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1.
Dissociation of a 110-kD peripheral membrane protein from the Golgi apparatus is an early event in brefeldin A action 总被引:17,自引:8,他引:9
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《The Journal of cell biology》1990,111(6):2295-2306
Brefeldin A (BFA) has a profound effect on the structure of the Golgi apparatus, causing Golgi proteins to redistribute into the ER minutes after drug treatment. Here we describe the dissociation of a 110-kD cytoplasmically oriented peripheral membrane protein (Allan, V. J., and T. E. Kreis. 1986. J. Cell Biol. 103:2229-2239) from the Golgi apparatus as an early event in BFA action, preceding other morphologic changes. In contrast, other peripheral membrane proteins of the Golgi apparatus were not released but followed Golgi membrane into the ER during BFA treatment. The 110-kD protein remained widely dispersed throughout the cytoplasm during drug treatment, but upon removal of BFA it reassociated with membranes during reformation of the Golgi apparatus. Although a 30-s exposure to the drug was sufficient to cause the redistribution of the 110-kD protein, removal of the drug after this short exposure resulted in the reassociation of the 110-kD protein and no change in Golgi structure. If cells were exposed to BFA for 1 min or more, however, a portion of the Golgi membrane was committed to move into and out of the ER after removal of the drug. ATP depletion also caused the reversible release of the 110-kD protein, but without Golgi membrane redistribution into the ER. These findings suggest that the interaction between the 110-kD protein and the Golgi apparatus is dynamic and can be perturbed by metabolic changes or the drug BFA. 相似文献
2.
Brefeldin A (BFA) causes rapid redistribution of Golgi proteins into the endoplasmic reticulum (ER), leaving no definable Golgi-apparatus, and blocks transport of proteins from the ER to distal secretory compartments of the cell. Using pulse-chase experiments the present study shows that BFA (1 microgram/ml) inhibits basal and CCK-stimulated protein secretion in isolated pancreatic acinar cells by 65 +/- 6% and 84 +/- 5%, respectively. In isolated permeabilized cells higher concentrations of BFA (30 micrograms/ml) were necessary to obtain inhibition of protein secretion. In parallel experiments protein secretion was stimulated by GTP (1 mM). BFA had no inhibitory effect on protein secretion in the presence of GTP, indicating that BFA might act on a GTP-binding protein. Investigating the effect of BFA on small molecular weight GTP-binding proteins we observed that [alpha-32P]GTP binding to a 21 kDa protein in a subcellular fraction enriched in ER was increased in the presence of BFA. We conclude that this 21 kDa and possibly also other GTP-binding proteins may be the molecular target of Brefeldin A in pancreatic acinar cells. 相似文献
3.
A novel 115-kD peripheral membrane protein is required for intercisternal transport in the Golgi stack 总被引:6,自引:0,他引:6
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We have used an in vitro Golgi protein transport assay dependent on high molecular weight (greater than 100 kD) cytosolic and/or peripheral membrane proteins to study the requirements for transport from the cis- to the medial-compartment. Fractionation of this system indicates that, besides the NEM-sensitive fusion protein (NSF) and the soluble NSF attachment protein (SNAP), at least three high molecular weight protein fractions from bovine liver cytosol are required. The activity from one of these fractions was purified using an assay that included the second and third fractions in a crude state. The result is a protein of 115-kD subunit molecular mass, which we term p115. Immunodepletion of the 115-kD protein from a purified preparation with mAbs removes activity. Peptide sequence analysis of tryptic peptides indicates that p115 is a "novel" protein that has not been described previously. Gel filtration and sedimentation analysis indicate that, in its native state, p115 is a nonglobular homo-oligomer. p115 is present on purified Golgi membranes and can be extracted with high salt concentration or alkaline pH, indicating that it is peripherally associated with the membrane. Indirect immunofluorescence indicates that p115 is associated with the Golgi apparatus in situ. 相似文献
4.
