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The Journal of Cell Biology Mar 1992
Review
Topics: Animals; Brefeldin A; Cell Compartmentation; Cyclopentanes; Endoplasmic Reticulum; Golgi Apparatus; Intracellular Membranes; Organelles
PubMed: 1740466
DOI: 10.1083/jcb.116.5.1071 -
Proceedings of the National Academy of... Feb 2003Like other guanine nucleotide-exchange proteins (GEPs) that activate ADP-ribosylation factor (ARF) GTPases, brefeldin A-inhibited GEP2, BIG2, contains an approximately...
Like other guanine nucleotide-exchange proteins (GEPs) that activate ADP-ribosylation factor (ARF) GTPases, brefeldin A-inhibited GEP2, BIG2, contains an approximately 200-aa Sec7 domain that is responsible for this catalytic activity and its inhibition by brefeldin A. The Sec7 domain is located near the center of the molecule and serves to accelerate replacement of GDP bound to ARF with GTP. To explore possible functions of the N-terminal region of BIG2 (1-832), we used three coding-region constructs as bait to screen a human heart cDNA library in a yeast two-hybrid system, retrieving two unique clones that encode a type I protein kinase A (PKA) regulatory subunit, RI alpha. Coimmunoprecipitation experiments confirmed interaction of in vitro translated BIG2 and RI alpha, as well as of the endogenous proteins in cytosol of cultured HepG2 cells. Using 28 deletion mutants, we found three regions of BIG2 that interacted with R subunits of PKA. Residues 27-48 (domain A) interacted with RI alpha and RI beta, 284-301 (domain B) interacted with RII alpha and RII beta, and 517-538 (domain C) interacted with RI alpha, RII alpha, and RII beta. Sequence analysis and helical wheel projection of amino acids in the three domains revealed potential amphipathic wheel structures characteristic for binding of PKA R subunits. Western blot analysis of subcellular fractions demonstrated translocation of BIG2 (and BIG1) from cytosol to the Golgi and other membrane structures after incubation of cells with 8-Br-cAMP or forskolin. All findings are consistent with a role for BIG2 as an A kinase-anchoring protein (or AKAP) that could coordinate cAMP and ARF regulatory pathways.
Topics: Amino Acid Sequence; Base Sequence; Binding Sites; Blotting, Western; Brefeldin A; Cell Line; Cell Membrane; Cloning, Molecular; Cyclic AMP-Dependent Protein Kinases; DNA Primers; DNA, Complementary; Guanine Nucleotide Exchange Factors; Humans; Precipitin Tests; Protein Transport; Two-Hybrid System Techniques
PubMed: 12571360
DOI: 10.1073/pnas.0337678100 -
The Journal of Biological Chemistry Oct 1996In this study, we have used immunocytochemical and fractionation approaches to provide a description of the localization of the mammalian Cdc42 protein (designated...
In this study, we have used immunocytochemical and fractionation approaches to provide a description of the localization of the mammalian Cdc42 protein (designated Cdc42Hs) in vivo. A specific anti-peptide antibody was generated against the C-terminal region of Cdc42Hs. Using affinity-purified preparations of this antibody in indirect immunofluorescence experiments, Cdc42Hs was found to be localized to the Golgi apparatus. Similar to the well-characterized non-clathrin coat proteins ADP-ribosylation factor (ARF) and beta-COP, the perinuclear clustering of Cdc42Hs is rapidly dispersed upon exposure of the cells to the drug brefeldin A, suggesting that it too may play a role in the processes of intracellular lipid and protein transport. Employing cell lines possessing inducible forms of ARF, we demonstrate here a tight coupling of the nucleotide-bound state of ARF and the subcellular localization of Cdc42Hs. Specifically, the expression of wild-type ARF had no effect on the brefeldin A sensitivity of Cdc42Hs while, as is the case for ARF and beta-COP, expression of a GTPase-deficient form of ARF (ARF(Q71L)) renders these Golgi-localized proteins resistant to brefeldin A treatment (; ). Moreover, the induced expression of a mutant form of ARF with a low affinity for nucleotide resulted in constitutive redistribution of Cdc42Hs in the absence of brefeldin A treatment. These results suggest that Cdc42Hs may play a role in cell morphogenesis by acting on targets in the Golgi that direct polarized growth at the plasma membrane.
Topics: Animals; Brefeldin A; Cell Cycle Proteins; Cyclopentanes; GTP-Binding Proteins; Golgi Apparatus; Microscopy, Fluorescence; Rabbits; cdc42 GTP-Binding Protein
PubMed: 8900167
DOI: 10.1074/jbc.271.43.26850 -
Nature Reviews. Cancer Dec 2010There is now considerable and increasing evidence for a causal role for aberrant activity of the Ras superfamily of small GTPases in human cancers. These GTPases... (Review)
Review
There is now considerable and increasing evidence for a causal role for aberrant activity of the Ras superfamily of small GTPases in human cancers. These GTPases function as GDP-GTP-regulated binary switches that control many fundamental cellular processes. A common mechanism of GTPase deregulation in cancer is the deregulated expression and/or activity of their regulatory proteins, guanine nucleotide exchange factors (GEFs) that promote formation of the active GTP-bound state and GTPase-activating proteins (GAPs) that return the GTPase to its GDP-bound inactive state. In this Review, we assess the association of GEFs and GAPs with cancer and their druggability for cancer therapeutics.
