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1.
Clathrin-coated vesicular transport of secretory proteins during the formation of ACTH-containing secretory granules in AtT20 cells 总被引:50,自引:35,他引:15
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We have studied by electron microscopy and immunocytochemistry the formation of secretory granules containing adrenocorticotropic hormone (ACTH) in murine pituitary cells of the AtT20 line. The first compartment in which condensed secretory protein appears is a complex reticular network at the extreme trans side of the Golgi stacks beyond the TPPase-positive cisternae. Condensed secretory protein accumulates in dilated regions of this trans Golgi network. Examination of en face and serial sections revealed that "condensing vacuoles" are in fact dilations of the trans Golgi network and not detached vacuoles. Only after presumptive secretory granules have reached an advanced stage of morphological maturation do they detach from the trans Golgi network. Frequently both the dilations of the trans Golgi network containing condensing secretory protein and the detached immature granules in the peri-Golgi region have surface coats which were identified as clathrin by immunocytochemistry. Moreover both are the site of budding (or fusion) of coated vesicles, some of which contain condensed secretory protein. The mature granules below the plasma membrane do not, however, have surface coats. Immunoperoxidase labeling with an antiserum specific for ACTH and its precursor polypeptide confirmed that many of the coated vesicles associated with the trans Golgi network contain ACTH. The involvement of the trans Golgi network and coated vesicles in the formation of secretory granules is discussed. 相似文献
2.
Sandra Isabel Vieira Sandra Rebelo Sara Catarina Domingues Edgar F. da Cruz e Silva Odete A. B. da Cruz e Silva 《Molecular and cellular biochemistry》2009,328(1-2):145-154
Cellular protein phosphorylation regulates proteolytic processing of the Alzheimer’s Amyloid Precursor Protein (APP). This appears to occur both indirectly and directly via APP phosphorylation at residues within cytoplasmic motifs related to targeting and protein–protein interactions. The sorting signal 653YTSI656 comprises the S655 residue that can be phosphorylated by PKC, particularly in mature APP molecules. The YTSI domain has been associated with APP internalization and Golgi polarized sorting, but no functional significance has been attributed to S655 phosphorylation thus far. Using APP695-GFP S655 phosphomutants we show that S655 phosphorylation is a signal that positively modulates APP secretory traffic. The phosphomimicking and dephosphomimicking S655 mutants exhibited contrasting Golgi dynamics, which correlated with differential Golgi vesicular exit and secretory cleavage to sAPP. The role of S655 phosphorylation in APP trafficking at sorting stations, such as the Golgi, its contribution toward cytoprotective alpha sAPP production, and implications for Alzheimer’s disease are discussed. 相似文献
3.
Summary Since the advent of green-fluorescent protein (GFP) technology there has been an explosion of interest in applying this molecule
to cell biology. This review summarizes new insights in secretory membrane traffic obtained by the use of GFP fusion proteins.
Transport steps between the endoplasmic reticulum and the Golgi apparatus, intra-Golgi traffic, and transport from the Golgi
to the plasma membrane are discussed. In addition, insights into the dynamics of the Golgi compartment in plant cells and
in mitotic mammalian cells have been included. We conclude that membrane traffic in the secretory pathway appears to be much
more dynamic and diverse than previously thought and that GFP promises to be a powerful means to unravel these complex processes. 相似文献
4.
Since the advent of green-fluorescent protein (GFP) technology there has been an explosion of interest in applying this molecule to cell biology. This review summarizes new insights in secretory membrane traffic obtained by the use of GFP fusion proteins. Transport steps between the endoplasmic reticulum and the Golgi apparatus, intra-Golgi traffic, and transport from the Golgi to the plasma membrane are discussed. In addition, insights into the dynamics of the Golgi compartment in plant cells and in mitotic mammalian cells have been included. We conclude that membrane traffic in the secretory pathway appears to be much more dynamic and diverse than previously thought and that GFP promises to be a powerful means to unravel these complex processes. 相似文献
5.