Golgi apparatus dynamics during mouse oocyte in vitro maturation: effect of the membrane trafficking inhibitor brefeldin A 总被引:1,自引:0,他引:1
We have studied Golgi apparatus dynamics during mouse oocyte in vitro maturation, employing both live imaging with the fluorescent lipid BODIPY-ceramide and immunocytochemistry using several specific markers (beta-COP, giantin, and TGN38). In germinal vesicle oocytes the Golgi consisted of a series of structures, possibly cisternal stacks, dispersed in the ooplasm, but slightly more concentrated in the interior than at the cortex. A similar pattern was detected in rhesus monkey germinal vesicle oocytes. These "mini-Golgis" were functionally active because they were reversibly disrupted by the membrane trafficking inhibitor brefeldin A. However, the drug had no visible effect if the oocytes had been previously microinjected with GTP-gamma-S. During in vitro maturation the large Golgi apparatus structures fragmented at germinal vesicle breakdown, and dispersed homogenously throughout the ooplasm, remaining in a fragmented state in metaphase-II oocytes. Similarly to what has been reported using protein synthesis inhibitors, the presence of brefeldin A blocked maturation at the germinal vesicle breakdown stage before the assembly of the metaphase-I spindle. These results suggest that progression of murine oocyte maturation may require functional membrane trafficking. 相似文献
5.
Association of a dynamin-like protein with the Golgi apparatus in mammalian cells 总被引:14,自引:4,他引:10
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《The Journal of cell biology》1996,133(4):761-775
Dynamins are a family of 100-kD GTPases comprised of at least three distinct gene products and multiple alternatively spliced variants. Homologies with the shibire gene product in Drosophila melanogaster and with Vps1p and Dnm1p in Saccharomyces cerevisiae suggest that dynamins play an important role in vesicular transport. Morphological studies have localized brain dynamin to coated pits and tubular invaginations at the plasma membrane, where it is believed to facilitate the formation of endocytic vesicles. Because similar membrane-budding events occur at the Golgi apparatus and multiple dynamin isoforms exist, we have studied the distribution of dynamins in mammalian cells. To this end, we generated and characterized peptide-specific antibodies directed against conserved regions of the dynamin family. By immunoblot analysis, these antibodies reacted specifically with a 100-kD protein in fibroblasts that sedimented with membranes and microtubules in vitro in a manner similar to brain dynamin. By immunofluorescence microscopy, these antibodies strongly labeled the Golgi complex in cultured fibroblasts and melanocytes, as confirmed by double labeling with a Golgi-specific antibody. Furthermore, Western blot analysis showed significant enrichment of a 100-kD dynamin band in Golgi fractions isolated from the liver. To substantiate these findings, we use a specific antidynamin antibody to immunoisolate Golgi membranes from subcellular Golgi fractions, as determined by EM and immunoblot analysis. This study provides the first morphological and biochemical evidence that a dynamin-like protein associates with the Golgi apparatus in mammalian cells, and suggests that dynamin-related proteins may have multiple cytoplasmic distributions. The potential contributions of dynamin to the secretory and endocytic pathways are discussed. 相似文献
6.
Rapid redistribution of Golgi proteins into the ER in cells treated with brefeldin A: evidence for membrane cycling from Golgi to ER 总被引:194,自引:0,他引:194
In cells treated with brefeldin A (BFA), movement of newly synthesized membrane proteins from the endoplasmic reticulum (ER) to the Golgi apparatus was blocked. Surprisingly, the glycoproteins retained in the ER were rapidly processed by cis/medial Golgi enzymes but not by trans Golgi enzymes. An explanation for these observations was provided from morphological studies at both the light and electron microscopic levels using markers for the cis/medial and trans Golgi. They revealed a rapid and dramatic redistribution to the ER of components of the cis/medial but not the trans Golgi in response to treatment with BFA. Upon removal of BFA, the morphology of the Golgi apparatus was rapidly reestablished and proteins normally transported out of the ER were efficiently and rapidly sorted to their final destinations. These results suggest that BFA disrupts a dynamic membrane-recycling pathway between the ER and cis/medial Golgi, effectively blocking membrane transport out of but not back to the ER. 相似文献
7.
