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At least 253 records · Page 14Linked to original sources
PubMed1998

Involvement of long chain fatty acid elongation in the trafficking of secretory vesicles in yeast.

Members of the synaptobrevin/VAMP family of v-SNAREs are thought to be essential for vesicle docking and exocytosis in both lower and higher eukaryotes. Here, we describe yeast mutants that appear to bypass the known v-SNARE requirement in secretion. Recessive mutations in either VBM1 or VBM2, which encode related ER-localized membrane proteins, allow yeast to grow normally and secrete in the absence of Snc v-SNAREs. These mutants show selective alterations in protein transport, resulting in the differential trafficking and secretion of certain protein cargo. Yet, processing of the vacuolar marker, carboxypeptidase Y, and the secreted protein, invertase, appear normal in these mutants indicating that general protein trafficking early in the pathway is unaffected. Interestingly, VBM1 and VBM2 are allelic to ELO3 and ELO2, two genes that have been shown recently to mediate the elongation of very long chain fatty acids and subsequent ceramide and inositol sphingolipid synthesis. Thus, the v-SNARE requirement in constitutive exocytosis is abrogated by mutations in early components of the secretory pathway that act at the level of lipid synthesis to affect the ability of secretory vesicles to sort and deliver protein cargo.

Alleles↗
PubMed1995

In vitro processing of anthrax toxin protective antigen by recombinant PC1 (SPC3) and bovine intermediate lobe secretory vesicle membranes.

Protective antigen (PA), an 83-kDa protein produced by Bacillus anthracis, requires proteolytic activation at a tetrabasic site (RKKR167) before it can combine with either edema factor or lethal factor on the cell surface. The complex is then endocytosed and the target cell intoxicated. Previous work has demonstrated that furin, a ubiquitously distributed, subtilisin-like protease, can perform this cleavage. In this study, another member of the furin family, PC1 (SPC3), was tested as a putative processing enzyme for PA. Recombinant PC1, partially purified from the medium of stably transfected L-cells, cleaved PA to a 63-kDa fragment (PA63) and a 20-kDa fragment (PA20). Amino-terminal sequence analysis of the 63 kDa product demonstrated that cleavage occurred between Arg167 and Ser168. The pH optimum for in vitro PA cleavage was 6.0 and the enzymatic activity was calcium-dependent. Medium from untransfected L-cells did not cleave PA. Site-directed mutagenesis of the tetrabasic cleavage site revealed that PC1 preferred to cleave sequences containing basic residues at positions -1 and -4 relative to the wild-type cleavage site, demonstrating that PC1 can cleave substrates at a monobasic residue site in vitro. Substrates having basic residues at the -1 and -2 positions were cleaved with approximately twofold less efficiency than wild-type PA. Mutants of PA containing basic residues in positions -1 and either -2 or -4 of the cleavage site were predicted to be substrates for PC1 and were more toxic to L-cells expressing PC1 than to untransfected L-cells. These results demonstrate that PA is cleaved by PC1 in vivo. Membranes from bovine intermediate lobe secretory vesicles which contain both prohormone convertases, PC1 and PC2, also cleaved PA to PA63 with a pH optimum of 5.5. Immunodepletion studies using antisera against PC1 and PC2 showed that these are the enzymes primarily responsible for the cleavage of PA in the membrane preparation. Thus, both recombinant PC1 and a membrane preparation containing endogenous PC1 can activate PA.

Amino Acid Sequence↗
PubMed2001

Actin filament nucleation by endosomes, lysosomes and secretory vesicles.

Intracellular pathogens such as Listeria monocytogenes and vaccinia virus propel themselves through the cytoplasm of mammalian cells by nucleating actin filaments. Recently, actin assembly has also been shown to power the movement of intracellular vesicles, and this may be a mechanism underlying endomembrane movement in a variety of physiological contexts. Surprisingly, class I myosins have been found to play important roles in both actin nucleation and endomembrane trafficking.

Actin Cytoskeleton↗
PubMed2002

Beta-amyloid peptide in regulated secretory vesicles of chromaffin cells: evidence for multiple cysteine proteolytic activities in distinct pathways for beta-secretase activity in chromaffin vesicles.