The Golgi complex plays a central role in protein secretion by regulating cargo sorting and trafficking. As these processes are of functional importance to cell polarity, motility, growth, and division, there is considerable interest in achieving a comprehensive understanding of Golgi complex biology. However, the unique stack structure of this organelle has been a major hurdle to our understanding of how proteins are secreted through the Golgi apparatus. Herein, we summarize available relevant research to gain an understanding of protein secretion via the Golgi complex. This includes the molecular mechanisms of intra-Golgi trafficking and cargo export in the trans-Golgi network. Moreover, we review recent insights on signaling pathways regulated by the Golgi complex and their physiological significance. 相似文献
6.
Protein myristoylation was investigated in the yeast secretory pathway. Conditional secretory mutations were used to accumulate inteRmediaries in the pathway between the endoplasmic reticulum and Golgi (sec 18, 20), within the Golgi (sec 7), and between the Golgi and plasma membrane (sec 1, 3, 4, 5, 6, 8, 9). The accumulation of vesicles was paralleled by the enrichment of a defined subset of proteins modified either via ester or amide linkages to myristic acid: Myristoylated proteins of 21, 32, 49, 56, 75, and 136 kDa were enriched between the endoplasmic reticulum and Golgi; proteins of 21, 32, 45, 56, 75, 136 kDa were enriched by blocks within the Golgi; and proteins of 18, 21, 32, 36, 49, 68, and 136 kDa were trapped in a myristoylated form by blocks between the Golgi and plasma membrane. This enrichment of myristoylated proteins was reversed upon returning the cells to the permissive temperature for secretion. The fatty acid was linked to the 21-kDa protein via a hydroxylamine-resistant amide linkage (N-myristoylation) and to the proteins of 24, 32, 49, 56, 68, 136 kDa via hydroxylamine-labile ester linkage (E-myristoylation). In addition, myristoylated proteins of 21, 56, and 136 kDa were glycosylated via amino linkages to asparagine. This suggests they are exposed to the lumen of the secretory pathway. Three proteins (24, 32, and 56) were E-myristoylated in the presence of protein synthesis inhibitors, indicating this modification can occur posttranslationally. After using cycloheximide to clear protein passengers from the secretory pathway the 21-, 32-, and 56-kDa proteins continued to accumulate in a myristoylated form when vesicular transport was blocked between the Golgi and plasma membrane. These data suggest that myristoylation occurs on a component of the secretory machinery rather than on a passenger protein. 相似文献
7.
Sorting of three secretory proteins to distinct secretory granules in acidophilic cells of cow anterior pituitary 总被引:12,自引:5,他引:7
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The distribution of three proteins discharged by regulated exocytosis--growth hormone (GH), prolactin (PRL), and secretogranin II (SgII)--was investigated by double immunolabeling of ultrathin frozen sections in the acidophilic cells of the bovine pituitary. In mammotrophs, heavy PRL labeling was observed over secretory granule matrices (including the immature matrices at the trans Golgi surface) and also over Golgi cisternae. In contrast, in somatotrophs heavy GH labeling was restricted to the granule matrices; vesicles and tubules at the trans Golgi region showed some and the Golgi cisternae only sparse labeling. All somatotrophs and mammotrophs were heavily positive for GH and PRL, respectively, and were found to contain small amounts of the other hormone as well, which, however, was almost completely absent from granules, and was more concentrated in the Golgi complex, admixed with the predominant hormone. Mixed somatomammotrophs (approximately 26% of the acidophilic cells) were heavily positive for both GH and PRL. Although admixed within Golgi cisternae, the two hormones were stored separately within distinct granule types. A third type of granule was found to contain SgII. Spillage of small amounts of each of the three secretory proteins into granules containing predominantly another protein was common, but true intermixing (i.e., coexistence within single granules of comparable amounts of two proteins) was very rare. It is concluded that in the regulated pathway of acidophilic pituitary, cell mechanisms exist that cause sorting of the three secretory proteins investigated. Such mechanisms operate beyond the Golgi cisternae, possibly at the sites where condensation of secretion products into granule matrices takes place. 相似文献
8.