PtK1 cells contain a nondiffusible, dominant factor that makes the Golgi apparatus resistant to brefeldin A 总被引:14,自引:5,他引:9
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Brefeldin A (BFA) was shown in earlier studies of numerous cell types to inhibit secretion, induce enzymes of the Golgi stacks to redistribute into the ER, and to cause the Golgi cisternae to disappear. Here, we demonstrate that the PtK1 line of rat kangaroo kidney cells is resistant to BFA. The drug did not disrupt the morphology of the Golgi complex in PtK1 cells, as judged by immunofluorescence using antibodies to 58- (58K) and 110-kD (beta-COP) Golgi proteins, and by fluorescence microscopy of live cells labeled with C6-NBD-ceramide. In addition, BFA did not inhibit protein secretion, not alter the kinetics or extent of glycosylation of the vesicular stomatitis virus (VSV) glycoprotein (G-protein) in VSV-infected PtK1 cells. To explore the mechanism of resistance to BFA, PtK1 cells were fused with BFA-sensitive CV-1 cells that had been infected with a recombinant SV-40 strain containing the gene for VSV G-protein and, at various times following fusion, the cultures were exposed to BFA. Shortly after cell fusion, heterokaryons contained one Golgi complex associated with each nucleus. Golgi membranes derived from CV-1 cells were sensitive to BFA, whereas those of PtK1 origin were BFA resistant. A few hours after fusion, most heterokaryons contained a single, large Golgi apparatus that was resistant to BFA and contained CV-1 galactosyltransferase. In unfused cells that had been perforated using nitrocellulose filters, retention of beta-COP on the Golgi was optimal in the presence of cytosol, ATP, and GTP. In perforated cell models of the BFA-sensitive MA104 line, BFA caused beta-COP to be released from the Golgi complex in the presence of nucleotides, and either MA104 or PtK1 cytosol. In contrast, when perforated PtK1 cells were incubated with BFA, nucleotides, and cytosol from either cell type, beta-COP remained bound to the Golgi complex. We conclude that PtK1 cells contain a nondiffusible factor, which is located on or very close to the Golgi complex, and confers a dominant resistance to BFA. It is possible that this factor is homologous to the target of BFA in cells that are sensitive to the drug. 相似文献
8.
Dairman Matthew; Donofrio Nicole; Domozych David S. 《Journal of experimental botany》1995,46(2):181-186
The fungal-derived derivative, brefeldin A, was used to disruptthe Golgi apparatus (GA) of the green alga, Gloeomonas kupfferi.Upon short treatments (10 µg ml1 for 10 min orless), the Golgi bodies maintain their perinuclear positioning.However, the medial locus transforms from a tight stack of elongatecisternae to a network of swollen tubules. Upon longer treatments(60 min), swelling and vesiculation of cis face cisternae becomeapparent. Likewise, the edges of several trans face cisternaemay fuse with those of adjacent Golgi bodies leading to theformation of multiGolgi complexes. Key words: Brefeldin A, Golgi apparatus, Gloeomonas kupfferi 相似文献
9.
Incubation of a highly purified fraction derived from rat liver Golgi apparatus with [gamma-32P]ATP results in phosphorylation of several endogenous phosphoproteins. One phosphoprotein with an apparent Mr of 48,300 is radiolabeled to an apparent extent at least 5-fold higher than any other phosphoprotein as part of either the Golgi apparatus or highly purified rat liver fractions derived from the rough endoplasmic reticulum, mitochondria, plasma membrane, coated vesicles, cytosol, and total homogenate. Approximately 70% of the 48.3-kDa phosphoprotein appears to be a specific extrinsic Golgi membrane protein with the phosphorylated amino acid being threonine. The protein kinase which phosphorylates the 48.3-kDa protein is an intrinsic Golgi membrane protein and is dependent on Mg2+, independent of Ca2+, calmodulin, and cAMP, and is inhibited by N-ethylmaleimide. Preliminary evidence suggests that there are also intrinsic membrane protein kinases in the Golgi apparatus which are dependent on Ca2+ and cAMP. The physiological role of the above phosphoproteins and protein kinases is not known. 相似文献
10.
High-pressure freezing and freeze-substitution were used to study Golgi ultrastructure and its brefeldin A-induced transformations in HepG2 human hepatoma cells. Cryoimmobilization arrested subcellular dynamics within milliseconds, thus considerably improving the temporal resolution in monitoring the very early effects of high brefeldin concentrations at the ultrastructural level (i.e., 20 microg/ml brefeldin applied for 35 s to 8 min). Moreover, this approach ruled out possible cumulative and/or synergistic effects of the drug and fixatives. Several findings differed from studies based on chemical fixation. In particular, Golgi breakdown did not proceed gradually but occurred in distinct steps. We found a conspicuous lag between the absence of nonclathrin coats on Golgi membranes after 30 s of brefeldin treatment and the disassembly of the stacks, which did not start until after 90 to 120 s. At this time, domains at the trans and cis faces separated from the stacks, starting tubulation and fragmentation. After 3-5 min the Golgi apparatus was completely replaced by loose meshworks of straight tubules of different sizes and staining properties; also frequent were bent tubules and vesicles forming glomerule-like structures. After 8 min all kinds of Golgi-derived structures had aggregated within huge clusters. The morphologically highly distinct structures found after brefeldin treatment could in part be correlated with particular Golgi domains in the control cells. 相似文献
11.
