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1.
The disulfide-bonded loop of chromogranin B mediates membrane binding and directs sorting from the trans-Golgi network to secretory granules 总被引:9,自引:0,他引:9
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The disulfide-bonded loop of chromogranin B (CgB), a regulated secretory protein with widespread distribution in neuroendocrine cells, is known to be essential for the sorting of CgB from the trans-Golgi network (TGN) to immature secretory granules. Here we show that this loop, when fused to the constitutively secreted protein alpha1-antitrypsin (AT), is sufficient to direct the fusion protein to secretory granules. Importantly, the sorting efficiency of the AT reporter protein bearing two loops (E2/3-AT-E2/3) is much higher compared with that of AT with a single disulfide-bonded loop. In contrast to endogenous CgB, E2/3-AT-E2/3 does not undergo Ca2+/pH-dependent aggregation in the TGN. Furthermore, the disulfide-bonded loop of CgB mediates membrane binding in the TGN and does so with 5-fold higher efficiency if two loops are present on the reporter protein. The latter finding supports the concept that under physiological conditions, aggregates of CgB are the sorted units of cargo which have multiple loops on their surface leading to high membrane binding and sorting efficiency of CgB in the TGN. 相似文献
2.
P Rosa F A Barr J C Stinchcombe C Binacchi W B Huttner 《European journal of cell biology》1992,59(2):265-274
The effects of brefeldin A (BFA) on membrane traffic between the trans-Golgi network (TGN) and the plasma membrane were investigated in intact PC12 cells and in a cell-free system derived from PC12 cells. In intact cells, BFA caused a virtually complete block of constitutive secretion, as indicated by the lack of release from, and accumulation in, the cells of a [35S]sulfate-labeled heparan sulfate proteoglycan (hsPG). Pulse-chase experiments with [35S]sulfate followed by subcellular fractionation showed that this block was due to the inhibition of formation of constitutive secretory vesicles (CSVs) from the TGN. BFA did not block the depolarization-induced release of [35S]sulfate-labeled chromogranin B (CgB) and secretogranin II (SgII) from secretory granules formed prior to the addition of the drug, showing that BFA does not block secretory granule fusion with the plasma membrane. The presence of BFA did, however, prevent the appearance of [35S]sulfate-labeled CgB and SgII in secretory granules, indicating that the drug inhibits the formation of secretory granules from the TGN. Evidence for a direct block of vesicle formation by BFA was obtained using a cell-free system derived from [35S]sulfate-labeled PC12 cells. In this system, low concentrations of BFA (5 micrograms/ml) inhibited the formation of the hsPG-containing CSVs and that of the SgII-containing secretory granules from the TGN to the same extent (50-60%) as, and in a non-additive manner with, the nonhydrolyzable GTP analogue GTP gamma S. Consistent with the inhibitory effects of BFA on vesicle formation from the TGN, BFA treatment of intact PC12 cells led to the hypersialylation of CgB, which presumably was due to the increased residence time of the protein in the TGN. In conclusion, our data are consistent with, and allow the generalization of, the concept that the BFA-induced block of anterograde membrane traffic results from the inhibition of vesicle formation from a donor compartment. 相似文献
3.
Trimeric G-proteins of the trans-Golgi network are involved in the formation of constitutive secretory vesicles and immature secretory granules. 总被引:10,自引:0,他引:10
Non-hydrolysable analogues of GTP, such as GTP gamma S and GMP-PNP, have previously been shown to inhibit the formation of constitutive secretory vesicles (CSVs) and immature secretory granules (ISGs) from the trans-Golgi network (TGN). Using a cell-free system, we show here that the formation of these vesicles is also inhibited by [A1F4]-, a compound known to act on trimeric G-proteins. Addition of highly purified G-protein beta gamma subunits stimulated, in a differential manner, the cell-free formation of both CSVs and ISGs. ADP-ribosylation experiments revealed the presence of a pertussis toxin-sensitive G-protein alpha subunit in the TGN. We conclude that trimeric G-proteins regulate the formation of secretory vesicles from the TGN. 相似文献
4.
Tylková L Novotová M Zahradník I Kiss A 《Analytical and quantitative cytology and histology / the International Academy of Cytology [and] American Society of Cytology》2008,30(1):53-59
OBJECTIVE: To evaluate the sensitivity and applicability of stereologic analysis for estimating changes in the secretory granule content of atrial myocytes. STUDY DESIGN: The content of secretory granules in the right atrial myocytes of Sprague-Dawley (SD) and Lewis (LW) rats was assessed using a stereologic analysis of electron microscopic images under control conditions and in response to forced wheel running. RESULTS: Volume density analysis revealed significant heterogeneity in the granule content of different strains of rats. The content of the dark secretory granules was significantly lower in control LW compared with control SD rats. The difference in pale granule content was opposite but less pronounced. Forced wheel running did not elicit statistically significant changes in the granule volume density. However, it changed significantly the number of dark granules in each rat strain, albeit in opposite directions, most likely due to changes in the number of small dark granules. No change was observed in the case of pale granules. This suggests higher sensitivity of dark granules to enhanced physical load. CONCLUSION: Both the volume and the number of secretory granules should be estimated in parallel to reveal responses of the atrial secretory system to different internal or external stimuli. 相似文献
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6.