Pulvirenti T Giannotta M Capestrano M Capitani M Pisanu A Polishchuk RS San Pietro E Beznoussenko GV Mironov AA Turacchio G Hsu VW Sallese M Luini A 《Nature cell biology》2008,10(8):912-922
As with other complex cellular functions, intracellular membrane transport involves the coordinated engagement of a series of organelles and machineries; however, the molecular basis of this coordination is unknown. Here we describe a Golgi-based signalling system that is activated by traffic and is involved in monitoring and balancing trafficking rates into and out of the Golgi complex. We provide evidence that the traffic signal is due to protein chaperones that leave the endoplasmic reticulum and reach the Golgi complex where they bind to the KDEL receptor. This initiates a signalling reaction that includes the activation of a Golgi pool of Src kinases and a phosphorylation cascade that in turn activates intra-Golgi trafficking, thereby maintaining the dynamic equilibrium of the Golgi complex. The concepts emerging from this study should help to understand the control circuits that coordinate high-order cellular functions. 相似文献
9.
The level of diacylglycerol (DAG) in the Golgi apparatus is crucial for protein transport to the plasma membrane. Studies in budding yeast indicate that Sec14p, a phosphatidylinositol (PI)-transfer protein, is involved in regulating DAG homeostasis in the Golgi complex. Here, we show that Nir2, a peripheral Golgi protein containing a PI-transfer domain, is essential for maintaining the structural and functional integrity of the Golgi apparatus in mammalian cells. Depletion of Nir2 by RNAi leads to substantial inhibition of protein transport from the trans-Golgi network to the plasma membrane, and causes a reduction in the DAG level in the Golgi apparatus. Remarkably, inactivation of cytidine [corrected] 5'-diphosphate (CDP)-choline pathway for phosphatidylcholine biosynthesis restores both effects. These results indicate that Nir2 is involved in maintaining a critical DAG pool in the Golgi apparatus by regulating its consumption via the CDP-choline pathway, demonstrating the interface between secretion from the Golgi and lipid homeostasis. 相似文献
10.
Biogenesis of secretory granules 总被引:1,自引:0,他引:1
Secretory granules of neuroendocrine cells store and release peptide hormones and neuropeptides in response to various stimuli. Generation of granules from the Golgi complex involves the aggregation of cargo proteins and their sorting from non-regulated secretory molecules. Recent findings on knockout mice lacking individual granule constituents have challenged the hypothesis that an 'essential' protein for the assembly of these organelles exists, while studies on polypyrimidine tract-binding protein and ICA512/IA-2 have provided insight into the mechanisms for adjusting granule production in relation to stimulation and secretory activity. 相似文献
11.
Newly synthesized proteins destined for delivery to the cell surface are inserted cotranslationally into the endoplasmic
reticulum (ER) and, after their correct folding, are transported out of the ER. During their transport to the cell surface,
cargo proteins pass through the various cisternae of the Golgi apparatus and, in the trans-most cisternae of the stack, are sorted into constitutive secretory vesicles that fuse with the plasma membrane. Simultaneously
with anterograde protein transport, retrograde protein transport occurs within the Golgi complex as well as from the Golgi
back to the ER. Vesicular transport within the early secretory pathway is mediated by two types of non-clathrin coated vesicles:
COPI- and COPII-coated vesicles. The formation of these carrier vesicles depends on the recruitment of cytosolic coat proteins
that are thought to act as a mechanical device to shape a flattened donor membrane into a spherical vesicle. A general molecular
machinery that mediates targeting and fusion of carrier vesicles has been identified as well. Beside a general overview of
the various coat structures known today, we will discuss issues specifically related to the biogenesis of COPI-coated vesicles:
(1) a possible role of phospholipase D in the formation of COPI-coated vesicles; (2) a functional role of a novel family of
transmembrane proteins, the p24 family, in the initiation of COPI assembly; and (3) the direction COPI-coated vesicles may
take within the early secretory pathway. Moreover, we will consider two alternative mechanisms of protein transport through
the Golgi stack: vesicular transport versus cisternal maturation.
Accepted: 24 October 1997 相似文献
12.