Lifen Wang Yi Zhan Eli Song Yong Yu Yaming Jiu Wen Du Jingze Lu Pingsheng Liu Pingyong Xu Tao Xu 《蛋白质与细胞》2011,2(1):74
12.
Identification of a 52-kD calmodulin-binding protein associated with the mitotic spindle apparatus in mammalian cells 总被引:2,自引:1,他引:1
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A pool of 10 calmodulin-binding proteins (CBPs) was isolated from Chinese hamster ovary (CHO) cells via calmodulin (CaM)-Sepharose affinity chromatography. One of these ten isolated CBPs with a molecular mass of 52 kD was also found to be present in isolated CHO cell mitotic spindles. Affinity-purified antibodies generated against this pool of isolated CBPs recognize a single 52-kD protein in isolated CHO cell mitotic spindles by immunoblot analysis. Immunofluorescence examination of CHO, 3T3, NRK, PTK-2, and HeLa cells resulted in a distinct pattern of mitotic spindle fluorescence. The localization pattern of this 52-kD CBP directly parallels that of CaM in the spindle apparatus throughout the various stages of mitosis. Interestingly, there was no association of this 52-kD CBP with cytoplasmic microtubules. As is the case with CaM, the localization pattern of the 52-kD CBP in interphase cells is diffuse within the cytoplasm and is not associated with any discrete, cellular structures. This 52-kD CBP appears to represent the first mitotic spindle-specific calmodulin-binding protein identified and represents an initial step toward the ultimate determination of CaM function in the mitotic spindle apparatus. 相似文献
13.
Disruption of the Golgi apparatus with brefeldin A does not destabilize the associated detyrosinated microtubule network. 总被引:2,自引:0,他引:2
Stable subsets of microtubules (MTs) are often enriched in detyrosinated alpha-tubulin. Recently it has been found that the Golgi apparatus is associated with a subset of relatively stable MTs and that detyrosinated MTs colocalize spatially and temporally with the Golgi apparatus in several cell lines. To determine whether the Golgi apparatus actively stabilizes associated MTs and thus allows their time-dependent detyrosination, we have used the drug brefeldin A (BFA) to disrupt the Golgi apparatus and have monitored changes in the Golgi apparatus and MT populations using simultaneous immunofluorescence and fluorescent lectin microscopy. We found that although BFA caused the Golgi apparatus to completely redistribute to the endoplasmic reticulum (ER), the detyrosinated MTs were not disrupted and remained in a juxtanuclear region. By Western blot analysis we found that even after 6 h of continuous exposure of cells to BFA, there was no detectable reduction in the level of detyrosinated alpha-tubulin. Simultaneous treatment with nocodazole and BFA led to a complete disruption of all MTs and normal Golgi structure/organization. Upon removal of nocodazole in the continued presence of BFA, we found that the detyrosinated MTs reformed in a compact juxtanuclear location in the absence of an intact Golgi complex. Finally, we found that the detyrosinated MTs colocalized precisely with a BFA-resistant structure that binds to the lectin, wheat germ agglutinin. We conclude that the juxtanuclear detyrosinated MTs are not actively stabilized by association with BFA-sensitive Golgi membranes. However, another closely associated structure which binds wheat germ agglutinin may serve to stabilize the juxtanuclear MTs. Alternatively, the MT organizing center (MTOC) and/or MT-associated proteins (MAPs) may organize and stabilize the juxtanuclear detyrosinated MTs. 相似文献
14.
Recruitment of coat proteins onto Golgi membranes in intact and permeabilized cells: effects of brefeldin A and G protein activators. 总被引:58,自引:0,他引:58
Brefeldin A (BFA) causes a rapid redistribution of coat proteins (e.g., gamma-adaptin) associated with the clathrin-coated vesicles that bud from the trans-Golgi network (TGN), while the clathrin-coated vesicles that bud from the plasma membrane are unaffected. gamma-Adaptin redistributes with the same kinetics as beta-COP, a coat protein associated with the non-clathrin-coated vesicles that bud from the Golgi complex. Upon removal of BFA, however, gamma-adaptin recovers its perinuclear distribution more rapidly. Redistribution of both proteins can be prevented by pretreating cells with AlF4-. Recruitment of adaptors from the cytosol onto the TGN membrane has been reconstituted in a permeabilized cell system and is increased by addition of GTP gamma S and blocked by addition of BFA. These results suggest a role for G proteins in the control of the clathrin-coated vesicle cycle at the TGN and further extend the similarities between clathrin-coated vesicles and non-clathrin-coated vesicles. 相似文献
15.