A key factor in Alzheimer's disease (AD) is the beta-secretase activity that is required for the production of beta-amyloid (Abeta) peptide from its amyloid precursor protein (APP) precursor. In this study, the majority of Abeta secretion from neuronal chromaffin cells was found to occur via the regulated secretory pathway, compared with the constitutive secretory pathway; therefore, beta-secretase activity in the regulated secretory pathway was examined for the production and secretion of Abeta in chromaffin cells obtained from in vivo adrenal medullary tissue. The presence of Abeta(1-40) in APP-containing chromaffin vesicles, which represent regulated secretory vesicles, was demonstrated by radioimmunoassay (RIA) and reverse-phase high-performance liquid chromatography. These vesicles also contain Abeta(1-42), measured by RIA. Significantly, regulated secretion of Abeta(1-40) from chromaffin cells represented the majority of secreted Abeta (> 95% of total secreted Abeta), compared with low levels of constitutively secreted Abeta(1-40). These results indicate the importance of Abeta production and secretion in the regulated secretory pathway as a major source of extracellular Abeta. Beta-secretase activity in isolated chromaffin vesicles was detected with the substrate Z-Val-Lys-Met-/MCA (methylcoumarinamide) that contains the beta-secretase cleavage site. Optimum beta-secretase activity in these vesicles required reducing conditions and acidic pH (pH 5-6), consistent with the in vivo intravesicular environment. Evidence for cysteine protease activity was shown by E64c inhibition of Z-Val-Lys-Met-MCA-cleaving activity, and E64c inhibition of Abeta(1-40) production in isolated chromaffin vesicles. Chromatography resolved the beta-secretase activity into two distinct proteolytic pathways consisting of: (i) direct cleavage of the beta-secretase site at Met-/Asp by two cysteine proteolytic activities represented by peaks Il-A and Il-B, and (ii) an aminopeptidase-dependent pathway represented by peak I cysteine protease activity that cleaves between Lys-/Met, followed by Met-aminopeptidase that would generate the beta-secretase cleavage site. Treatment of chromaffin cells in primary culture with the cysteine protease inhibitor E64d reduced the production of the beta-secretase product, a 12-14 kDa C-terminal APP fragment. In addition, BACE 1 and BACE 2 were detected in chromaffin vesicles; BACE 1 represented a small fraction of total beta-secretase activity in these vesicles. These results illustrate that multiple cysteine proteases, in combination with BACE 1, contribute to beta-secretase activity in the regulated secretory pathway. These results complement earlier findings for BACE 1 as beta3-secretase for Abeta production in the constitutive secretory pathway that provides basal secretion of Abeta into conditioned media. These findings suggest that drug inhibition of several proteases may be required for reducing Abeta levels as a potential therapeutic approach for AD.

Adrenal Medulla↗
PubMed1991

Inability of Tfm (testicular feminization) epithelial cells to express androgen-dependent seminal vesicle secretory proteins in chimeric tissue recombinants.

To assess the role of androgen receptors (ARs) in the expression of androgen-dependent seminal vesicle (SV) secretory proteins, tissue recombinants were prepared with rat seminal vesicle mesenchyme plus ureter epithelium of wild-type or Tfm mice (rat SVM plus wild-type mouse URE and rat SVM plus Tfm mouse URE, respectively). After growth in male hosts, both the wild-type and Tfm ureter epithelia were induced by SVM to differentiate into a simple columnar epithelium exhibiting the complex folded morphology characteristic of the SV. In SVM plus wild-type mouse URE recombinants, epithelial ARs were induced, and the epithelium expressed the full spectrum of SV secretory proteins. By contrast, in SVM plus Tfm mouse URE recombinants, the Tfm epithelium was genetically incapable of producing functional ARs and failed to produce SV secretory proteins. These data demonstrate in vivo that the induction of SV secretory proteins by androgens is an event requiring intraepithelial ARs. In contrast, androgen-dependent epithelial morphogenesis, columnar cytodifferentiation, and probably also proliferation can be expressed in Tfm epithelium grown in association with wild-type mesenchyme, strongly suggesting that these events are indirect effects on the epithelium mediated by mesenchymal ARs.

Androgens↗
PubMed1988

Processing of two protein precursors yields four mature guinea pig seminal vesicle secretory proteins.