Sorting within the regulated secretory pathway occurs in the trans-Golgi network 总被引:8,自引:8,他引:8
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Bioactive peptides cleaved from the egg-laying hormone precursor in the bag cell neurons of Aplysia are sorted into distinct dense core vesicle classes (DCVs). Bag cell prohormone processing can be divided into two stages, an initial cleavage occurring in a late Golgi compartment, which is not blocked by monensin, and later cleavages that occur within DCVs and are blocked by monensin. Prohormone intermediates are sorted in the trans-Golgi network. The large soma-specific DCVs turn over, while the small DCVs are transported to processes for regulated release. Thus, protein trafficking differentially regulates the levels and localization of multiple biologically active peptides derived from a common prohormone. 相似文献
7.
Cargo proteins moving along the secretory pathway are sorted at the TGN (trans-Golgi network) into distinct carriers for delivery to the plasma membrane or endosomes. Recent studies in yeast and mammals have shown that formation of these carriers is regulated by PtdIns(4)P. This phosphoinositide is abundant at the TGN and acts to recruit components required for carrier formation to the membrane. Other phosphoinositides are also present on the TGN, but the extent to which they regulate trafficking is less clear. Further characterization of phosphoinositide kinases and phosphatases together with identification of new TGN-associated phosphoinositide-binding proteins will reveal the extent to which different phosphoinositides regulate TGN trafficking, and help define the molecular mechanisms involved. 相似文献
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Summary Using an in situ tannic acid perfusion technique, this study presents evidence that the removal of membrane components from the rat atrial secretory granule membrane after granule exocytosis is mediated by coated vesicles. When tannic acid is used to arrest the post-fusion stages of granule release, coated pit formation occurs on granule membrane, which, although continuous with the sarcolemma, is easily recognised by the membrane omega profile and the continued presence of the granule core. Tannic acid perfusion, before aldehyde fixation, allows a degree of continued cell function, and granule fusions can persist after tannic acid has reached the cell. This results in an increase in the numbers of fusion profiles and the appearance of coated pits on granule membrane at these sites. The proportion of granules with coats increases with perfusion time, suggesting that endocytotic, as well exocytotic events, may be arrested by the action of tannic acid. Coated vesicles are also involved at earlier stages of the release pathway. In other types of secretory system this is considered to represent recycling of membrane proteins as part of the maturation process of the granule. Although arrested granules exhibiting this clathrin coat could have had the coat prior to fusion, as part of the maturation process, our results show that it is more likely to represent a second stage of membrane protein recycling; the postfusion reclamation of proteins from the sarcolemma. This facet of the tannic acid perfusion procedure suggests a general method for quantifying coated pit formation during secretory granule release. 相似文献
10.
Constitutive secretion is used to deliver newly synthesized proteins to the cell surface and to the extracellular milieu. The trans-Golgi network is a key station along this route that mediates sorting of proteins into distinct transport pathways, aided in part by clathrin and adaptor proteins. Subsequent movement of proteins to the plasma membrane can occur either directly or via the endocytic pathway. Moreover, multiple, parallel pathways from the trans-Golgi network to the plasma membrane appear to exist, not only in complex, polarized cells such as epithelial cells and neurons, but also in relatively simple cells such as fibroblasts. In addition to typical secretory vesicles, these pathways involve both small, pleiomorphic transport containers and relatively large tubular-saccular carriers that travel along cytoskeletal tracks. While production and movement of these membranous structures are typically described as constitutive, recent studies have revealed that these key steps in secretion are tightly regulated by Ras-superfamily GTPases, members of the protein kinase D family and tethering complexes such as the exocyst. 相似文献
11.