《The Journal of cell biology》1986,103(6):2229-2239
A monoclonal antibody (M3A5), raised against microtubule-associated protein 2 (MAP-2), recognized an antigen associated with the Golgi complex in a variety of non-neuronal tissue culture cells. In double immunofluorescence studies M3A5 staining was very similar to that of specific Golgi markers, even after disruption of the Golgi apparatus organization with monensin or nocodazole. M3A5 recognized one band of Mr approximately 110,000 in immunoblots of culture cell extracts; this protein, designated 110K, was enriched in Golgi stack fractions prepared from rat liver. The 110K protein has been shown to partition into the aqueous phase by Triton X-114 extraction of a Golgi-enriched fraction and was eluted after pH 11.0 carbonate washing. It is therefore likely to be a peripheral membrane protein. Proteinase K treatment of an isolated Golgi stack fraction resulted in complete digestion of the 110K protein, both in the presence and absence of Triton X-100. A the 110K protein is accessible to protease in intact vesicles in vitro, it is presumably located on the cytoplasmic face of the Golgi membrane in vivo. The 110K protein was able to interact specifically with taxol-polymerized microtubules in vitro. These results suggest that the 110K protein may serve to link the Golgi apparatus to the microtubule network and so may belong to a novel class of proteins: the microtubule-binding proteins. 相似文献
16.
Summary We have followed the action of brefeldin A (BFA) on the Golgi apparatus of developing pea cotyledons, the cells of which are actively engaged in the synthesis and deposition of storage proteins. The Golgi apparatus of normal cells is characterized by the presence of three different types of vesicle: smooth-surfaced secretory vesicles, dense vesicles which carry the storage proteins, and clathrin-coated vesicles (CCV). The dense vesicles originate at the cis cisternae and undergo a maturation as they pass through the Golgi stack, presumably as a result of cisternal progression. CCV bud off from dense and smooth vesicles, which may be attached to one another, at the trans pole of the Golgi apparatus. BFA eliminates the CCV and leads, initially, to an increase in the number and length of the cisternae. Dense vesicles are still to be seen, and many show an increase in diameter. Longer BFA treatments result in a trans-driven vesiculation and an accumulation of vesicles within the vicinity of single cisternae. The vesicles were sometimes seen to be connected to one another via a network of tubules. As judged by immunocytochemistry with gold-coupled legumin and vicilin antisera, some of the dilated vesicles originate directly from dense vesicles by swelling whereas others probably arise by dilation of Golgi cisternae since they possess a layer of flocculent storage proteins at their periphery. By contrast the centre of the dilated vesicles labels positively with antibodies against complex glycans, indicating that the ability to segregate storage proteins from cell wall or lytic vacuole glycoproteins is lost during extended BFA treatment. The effects of BFA are reversible when cotyledons are further incubated on Gamborg's medium for 5 h without the inhibitor.Dedicated to Professor R. Kollmann on the occasion of his 65th birthday. 相似文献
17.
The mucilage-secreting desmid, Closterium acerosum, is
sensitive to the secretory inhibiting drug, brefeldin A (BFA). After 5 min
of treatment with 5 g
ml-1 of BFA, the Golgi body displays the following
alterations: the number of cisternae decreases from 12-15 to 6-7;
peripheries of cisternae from the same Golgi body often fuse to yield
unique profiles; secretory vesicles still merge from the Golgi body; the
cisternal stack dissociates to form irregular masses in the alleys of
cytoplasm created by the lobes of the chloroplast. Fluoresbrite bead
labelling shows that mucilage production ceases in cells treated with BFA
even after only 5 min of treatment. Immunogold labelling using
anti-mucilage antibody reveals that mucilage production still occurs in the
Golgi body and associated vesicles. Helix pomatia
lectin-gold labelling shows that wall synthesis still occurs in BFA-treated
Golgi bodies and wall precursors accumulate in the perforate
cisternal/vesicular masses seen in the TGN region of the Golgi
stack. 相似文献
18.