The physiology of protein intracellular transport and secretion by cell types thought to be free from short-term control has been compared with that of the pancreatic acinar cell, using pulse-chase protocols to follow biosynthetically-labeled secretory products. Data previously obtained (Tartakoff, A.M., and P. Vassalli. J. Exp. Med. 146:1332-1345) has shown that plasma-cell immunoglobulin (Ig) secretion is inhibited by respiratory inhibitors, by partial Na/K equilibration effected by the carboxylic ionophore monensin, and by calcium withdrawal effected by the carboxylic ionophore A 23187 in the presence of ethylene glycol bis (beta-aminoethylether)-N,N,N'',N''-tetraacetic acid (EGTA) and absence of calcium. We report here that both inhibition of respiration and treatment with monensin slow secretion by fibroblasts, and also macrophages and slow intracellular transport (though not discharge per se) by the exocrine pancreatic cells. Attempted calcium withdrawal is inhibitory for fibroblasts but not for macrophages. The elimination of extracellular calcium or addition of 50 mM KCl has no major effect on secretory rate of either fibroblasts or macrophages. Electron microscopic examination of all cell types shows that monensin causes a rapid and impressive dilation of Golgi elements. Combined cell fractionation and autoradiographic studies of the pancreas show that the effect of monensin is exerted at the point of the exit of secretory protein from the Golgi apparatus. Other steps in intracellular transport proceed at normal rates. These observations suggest a common effect of the cytoplasmic Na/K balance at the Golgi level and lead to a model of intracellular transport in which secretory product obligatorily passes through Golgi elements (cisternae?) that are sensitive to monensin. Thus, intracellular transport follows a similar course in both regulated and nonregulated secretory cells up to the level of distal Golgi elements. 相似文献
13.
Towler MC Prescott AR James J Lucocq JM Ponnambalam S 《Experimental cell research》2000,259(1):167-179
The endoplasmic reticulum and Golgi apparatus play key roles in regulating the folding, assembly, and transport of newly synthesized proteins along the secretory pathway. We find that the divalent cation manganese disrupts the Golgi apparatus and endoplasmic reticulum (ER). The Golgi apparatus is fragmented into smaller dispersed structures upon manganese treatment. Golgi residents, such as TGN46, beta1,4-galactosyltransferase, giantin, and GM130, are still segregated and partitioned correctly into smaller stacked fragments in manganese-treated cells. The mesh-like ER network is substantially affected and peripheral ER elements are collapsed. These effects are consistent with manganese-mediated inhibition of motor proteins that link membrane organelles along the secretory pathway to the cytoskeleton. This divalent cation thus represents a new tool for studying protein secretion and membrane dynamics along the secretory pathway. 相似文献
14.
The early secretory pathway (ESP) consisting of the endoplasmic reticulum (ER), pre-Golgi intermediates and the Golgi stack links protein synthesis to folding and vesicle trafficking to generate the membrane architecture of the eukaryotic cell. The fundamental principles that contribute to organization of the ESP remain largely unknown. We raise the possibility that assembly of the ESP is largely built on a foundation that is influenced by the kinetic and thermodynamic properties of the protein fold. Folding energetics may provide an adjustable platform for adaptor-dependent interactions with the transport machinery, suggesting the possibility that protein cargo energetics plays a central role in directing both trafficking patterns and global compartmental organization of the ESP. In this view, cargo energetics likely coordinates the composition and maturation of ER and Golgi compartments with the physiological state of the cell in different tissue and environmental settings. 相似文献
15.
A. I. Fokin I. B. Brodsky A. V. Burakov E. S. Nadezhdina 《Biochemistry. Biokhimii?a》2014,79(9):879-893
This review summarizes the data describing the role of cellular microtubules in transportation of membrane vesicles — transport containers for secreted proteins or lipids. Most events of early vesicular transport in animal cells (from the endoplasmic reticulum to the Golgi apparatus and in the opposite recycling direction) are mediated by microtubules and microtubule motor proteins. Data on the role of dynein and kinesin in early vesicle transport remain controversial, probably because of the differentiated role of these proteins in the movements of vesicles or membrane tubules with various cargos and at different stages of secretion and retrograde transport. Microtubules and dynein motor protein are essential for maintaining a compact structure of the Golgi apparatus; moreover, there is a set of proteins that are essential for Golgi compactness. Dispersion of ribbon-like Golgi often occurs under physiological conditions in interphase cells. Golgi is localized in the leading part of crawling cultured fibroblasts, which also depends on microtubules and dynein. The Golgi apparatus creates its own system of microtubules by attracting γ-tubulin and some microtubule-associated proteins to membranes. Molecular mechanisms of binding microtubule-associated and motor proteins to membranes are very diverse, suggesting the possibility of regulation of Golgi interaction with microtubules during cell differentiation. To illustrate some statements, we present our own data showing that the cluster of vesicles induced by expression of constitutively active GTPase Sar1a[H79G] in cells is dispersed throughout the cell after microtubule disruption. Movement of vesicles in cells containing the intermediate compartment protein ERGIC53/LMANI was inhibited by inhibiting dynein. Inhibiting protein kinase LOSK/SLK prevented orientation of Golgi to the leading part of crawling cells, but the activity of dynein was not inhibited according to data on the movement of ERGIC53/LMANI-marked vesicles. 相似文献
16.