Milieu-induced, selective aggregation of regulated secretory proteins in the trans-Golgi network 总被引:26,自引:9,他引:17
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Regulated secretory proteins are thought to be sorted in the trans-Golgi network (TGN) via selective aggregation. The factors responsible for this aggregation are unknown. We show here that two widespread regulated secretory proteins, chromogranin B and secretogranin II (granins), remain in an aggregated state when TGN vesicles from neuroendocrine cells (PC12) are permeabilized at pH 6.4 in 1-10 mM calcium, conditions believed to exist in this compartment. Permeabilization of immature secretory granules under these conditions allowed the recovery of electron dense cores. The granin aggregates in the TGN largely excluded glycosaminoglycan chains which served as constitutively secreted bulk flow markers. The low pH, high calcium milieu was sufficient to induce granin aggregation in the RER. In the TGN of pituitary GH4C1 cells, the proportion of granins conserved as aggregates was higher upon hormonal treatment known to increase secretory granule formation. Our data suggest that a decrease in pH and an increase in calcium are sufficient to trigger the selective aggregation of the granins in the TGN, segregating them from constitutive secretory proteins. 相似文献
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Tsuboi T Kikuta T Warashina A Terakawa S 《Biochemical and biophysical research communications》2001,282(2):621-628
To elucidate the mechanism for supplying secretory granules to the cell membrane, chromaffin cells isolated from the bovine adrenal medulla were observed by the evanescent wave microscopy after staining their granules with acridine orange. The secretory granules showed only a very small fluctuation, indicating their docking to the plasma membrane. The rate and range of movement increased greatly by application of botulinum toxin A or C. The number of secretory granules docked to the plasma membrane significantly decreased by botulinum toxin C. Conversely, the number increased greatly by activation of protein kinase C with phorbol 12,13-dibutyrate (PDBu). In the presence of an anti-actin reagent cytochalasin D, no increasing effect of PDBu on the number of docked granules was observed. While in the presence of an anti-mitotic reagent, colchicine, a clear increasing effect of PDBu was observed. The final step for supplying granules to the plasma membrane in endocrine cells is concluded to be mediated by a phosphorylation-dependent and actin-based transport system. 相似文献
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15.
Wylie FG Lock JG Jamriska L Khromykh T Brown DL Stow JL 《Traffic (Copenhagen, Denmark)》2003,4(3):175-189
Galpha interacting protein (GAIP) is a regulator of G protein signaling protein that associates dynamically with vesicles and has been implicated in membrane trafficking, although its specific role is not yet known. Using an in vitro budding assay, we show that GAIP is recruited to a specific population of trans -Golgi network-derived vesicles and that these are distinct from coatomer or clathrin-coated vesicles. A truncation mutant (NT-GAIP) encoding only the N-terminal half of GAIP is recruited to trans -Golgi network membranes during the formation of vesicle carriers. Overexpression of NT-GAIP induces the formation of long, coated tubules, which are stabilized by microtubules. Results from the budding assay and from imaging in live cells show that these tubules remain attached to the Golgi stack rather than being released as carrier vesicles. NT-GAIP expression blocks membrane budding and results in the accumulation of tubular carrier intermediates. NT-GAIP-decorated tubules are competent to load vesicular stomatitis virus protein G-green fluorescent protein as post-Golgi, exocytic cargo and in cells expressing NT-GAIP there is reduced surface delivery of vesicular stomatitis virus protein G-green fluorescent protein. We conclude that GAIP functions as an essential part of the membrane budding machinery for a subset of post-Golgi exocytic carriers derived from the trans -Golgi network. 相似文献
16.
Wu X Zhao X Puertollano R Bonifacino JS Eisenberg E Greene LE 《Molecular biology of the cell》2003,14(2):516-528
We previously demonstrated, using fluorescence recovery after photobleaching, that clathrin in clathrin-coated pits at the plasma membrane exchanges with free clathrin in the cytosol, suggesting that clathrin-coated pits are dynamic structures. We now investigated whether clathrin at the trans-Golgi network as well as the clathrin adaptors AP2 and AP1 in clathrin-coated pits at the plasma membrane and trans-Golgi network, respectively, also exchange with free proteins in the cytosol. We found that when the budding of clathrin-coated vesicle is blocked without significantly affecting the structure of clathrin-coated pits, both clathrin and AP2 at the plasma membrane and clathrin and AP1 at the trans-Golgi network exchange rapidly with free proteins in the cytosol. In contrast, when budding of clathrin-coated vesicles was blocked at the plasma membrane or trans-Golgi network by hypertonic sucrose or K(+) depletion, conditions that markedly affect the structure of clathrin-coated pits, clathrin exchange was blocked but AP2 at the plasma membrane and both AP1 and the GGA1 adaptor at the trans-Golgi network continue to rapidly exchange. We conclude that clathrin-coated pits are dynamic structures with rapid exchange of both clathrin and adaptors and that adaptors are able to exchange independently of clathrin when clathrin exchange is blocked. 相似文献
17.