The translation of the two most abundant guinea pig seminal vesicle epithelium mRNAs (1800 nucleotides and 950 nucleotides) and the subsequent processing of their protein products were studied in an effort to elucidate the mechanism by which the four mature guinea pig seminal vesicle epithelium (GPSVE) secretory proteins are produced. The primary translation products of the 1800 nt and 950 nt mRNAs are two secretory protein precursors of 45 kDa and 20 kDa, respectively. Removal of signal peptides from these two precursors produces proteins of 43 kDa and 18.5 kDa, which are recognized by polyclonal antisera directed against the four mature secretory proteins. The existence of further processing intermediates in the production of the secretory proteins is suggested by the appearance of other immunoreactive polypeptides following incubation of GPSVE in nutrient medium containing [3H] leucine. Immunological and pulse-chase analysis strongly suggests that the 43-kDa protein gives rise to SVP-1, -3, and -4 and that SVP-2 is derived from the 18.5-kDa protein.

Animals↗
PubMed2000

Enhancement of the dense-core vesicle secretory cycle by glucocorticoid differentiation of PC12 cells: characteristics of rapid exocytosis and endocytosis.

The secretory cycle of dense-core vesicles (DCVs) in physiologically stimulated patch-clamped PC12 cells was analyzed using both amperometry and capacitance measurements. Untreated cells had low or undetectable Ca currents and sparse secretory responses to short depolarizations. Dexamethasone (5 microM) treatment for 5-7 d tripled Ca current magnitude and dramatically increased quantal secretion in response to depolarization with action potentials. Such cells expressed L-, N-, and P-type Ca channels, and depolarization evoked rapid catecholamine secretion recorded as amperometric spikes; the average latency was approximately 50 msec. These spikes were much smaller and shorter than those of primary adrenal chromaffin cells, reflecting the smaller size of DCVs in PC12 cells. Depolarizing pulse trains also elicited a rapid increase in membrane capacitance corresponding to exocytosis in differentiated but not in naïve cells. On termination of stimulation, membrane capacitance declined within 20 sec to baseline indicative of rapid endocytosis (RE). RE did not take place when secretion was stimulated in the presence of Ba or Sr, indicating that RE is Ca-specific. RE was blocked when either anti-dynamin antibodies or the pleckstrin homology domain of dynamin-1 was loaded into the cell via the patch pipette. These studies indicate that neuroendocrine differentiation of PC12 cells with glucocorticoids enhances the development of the excitable membrane and increases the coupling between Ca channels and vesicle release sites, leading to rapid exocytosis and endocytosis. Slow catecholamine secretion in undifferentiated cells may be caused in part by a lack of localized secretory machinery rather than being an intrinsic property of dense-core vesicles.

Animals↗
PubMed1988

Ampicillin inhibits the movement of biliary secretory vesicles in rat hepatocytes.

A number of biliary secretory processes are inhibited by administration of ampicillin to isolated perfused rat livers. Reduction in output was observed for phospholipid, cholesterol, the endogenous protein rat serum albumin and the exogenous protein bovine serum albumin, whilst secretin of bile salts was virtually unaffected. All of the affected materials are secreted by processes involving vesicles which are brought to the appropriate pole of the hepatocyte, and the observed inhibitory effects of ampicillin may, therefore, possibly be due to a blockage in the transport of these substances. The effects of ampicillin were much less marked on materials secreted at the sinusoidal pole of the cell.

Ampicillin↗
PubMed1988

Targeting of secretory vesicles to cytoplasmic domains in AtT-20 and PC-12 cells.

Organelles are not uniformly distributed throughout the cytoplasm but have preferred locations that vary between tissues and during development. To investigate organelle targeting to cytoplasmic domains we have taken advantage of the mouse pituitary cell line, AtT-20, which, when induced to extend long processes, accumulates dense core secretory granules at the tips of the processes. During mitosis, these secretory granules accumulate along the plane of division. Protein synthesis is not mandatory for such redistribution of secretory granules. To explore the specificity of the redistribution we have used transfected AtT-20 cells that express the immunoglobulin kappa light chain. While the endogenous hormone ACTH is found in secretory granules, the kappa chain is a marker for organelles involved in constitutive secretion. By immunofluorescence, kappa also accumulates at the tips of growing processes, and along the midline of dividing cells, suggesting that the redistribution of vesicles is not specific for dense-core secretory granules. Since there is evidence for selective organelle transport along processes in neuronal cells, the rat pheochromocytoma cell PC-12 was transfected with DNA encoding markers for regulated and constitutive secretory vesicles. Again regulated and constitutive vesicles co-distribute, even in cells grown in the presence of nerve growth factor. We suggest that at least in the cells studied here, cytoskeletal elements normally carry exocytotic organelles to the surface; when the cytoskeletal elements coalesce in an extending process, exocytotic organelles of both the constitutive and regulated pathway are transported nonselectively to the tips of the cytoskeletal elements where they accumulate.