Quantitative proteomics analysis of the secretory pathway 总被引:11,自引:0,他引:11
Gilchrist A Au CE Hiding J Bell AW Fernandez-Rodriguez J Lesimple S Nagaya H Roy L Gosline SJ Hallett M Paiement J Kearney RE Nilsson T Bergeron JJ 《Cell》2006,127(6):1265-1281
We report more than 1400 proteins of the secretory-pathway proteome and provide spatial information on the relative presence of each protein in the rough and smooth ER Golgi cisternae and Golgi-derived COPI vesicles. The data support a role for COPI vesicles in recycling and cisternal maturation, showing that Golgi-resident proteins are present at a higher concentration than secretory cargo. Of the 1400 proteins, 345 were identified as previously uncharacterized. Of these, 230 had their subcellular location deduced by proteomics. This study provides a comprehensive catalog of the ER and Golgi proteomes with insight into their identity and function. 相似文献
17.
The secretory pathway is important in actively transporting proteins into the extracellular environment of eucaryotic cells. In this study a green fluorescent protein (GFP) mutant engineered to contain a secretion signal was used as a model protein in order to visualize the secretion process inside insect cells. Fluorescent microscopy indicated that significant amounts of secreted green fluorescent protein (sGFP) accumulated in High-Five, Trichoplusia ni, cells following infection with a baculovirus vector containing the gene under the polyhedrin promoter. Laser scanning confocal microscopy was used to reconstruct whole cell images of the infected High-Five cells at multiple days postinfection. While the protein was widely distributed at 2 days postinfection, certain intracellular regions appeared to contain higher or lower concentrations of the sGFP. A layer by layer examination indicated pockets in which sGFP was absent, and these appear to be vesicles that have recently released the sGFP or are not yet accumulating sGFP. By 3 days postinfection, the sGFP in some cells was concentrated in a number of widely dispersed globules, which may represent the vesicle remnants of a deteriorating secretory pathway. In contrast, nonsecreted GFP was more uniformly distributed in the cells than sGFP and did not accumulate in vesicles. In addition to GFP, the lectins wheat germ agglutinin (WGA) and concanavalin A (ConA), which have affinities for sugar residues, were used to examine the secretory pathway. The WGA, which is a Golgi marker, was distributed around the nucleus prior to infection but then was found to be polarized in one region of the cell following the baculovirus infection. The expansion of other cellular compartments following the baculovirus infection may have caused a change in intracellular distribution of the Golgi. While some of the sGFP was found to colocalize with the WGA label, much of the sGFP was outside this Golgi region. In contrast, ConA labeling, which was not as specific as WGA, was found throughout the cell both before and after infection similar to the sGFP distribution. These studies demonstrate that confocal visualization of fluorescent proteins can be used as an in vivo tool for examining secretory processing in insect cells. 相似文献
18.