Robin W. Klemm Christer S. Ejsing Michal A. Surma Hermann-Josef Kaiser Mathias J. Gerl Julio L. Sampaio Quentin de Robillard Charles Ferguson Tomasz J. Proszynski Andrej Shevchenko Kai Simons 《The Journal of cell biology》2009,185(4):601-612
The trans-Golgi network (TGN) is the major sorting station in the secretory pathway of all eukaryotic cells. How the TGN sorts proteins and lipids to generate the enrichment of sphingolipids and sterols at the plasma membrane is poorly understood. To address this fundamental question in membrane trafficking, we devised an immunoisolation procedure for specific recovery of post-Golgi secretory vesicles transporting a transmembrane raft protein from the TGN to the cell surface in the yeast Saccharomyces cerevisiae. Using a novel quantitative shotgun lipidomics approach, we could demonstrate that TGN sorting selectively enriched ergosterol and sphingolipid species in the immunoisolated secretory vesicles. This finding, for the first time, indicates that the TGN exhibits the capacity to sort membrane lipids. Furthermore, the observation that the immunoisolated vesicles exhibited a higher membrane order than the late Golgi membrane, as measured by C-Laurdan spectrophotometry, strongly suggests that lipid rafts play a role in the TGN-sorting machinery. 相似文献
18.
We examined the effects of disassembly of microtubules (MT) on the structure and the functions of the Golgi apparatus (GA) in cultured atrial myocytes. MT disassembly with nocodazole led to fragmentation of the GA into small units. The fragmented Golgi units retained their cis-trans polarity and post-cisternal elements, including the trans-Golgi network (TGN). Neither endocytosis of lectin-labeled membrane nor its delivery to the fragmented Golgi units was interrupted by fragmentation of the GA after MT disassembly with nocodazole treatment. A fraction of the secretory granules associated with the fragmented Golgi units was also labeled with the internalized tracer. These results suggest that in nocodazole-treated cultured atrial myocytes, the fragmented Golgi units appear to be structurally and functionally intact despite the altered geometric arrangement of the GA in the cells. 相似文献
19.
Garver WS Krishnan K Gallagos JR Michikawa M Francis GA Heidenreich RA 《Journal of lipid research》2002,43(4):579-589
The Niemann-Pick C1 (NPC1) protein regulates cholesterol transport from late endosomes-lysosomes to other intracellular compartments. In this article, cholesterol transport to caveolin-1 and caveolin-2 containing compartments, such as the trans-Golgi network (TGN) and plasma membrane caveolae, was examined in normal (NPC+/+), NPC heterozygous (NPC+/-), and NPC homozygous (NPC-/-) human fibroblasts. The expression and distribution of NPC1 in each cell type were similar, and characterized by a finely dispersed, granular staining pattern. The expression of caveolin-1 and caveolin-2 was increased in NPC+/- and NPC-/- fibroblasts, although the distribution in each cell type was similar and characterized by predominant staining of the TGN and plasma membrane. The TGN in NPC+/+ fibroblasts was relatively cholesterol-enriched, whereas the TGN in NPC+/- and NPC-/- fibroblasts was partially or completely cholesterol-deficient, respectively. Consistent with studies demonstrating the transport of cholesterol from the TGN to plasma membrane caveolae, the concentration of cholesterol in plasma membrane caveolae isolated from NPC+/- and NPC-/- fibroblasts was significantly decreased, even though the total concentration of plasma membrane cholesterol in each cell type was similar.These studies demonstrate that NPC1 regulates cholesterol transport to caveolin-1 and caveolin-2 containing compartments such as the TGN and plasma membrane caveolae. 相似文献
20.
Steveson TC Zhao GC Keutmann HT Mains RE Eipper BA 《The Journal of biological chemistry》2001,276(43):40326-40337
Peptidylglycine alpha-amidating monooxygenase (PAM), an integral membrane protein essential for the biosynthesis of amidated peptides, was used to assess the role of cytosolic acidic clusters in trafficking to regulated secretory granules. Casein kinase II phosphorylates Ser(949) and Thr(946) of PAM, generating a short, cytosolic acidic cluster. P-CIP2, a protein kinase identified by its ability to interact with several juxtamembrane determinants in the PAM cytosolic domain, also phosphorylates Ser(949). Antibody specific for phospho-Ser(949)-PAM-CD demonstrates that a small fraction of the PAM-1 localized to the perinuclear region bears this modification. Pituitary cell lines expressing PAM-1 mutants that mimic (TS/DD) or prevent (TS/AA) phosphorylation at these sites were studied. PAM-1 TS/AA yields a lumenal monooxygenase domain that enters secretory granules inefficiently and is rapidly degraded. In contrast, PAM-1 TS/DD is routed to regulated secretory granules more efficiently than wild-type PAM-1 and monooxygenase release is more responsive to secretagogue. Furthermore, this acidic cluster affects exit of internalized PAM-antibody complexes from late endosomes; internalized PAM-1 TS/DD accumulates in a late endocytic compartment instead of the trans-Golgi network. The increased ability of solubilized PAM-1 TS/DD to aggregate at neutral pH may play an important role in its altered trafficking. 相似文献