Identification, by a monoclonal antibody, of a 53-kD protein associated with a tubulo-vesicular compartment at the cis-side of the Golgi apparatus 总被引:72,自引:45,他引:27
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A Schweizer J A Fransen T B?chi L Ginsel H P Hauri 《The Journal of cell biology》1988,107(5):1643-1653
Purified Golgi membranes of the human intestinal adenocarcinoma cell line Caco-2 were used as an antigen to produce a monoclonal antibody, G1/93, which specifically labels a tubulovesicular compartment near the cis side of the Golgi apparatus, including the first cis-cisterna itself, as visualized by single and double immunoelectron microscopy with antibodies against galactosyltransferase. The antigen recognized by G1/93 was identified as a protein with a subunit size of 53 kD. Pulse-chase experiments revealed that the 53-kD protein dimerizes immediately after synthesis followed by formation of oligomers of approximately 310 kD, probably homohexamers. The protein has a transmembrane topology with only a short cytoplasmic segment as assessed by protease protection experiments. Glycosidase digestion studies indicated that the protein is probably not glycosylated. The unique subcellular distribution of the G1/93 antigen in close vicinity to the cis-Golgi is in line with the notion that this protein may delineate the biosynthetic transport pathway from the endoplasmic reticulum to the Golgi apparatus. Moreover, G1/93 is a useful marker to identify the cis side of the Golgi apparatus in a variety of human cells. 相似文献
19.
A Golgi apparatus-rich fraction and a plasma membrane-rich fraction were isolated from a common homogenate of rat liver. Their respective buovant densities, appearances in the electron microscope and 5'-nucleotidase and UDP-galactose ovalbumin galactosyltransferase activities were in accord with published data on separately isolated Golgi apparatus-rich and plasma membrane-rich fractions. Contamination by endoplasmic reticulum and mitochondria was low. Gel electrophoresis of the membrane proteins of the Golgi apparatus-rich and plasma membrane-rich fractions (separately and mixed) showed a close similarity. After Neville's demonstration that electrophoretic patterns of membrane protein subunits from different subcellular fractions are easily distinguishable, the present work demonstrates an unusually close relationship between the Golgi apparatus membrane and the cell membrane. It is possible that membrane similarity may be mediated by the transfer of membrane-bound vesicles from the Golgi apparatus to the cell membrane. 相似文献
20.
AMP-activated protein kinase phosphorylates Golgi-specific brefeldin A resistance factor 1 at Thr1337 to induce disassembly of Golgi apparatus 总被引:3,自引:0,他引:3
Miyamoto T Oshiro N Yoshino K Nakashima A Eguchi S Takahashi M Ono Y Kikkawa U Yonezawa K 《The Journal of biological chemistry》2008,283(7):4430-4438
Sufficiency and depletion of nutrients regulate the cellular activities through the protein phosphorylation reaction; however, many protein substrates remain to be clarified. GBF1 (Golgi-specific brefeldin A resistance factor 1), a guanine nucleotide exchange factor for the ADP-ribosylation factor family associated with the Golgi apparatus, was isolated as a phosphoprotein from the glucose-depleted cells by using the phospho-Akt-substrate antibody, which recognizes the substrate proteins of several protein kinases. The phosphorylation of GBF1 was induced by 2-deoxyglucose (2-DG), which blocks glucose utilization and increases the intracellular AMP concentration, and by AICAR, an AMP-activated protein kinase (AMPK) activator. This phosphorylation was observed in the cells expressing the constitutively active AMPK. The 2-DG-induced phosphorylation of GBF1 was suppressed by Compound C, an AMPK inhibitor, and by the overexpression of the kinase-negative AMPK. Analysis using the deletion and point mutants identified Thr(1337) as the 2-DG-induced phosphorylation site in GBF1, which is phosphorylated by AMPK in vitro. ATP depletion is known to provoke the Golgi apparatus disassembly. Immunofluorescent microscopic analysis with the Golgi markers indicated that GBF1 associates with the fragmented Golgi apparatus in the cells treated with 2-DG and AICAR. The expression of the kinase-negative AMPK and the GBF1 mutant replacing Thr(1337) by Ala prevented the 2-DG-induced Golgi disassembly. These results indicate that GBF1 is a novel AMPK substrate and that the AMPK-mediated phosphorylation of GBF1 at Thr(1337) has a critical role, presumably by attenuating the function of GBF1, in the disassembly of the Golgi apparatus induced under stress conditions that lower the intracellular ATP concentration. 相似文献