M. S. Vildanova M. A. Savitskaya G. E. Onishchenko E. A. Smirnova 《Cell and Tissue Biology》2014,8(5):407-415
Plant hormones play a key role in plant growth and differentiation. Certain plant hormones are known to be potential antitumor agents, affect the secretory activity of animal cells, and are produced by mammalian cells as proinflammatory cytokines. The goal of this research was to study the effect of abscisic and gibberellic acids on the secretory system of human epidermoid A431 carcinoma cells and HaCaT keratinocytes. Immunocytochemical and morphometric analysis showed that a subtoxic concentration of abscisic and gibberellic acids induced extension of the ER network and increased the size of the Golgi complex. Electron-microscope studies confirmed the hypertrophic changes of the Golgi complex: swelling of cisternae in the trans-Golgi compartment after exposure to abscisic acid and swelling of cis- and trans-compartments after exposure to gibberellic acid. The Click-iT technique revealed elevation of total protein synthesis only in A431 cells exposed to abscisic acid. Our data suggest that the hypertrophy of Golgi may reflect enhanced secretory activity in A431 cells exposed to abscisic acid. In other experiments, Golgi hypertrophy was not accompanied with increased protein synthesis that suggested the stress-related changes of ER and Golgi complex. Our results demonstrate that morphologically similar reaction manifested in hypertrophy of Golgi complex, in response to plant hormones, is the result of different functional activities, and that molecular mechanisms underlying induced changes need further investigations. 相似文献
19.
Cruz-García D Díaz-Ruiz A Rabanal-Ruiz Y Peinado JR Gracia-Navarro F Castaño JP Montero-Hadjadje M Tonon MC Vaudry H Anouar Y Vázquez-Martínez R Malagón MM 《The Biochemical journal》2012,443(2):387-396
Golgi-associated long coiled-coil proteins, often referred to as golgins, are involved in the maintenance of the structural organization of the Golgi apparatus and the regulation of membrane traffic events occurring in this organelle. Little information is available on the contribution of golgins to Golgi function in cells specialized in secretion such as endocrine cells or neurons. In the present study, we characterize the intracellular distribution as well as the biochemical and functional properties of a novel long coiled-coil protein present in neuroendocrine tissues, NECC1 (neuroendocrine long coiled-coil protein 1). The present study shows that NECC1 is a peripheral membrane protein displaying high stability to detergent extraction, which distributes across the Golgi apparatus in neuroendocrine cells. In addition, NECC1 partially localizes to post-Golgi carriers containing secretory cargo in PC12 cells. Overexpression of NECC1 resulted in the formation of juxtanuclear aggregates together with a slight fragmentation of the Golgi and a decrease in K+-stimulated hormone release. In contrast, NECC1 silencing did not alter Golgi architecture, but enhanced K+-stimulated hormone secretion in PC12 cells. In all, the results of the present study identify NECC1 as a novel component of the Golgi matrix and support a role for this protein as a negative modulator of the regulated trafficking of secretory cargo in neuroendocrine cells. 相似文献
20.
Bubeck J Scheuring D Hummel E Langhans M Viotti C Foresti O Denecke J Banfield DK Robinson DG 《Traffic (Copenhagen, Denmark)》2008,9(10):1629-1652
Overexpression of the Golgi and endoplasmic reticulum (ER) syntaxins SYP31 and SYP81 strongly inhibits constitutive secretion. By comparing the secreted reporter alpha-amylase with the ER-retained reporter alpha-amylase-HDEL, it was concluded that SYP81 overexpression inhibits both retrograde and anterograde transport, while SYP31 overexpression mainly affected anterograde transport. Of the other interacting SNAREs investigated, only the overexpression of MEMB11 led to an inhibition of protein secretion. Although the position of a fluorescent tag does not influence the correct localization of the fusion protein, only N-terminal-tagged SYP31 retained the ability of the untagged SNARE to inhibit transport. C-terminal-tagged SYP31 failed to exhibit this effect. Overexpression of both wild-type and N-terminal-tagged syntaxins caused standard Golgi marker proteins to redistribute into the ER. Nevertheless, green fluorescent protein (GFP)-SYP31 was still visible as fluorescent punctae, which, unlike SYP31-GFP, were resistant to brefeldin A treatment. Immunogold electron microscopy showed that endogenous SYP81 is not only present at the ER but also in the cis Golgi, indicating that this syntaxin cycles between these two organelles. However, when expressed at non-inhibitory levels, YFP-SYP81 was seen to locate principally to subdomains of the ER. These punctate structures were physically separated from the Golgi, suggesting that they might possibly reflect the position of ER import sites. 相似文献