Adrenocorticotropic Hormone↗
PubMed2005

Exocytosis of lung surfactant: from the secretory vesicle to the air-liquid interface.

Exocytosis is fundamental in biology and requires an orchestra of proteins and other constituents to fuse a vesicle with the plasma membrane. Although the molecular fusion machinery appears to be well conserved in evolution, the process itself varies considerably with regard to the diversity of physico-chemical and structural factors that govern the delay between stimulus and fusion, the expansion of the fusion pore, the release of vesicle content, and, finally, its extracellular dispersion. Exocytosis of surfactant is unique in many of these aspects. This review deals with the secretory pathway of pulmonary surfactant from the type II cell to the air-liquid interface, with focus on the distinct mechanisms and regulation of lamellar body (LB) fusion and release. We also discuss the fate of secreted material until it is rearranged into units that finally function to reduce the surface tension in the lung.

Animals↗
PubMed2005

Calcium dynamics in catecholamine-containing secretory vesicles.

We have used an aequorin chimera targeted to the membrane of the secretory granules to monitor the free [Ca(2+)] inside them in neurosecretory PC12 cells. More than 95% of the probe was located in a compartment with an homogeneous [Ca(2+)] around 40 microM. Cell stimulation with either ATP, caffeine or high-K(+) depolarization increased cytosolic [Ca(2+)] and decreased secretory granule [Ca(2+)] ([Ca(2+)](SG)). Inositol-(1,4,5)-trisphosphate, cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate were all ineffective to release Ca(2+) from the granules. Changes in cytosolic [Na(+)] (0-140 mM) or [Ca(2+)] (0-10 microM) did not modify either ([Ca(2+)](SG)). Instead, [Ca(2+)](SG) was highly sensitive to changes in the pH gradient between the cytosol and the granules. Both carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) and nigericin, as well as cytosolic acidification, reversibly decreased [Ca(2+)](SG), while cytosolic alcalinization reversibly increased [Ca(2+)](SG). These results are consistent with the operation of a H(+)/Ca(2+) antiporter in the vesicular membrane. This antiporter could also mediate the effects of ATP, caffeine and high-K(+) on [Ca(2+)](SG), because all of them induced a transient cytosolic acidification. The FCCP-induced decrease in [Ca(2+)](SG) was reversible in 10-15 min even in the absence of cytosolic Ca(2+) or ATP, suggesting that most of the calcium content of the vesicles is bound to a slowly exchanging Ca(2+) buffer. This large store buffers [Ca(2+)](SG) changes in the long-term but allows highly dynamic free [Ca(2+)](SG) changes to occur in seconds or minutes.

Aequorin↗
PubMed2004

Novel secretory vesicle proteins essential for membrane fusion display extracellular-matrix domains.

Exocytotic mutants can be obtained in Paramecium that affect the organization of the fusion machinery, visible by electron microscopy. The site of action of the genes in the plasma membrane, cytosol or secretory compartment can easily be determined in such mutants. Functional complementation cloning of exocytotic mutants specifically affected in the secretory compartment, nd2-1 and nd169-1, reported here, and the previously studied nd7-1, led to the discovery of a set of novel proteins that display PSI and EGF domains, normally found in extracellular matrix proteins and involved in transmembrane signaling. The structure of one of these proteins, Nd2p, and of the product of a paralog found in the genome Nd22p, corresponds to that of type I membrane receptors, generally involved in protein and vesicle sorting. Our characterization suggests that the proteins we have identified are required to indicate the presence of a mature secretory vesicle to the plasma membrane, to prepare the machinery for fusion. We propose to name this novel subclass of receptors VEMIF, for Vesicular Extracellular-Matrix-like proteins Involved in preparing membrane Fusion.

