretes an extracellular cyst wall using a poorly understood process. An encystation-specific secretory vesicle (ESV) was previously described containing cyst wall proteins. The process of release of these vesicles has been suggested to occur after fragmentation of large ESV in small secretory vesicles, followed by exocytosis, but it was not demonstrated. The release of the ESV was studied by transmission electron microscopy. It was observed: (1) the moment of vesicle release; (2) that a large vesicle is exocytosed and does not fragment into small vesicles; (3) membrane fusion is distinct from traditional exocytosis since it is incomplete; (4) the occurrence of membrane fragmentation and that those membranes reseal to form ghosts; (5) these membrane ghosts may be endocytosed, adhered to flagellar surface or/and form empty vesicles in the extracellular medium.

Animals↗

PubMed1996

ADP-ribosylation factor-1 stimulates formation of nascent secretory vesicles from the trans-Golgi network of endocrine cells.

ADP-ribosylation factor (ARF) is a small GTP-binding protein that has been implicated in intracellular vesicular transport. ARF regulates the budding of vesicles that mediate endoplasmic reticulum to Golgi and intra-Golgi transport. It also plays an important role in maintaining the function and morphology of the Golgi apparatus. Using a permeabilized cell system derived from GH3 cells, we provide evidence that ARF-1 regulates the formation of nascent secretory vesicles from the trans-Golgi network. Both myristoylated and non-myristoylated forms of recombinant human ARF-1 enhanced secretory vesicle budding about 2-fold. A mutant lacking the first 17 N-terminal residues, as well as one that preferentially binds GDP (T31N) did not stimulate vesicle formation. In contrast, a mutant defective in GTP hydrolysis (Q71L) promoted vesicle budding. Strikingly, a peptide corresponding to the N terminus of human ARF-1 (amino acids 2-17) also stimulated vesicle budding from the trans-Golgi network, in marked contrast to its inhibitory effect on vesicular transport from the endoplasmic reticulum to Golgi. These data demonstrate that in endocrine cells, ARF-1 and in particular its N terminus play an essential role in the formation of secretory vesicles.

ADP-Ribosylation Factors↗

PubMed2002

Imaging secretory vesicles by fluorescent protein insertion in propeptide rather than mature secreted peptide.

We combined confocal and live-cell imaging with a novel molecular strategy aimed at revealing mechanisms underlying glucose-regulated insulin vesicle secretion. The 'Ins-C-GFP' reporter monitors secretory peptide targeting, trafficking, and exocytosis without directly tagging the mature secreted peptide. We trapped a green fluorescent protein (GFP) reporter in equimolar quantity within the secretory vesicle by fusing it within the C peptide of proinsulin which only after nascent vesicle sealing and acidification is cleaved from the mature secreted A and B chains of insulin. Ins-C-GFP expression in mouse islets without fail exhibited punctate distribution of green fluorescence by confocal microscopy. Ins-C-GFP colocalized GFP with insulin at vesicle dense cores by immuno-electron microscopy. Glucose stimulation decreased vesicle fluorescence coordinately with enhanced secretion from islets of C-GFP detected by anti-GFP Western blots, and of insulin detected by anti-insulin radioimmunoassay. An insulin secretagogue with a red fluorescent label, glibenclamide BODIPY TR, was applied to islets expressing Ins-C-GFP. The stimulus response was imaged as a rise in red secretagogue leading to marked loss in green granules. Since neuropeptides as well as peptide hormones are processed from propeptides after sealing of secretory granules, vesicle trapping likely is widely applicable for studies on targeting, trafficking, and regulated release of secretory peptides.

Animals↗

PubMed2005

Myosin Va transports dense core secretory vesicles in pancreatic MIN6 beta-cells.

The role of unconventional myosins in neuroendocrine cells is not fully understood, with involvement suggested in the movement of both secretory vesicles and mitochondria. Here, we demonstrate colocalization of myosin Va (MyoVa) with insulin in pancreatic beta-cells and show that MyoVa copurifies with insulin in density gradients and with the vesicle marker phogrin-enhanced green fluorescent protein upon fluorescence-activated sorting of vesicles. By contrast, MyoVa immunoreactivity was poorly colocalized with mitochondrial or other markers. Demonstrating an important role for MyoVa in the recruitment of secretory vesicles to the cell surface, a reduction of MyoVa protein levels achieved by RNA interference caused a significant decrease in glucose- or depolarization-stimulated insulin secretion. Similarly, expression of the dominant-negative-acting globular tail domain of MyoVa decreased by approximately 50% the number of vesicles docked at the plasma membrane and by 87% the number of depolarization-stimulated exocytotic events detected by total internal reflection fluorescence microscopy. We conclude that MyoVa-driven movements of vesicles along the cortical actin network are essential for the terminal stages of regulated exocytosis in beta-cells.

Animals↗

PubMed1995

Neutrophil alkaline phosphatase activity increase in bacterial infections is not associated with a general increase in secretory vesicle membrane components.