Amino Acid Sequence↗
PubMed2004

Kiss and run exocytosis of dense core secretory vesicles.

The mechanisms by which large, dense core, peptide-containing vesicles release their cargo from neuroendocrine cells has been the subject of intense debate during the past few years. Thanks to recent studies of the vesicle membrane post fusion, the view that exocytosis is obligatorily linked to the complete collapse of these vesicles at the cell surface is gradually being revised. We discuss here the evidence supporting a model in which (1) exocytosis and endocytosis of dense core vesicles are closely coupled, and (2) the release of peptides occurs from largely intact vesicles via the formation of a large, but transient, fusion pore.

Animals↗
PubMed2003

Association of Caldendrin splice isoforms with secretory vesicles in neurohypophyseal axons and the pituitary.

Caldendrin is a neuronal calcium-binding protein, which is highly enriched in the postsynaptic density fraction and exhibits a prominent somato-dendritic distribution in brain. Two additional splice variants derive from the caldendrin gene, which have unrelated N-termini and were previously only detected in the retina. We now show that these isoforms are present in neurohypophyseal axons and on secretory granules of endocrine cells. In light of the described interaction of the Caldendrin C-terminus with Q-type Ca(v)2.1 calcium channels these data suggest that this interaction takes place in neurohypophyseal axons and pituitary cells indicating functions of the short splice variants in triggering Ca2+ transients to a vesicular target interaction.

Alternative Splicing↗
PubMed2001

Different secretory vesicles can be involved in depolarization-evoked exocytosis.

The relationship between Ca(2+) influx through voltage-activated Ca(2+) channels, resting intracellular Ca(2+) level (Ca(i)) and Ca(2+)-dependent exocytosis was studied in bovine adrenal chromaffin cells by using patch-clamp, capacitance, and fluorescent measurements. It was established that depolarization-induced exocytosis passed over two steps, both of which linearly depend on Ca(i). At Ca(i) lying below critical point (200-300 nM) the slope of the relationship was 4.43 and at Ca(i) exceeding the critical point the slope was equal to 31.63. The vesicular mechanism describing experimental two-step dependence of exocytosis on intracellular Ca(2+) (Ca(i)) is proposed. According to the model at Ca(i) below critical point only small-sized vesicles fuse with plasma membrane, whereas at higher Ca(i), larger vesicles started to fuse.

Animals↗
PubMed2001

Cell-specific targeting of caveolin-1 to caveolae, secretory vesicles, cytoplasm or mitochondria.

In commonly used tissue culture cells, caveolin-1 is embedded in caveolae membranes. It appears to reach this location after being cotranslationally inserted into ER membranes, processed in the Golgi and shipped to the cell surface. We now report that caveolae are not the preferred location for caveolin-1 in all cell types. Skeletal muscle cells and keratinocytes target caveolin-1 to the cytosol while in exocrine and endocrine cells it accumulates in the secretory pathway. We also found that airway epithelial cells accumulate caveolin-1 in modified mitochondria. The cytosolic and the secreted forms appear to be incorporated into a soluble, lipid complex. We conclude that caveolin-1 can be targeted to a variety of intracellular destinations, which suggests a novel mechanism for the intracellular traffic of this protein.

Adult↗
PubMed2002

Complexities in ETS-domain transcription factor function and regulation: lessons from the TCF (ternary complex factor) subfamily. The Colworth Medal Lecture.

The ETS-domain transcription factor family can be divided into a series of subfamilies. Elk-1 represents the founding member of the ternary complex factor (TCF) subfamily. By focusing on the TCF subfamily, we can demonstrate the complexities that exist in the function and regulation of ETS-domain transcription factors. This article focuses on Elk-1 in detail and summarizes the functions of other TCFs. The key themes covered include the domain structure of the TCFs, the mechanisms of complex formation with serum response factor, regulation of TCFs by mitogen-activated protein kinase cascades, and transcriptional regulatory properties of the TCFs. Finally, the emerging role of the TCFs in vivo is discussed. A picture is developing indicating that, while these proteins exhibit significant sequence and functional conservation, key differences in their structure and regulation are being identified which may relate to unique functions of these proteins in vivo.

Amino Acid Sequence↗