The content of alkaline phosphatase (ALP) was determined in neutrophils isolated from patients with acute bacterial infections by a standard enzyme assay. Compared with control cells, patient cells exhibited about a fivefold increase in ALP activity. There was no difference between the ALP Km values of control and patient cells, which indicates that the elevated activity in patient cells was due to the presence of increased amounts of the enzyme. The ALP isozyme in both cell types was determined to be the tissue-unspecific ALP. The fact that much of the ALP activity was measurable only in the presence of detergent suggested that the enzyme was localized in the secretory vesicles, a putative reservoir of plasma membrane components. The amount and subcellular distribution of two other secretory vesicle membrane proteins, i.e., cytochrome b and complement receptor 3, were not altered; hence, we conclude that there was no general increase in amounts of secretory vesicle membrane constituents in the patient cells.

Alkaline Phosphatase↗

PubMed1997

The androgen-dependent mouse seminal vesicle secretory protein of 99 amino acids (MSVSP99): regulation of the mRNA and preliminary characterization of the promoter.

MSVSP99 (mouse seminal vesicle secretory protein of 99 amino acids) is a member of the rat and mouse seminal vesicle secretory protein (SVS) family. In order to characterize its androgenic regulation, the cloned cDNA and gene encoding MSVSP99 have been used. At adulthood, the MSVSP99 mRNA represents from 3 to 7% of the total mRNA population. This mRNA accumulation is under androgenic control because it is abolished by castration and restored in castrated mice by heptylate testosterone injection. During ontogenesis, MSVSP99 mRNA is just detectable in 10-day-old mice, and reaches adult levels at 30 days. Neonatal castration abolishes MSVSP99 mRNA accumulation in 20-day-old mice. Transcription elongation assays show that androgens act mainly on the MSVSP99 gene transcription. In an attempt to obtain information about the mechanism of androgen action on transcription, preliminary transient transfection experiments in CV-1 cells permitted us to define a promoter region (-387/ + 16), the activity of which is enhanced by dihydrotestosterone.

Androgens↗

PubMed1987

Purification and characterization of constitutive secretory vesicles from yeast.

We have developed a purification procedure for the isolation of constitutive post-Golgi secretory vesicles from Saccharomyces cerevisiae. Although the post-Golgi stage of the secretion pathway is normally very rapid, we have used a temperature-sensitive secretory mutant, sec 6-4, to greatly expand the population of secretory vesicles. Following invertase as a marker, intact vesicles are enriched 36-fold from the crude lysate. The final preparation contains few contaminants as assessed by morphologic and biochemical examination. Three proteins (110, 40-45, and 18 kD) co-purify with the vesicle marker enzyme invertase. Metabolic labeling experiments indicate that these vesicle-associated proteins are synthesized during the period of vesicle accumulation. They are not apparent in the corresponding fractions from wild-type cells. Analysis of these proteins indicates that the 110-kD protein is a major glycoprotein residing in the vesicle lumen, while the 40-45- and 18-kD proteins are not glycosylated and are firmly associated with the vesicle membrane, each with at least one domain exposed on the cytoplasmic surface.

Cell Fractionation↗

PubMed1999

Imaging Ca2+ concentration changes at the secretory vesicle surface with a recombinant targeted cameleon.

Regulated exocytosis involves the Ca(2+)-triggered fusion of secretory vesicles with the plasma membrane, by activation of vesicle membrane Ca(2+)-binding proteins [1]. The Ca(2+)-binding sites of these proteins are likely to lie within 30 nm of the vesicle surface, a domain in which changes in Ca2+ concentration cannot be resolved by conventional fluorescence microscopy. A fluorescent indicator for Ca2+ called a yellow 'cameleon' (Ycam2) - comprising a fusion between a cyan-emitting mutant of the green fluorescent protein (GFP), calmodulin, the calmodulin-binding peptide M13 and an enhanced yellow-emitting GFP - which is targetable to specific intracellular locations, has been described [2]. Here, we generated a fusion between phogrin, a protein that is localised to secretory granule membranes [3], and Ycam2 (phogrin-Ycam2) to monitor changes in Ca2+ concentration ([Ca2+]) at the secretory vesicle surface ([Ca2+]gd) through alterations in fluorescence resonance energy transfer (FRET) between the linked cyan and yellow fluorescent proteins (CFP and YFP, respectively) in Ycam2. In both neuroendocrine PC12 and MIN6 pancreatic beta cells, apparent resting values of cytosolic [Ca2+] and [Ca2+](gd) were similar throughout the cell. In MIN6 cells following the activation of Ca2+ influx, the minority of vesicles that were within approximately 1 microm of the plasma membrane underwent increases in [Ca2+](gd) that were significantly greater than those experienced by deeper vesicles, and greater than the apparent cytosolic [Ca2+] change. The ability to image both global and compartmentalised [Ca2+] changes with recombinant targeted cameleons should extend the usefulness of these new Ca2+ probes.

Animals↗

PubMed2002

Dynamin and clathrin are required for the biogenesis of a distinct class of secretory vesicles in yeast.

Yeast produce two classes of secretory vesicles (SVs) that differ in both density and cargo protein content. In late-acting secretory mutants (e.g. snc1(ala43) and sec6-4), both low- (LDSV) and high-density (HDSV) classes of vesicles accumulate at restrictive temperatures. Here, we have found that disruptions in the genes encoding a dynamin-related protein (VPS1) or clathrin heavy chain (CHC1) abolish HDSV production, yielding LDSVs that contain all secreted cargos. Interestingly, disruption of the PEP12 gene, which encodes the t-SNARE that mediates all Golgi to pre-vacuolar compartment (PVC) transport, also abolishes HDSV production. In contrast, deletions in genes that selectively confer vacuolar hydrolase sorting to the PVC or protein transport to the vacuole (i.e. VPS34 and VAM3, respectively) have no effect. Thus, one branch of the secretory pathway in yeast involves an intermediate sorting compartment and has a specific requirement for clathrin and a dynamin-related protein in SV biogenesis.

Carrier Proteins↗

PubMed2003

Functional and spatial segregation of secretory vesicle pools according to vesicle age.

Synaptic terminals and neuroendocrine cells are packed with secretory vesicles, only a few of which are docked at the plasma membrane and readily releasable. The remainder are thought to constitute a large cytoplasmic reserve pool awaiting recruitment into the readily releasable pool (RRP) for exocytosis. How vesicles are prioritized in recruitment is still unknown: the choice could be random, or else the oldest or the newest ones might be favoured. Here we show, using a fluorescent cargo protein that changes colour with time, that vesicles in bovine adrenal chromaffin cells segregate into distinct populations, based on age. Newly assembled vesicles are immobile (morphologically docked) at the plasma membrane shortly after biogenesis, whereas older vesicles are mobile and located deeper in the cell. Different secretagogues selectively release vesicles from the RRP or, surprisingly, selectively from the deeper cytoplasmic pool. Thus, far from being equal, vesicles are segregated functionally and spatially according to age.

Adrenal Glands↗

PubMed1995

Processing of prothyrotropin-releasing hormone (Pro-TRH) by bovine intermediate lobe secretory vesicle membrane PC1 and PC2 enzymes.

TRH is synthesized from a larger 26-kilodalton (kDa) prohormone (pro-TRH). Rat pro-TRH contains five copies of the TRH progenitor sequence (Gln-His-Pro-Gly) and seven other cryptic peptides. Each of the five TRH progenitor sequences is flanked by pairs of basic amino acids. We used a bovine intermediate lobe secretory vesicle membrane preparation, which contains the prohormone convertases (PCs) PC1 and PC2, to study the in vitro processing of pro-TRH. Pro-TRH was radiolabeled using [3H]Leu in AtT20 cells transfected with prepro-TRH complementary DNA, and the labeled 26-kDa pro-TRH was isolated from the cell extract by preparative sodium dodecyl sulfate-gel electrophoresis. Incubation of [3H]pro-TRH with the intermediate lobe secretory vesicle membrane preparation was followed by immunoprecipitation with antibodies specific for various regions of the pro-TRH sequence, and the immunoprecipitates were analyzed by sodium dodecyl sulfate-gel electrophoresis. Immunoprecipitation of the reaction mixture with anti-pCC10 antibody (an antibody that recognizes the intact precursor and amino-terminal intermediate products of processing) showed a time-dependent appearance of a 15-kDa and a 6-kDa peptide and, at times, a 3.8-kDa peptide with diminution of the 26-kDa substrate. Immunoprecipitation of the incubate with the C-terminal-directed antibody, pYE17 (an antibody that recognizes the intact precursor and C-terminal intermediate products of processing), showed the generation of 16.5-, 10-, and 5.4-kDa products in a time-dependent manner, with disappearance of the substrate. Western blot analysis demonstrated that the secretory vesicle membrane preparation contains PC1 and PC2. Immunodepletion studies with antiserum specific for PC1 or PC2 demonstrated that PC1 and PC2 can process pro-TRH to these intermediate products. An initial site of cleavage appeared to be either at the 152-153 or the 158-159 pair of basic residues to yield a 15-kDa N-terminal fragment that was then processed to the 6-kDa [TRH-(25-74)] and 3.8-kDa [TRH-(83-112)] forms. The 10-kDa C-terminal peptide generated by this cleavage was then processed to a 5.4-kDa peptide [TRH-(208-255)]. Alternatively, an initial cleavage at the 107-108 or the 112-113 bonds was also observed, yielding a 16.5-kDa C-terminal product that was further processed to the 5.4-kDa peptide. The pH profile for the appearance of both C- and N-terminal products showed a bimodal distribution, with optima at both 5.5 and 7.5. The cleavage of pro-TRH was enhanced by Ca2+ and partially inhibited by Zn2+.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