Topics: Animals; Brefeldin A; Drug Discovery; GTPase-Activating Proteins; Guanine Nucleotide Exchange Factors; Humans; Neoplasms; Proto-Oncogene Proteins; T-Lymphoma Invasion and Metastasis-inducing Protein 1
PubMed: 21102635
DOI: 10.1038/nrc2960 -
The Journal of Biological Chemistry Mar 1993Brefeldin A has proven to be a useful pharmacologic tool, which, when added to mammalian cells, results in a block in secretion as well as the structural disruption of...
Brefeldin A has proven to be a useful pharmacologic tool, which, when added to mammalian cells, results in a block in secretion as well as the structural disruption of specific intracellular organelles. In spite of our understanding of some of the biochemistry underlying the action of brefeldin A, the most proximal molecular target(s) of the drug remain elusive. In attempting to address this problem, a genetic approach will undoubtedly prove useful and complementary to the biochemical identification of such a site(s). As a result of the relatively resistant nature of wild-type Saccharomyces cerevisiae to brefeldin A, an approach utilizing yeast genetics has not been possible. We report the selective sensitivity of three drug-sensitive strains of S. cerevisiae (ise-1, ISE-2, and erg6) with enhanced membrane permeability allowing uptake of brefeldin A. Upon addition of the drug, growth is dramatically inhibited and invertase secretion is rapidly, specifically, and reversibly blocked at the level of the endoplasmic reticulum. In addition, only structural analogues of brefeldin A effective in mammalian cells are active in these yeast strains.
Topics: Antifungal Agents; Brefeldin A; Cyclopentanes; Electrophoresis, Polyacrylamide Gel; Glycoside Hydrolases; Microbial Sensitivity Tests; Precipitin Tests; Saccharomyces cerevisiae; beta-Fructofuranosidase
PubMed: 8449896
DOI: No ID Found -
The Journal of Biological Chemistry Jun 1999A brefeldin A (BFA)-inhibited guanine nucleotide-exchange protein (GEP) for ADP-ribosylation factors (ARF) was purified earlier from bovine brain cytosol. Cloning and...
A brefeldin A (BFA)-inhibited guanine nucleotide-exchange protein (GEP) for ADP-ribosylation factors (ARF) was purified earlier from bovine brain cytosol. Cloning and expression of the cDNA confirmed that the recombinant protein (p200) is a BFA-sensitive ARF GEP. p200 contains a domain that is 50% identical in amino acid sequence to a region in yeast Sec7, termed the Sec7 domain. Sec7 domains have been identified also in other proteins with ARF GEP activity, some of which are not inhibited by BFA. To identify structural elements that influence GEP activity and its BFA sensitivity, several truncated mutants of p200 were made. Deletion of sequence C-terminal to the Sec7 domain did not affect GEP activity. A protein lacking 594 amino acids at the N terminus, as well as sequence following the Sec7 domain, also had high activity. The mutant lacking 630 N-terminal amino acids was, however, only 1% as active, as was the Sec7 domain itself (mutant lacking 697 N-terminal residues). It appears that the Sec7 domain of p200 contains the catalytic site but additional sequence (perhaps especially that between positions 595 and 630) modifies activity dramatically. Myristoylated recombinant ARFs were better than non-myristoylated as substrates; ARFs 1 and 3 were better than ARF5, and no activity was detected with ARF6. Physical interaction of the Sec7 domain with an ARF1 mutant was demonstrated, but it was much weaker than that of the cytohesin-1 Sec7 domain with the same ARF protein. Effects of BFA on p200 and all mutants with high activity were similar with approximately 50% inhibition at =50 microM. The inactive BFA analogue B36 did not inhibit the Sec7 domain or p200. Thus, the Sec7 domain of p200, like that of Sec7 itself (Sata, M., Donaldson, J. G., Moss, J., and Vaughan, M. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 4204-4208), plays a role in BFA inhibition as well as in GEP activity, although the latter is markedly modified by other structural elements.
Topics: ADP-Ribosylation Factor 1; ADP-Ribosylation Factors; Animals; Brain; Brefeldin A; Cattle; Fungal Proteins; GTP-Binding Proteins; Guanine Nucleotide Exchange Factors; Guanosine 5'-O-(3-Thiotriphosphate); Kinetics; Magnesium; Mutation; Phosphatidylserines; Protein Binding; Proteins; Recombinant Proteins; Sequence Deletion; Substrate Specificity
PubMed: 10364170
DOI: 10.1074/jbc.274.25.17417 -
The Journal of Biological Chemistry Feb 2013The GDP/GTP nucleotide exchange of Arf1 is catalyzed by nucleotide exchange factors (GEF), such as Arno, which act through their catalytic Sec7 domain. This exchange is...
Kinetics of interaction between ADP-ribosylation factor-1 (Arf1) and the Sec7 domain of Arno guanine nucleotide exchange factor, modulation by allosteric factors, and the uncompetitive inhibitor brefeldin A.
The GDP/GTP nucleotide exchange of Arf1 is catalyzed by nucleotide exchange factors (GEF), such as Arno, which act through their catalytic Sec7 domain. This exchange is a complex mechanism that undergoes conformational changes and intermediate complex species involving several allosteric partners such as nucleotides, Mg(2+), and Sec7 domains. Using a surface plasmon resonance approach, we characterized the kinetic binding parameters for various intermediate complexes. We first confirmed that both GDP and GTP counteract equivalently to the free-nucleotide binary Arf1-Arno complex stability and revealed that Mg(2+) potentiates by a factor of 2 the allosteric effect of GDP. Then we explored the uncompetitive inhibitory mechanism of brefeldin A (BFA) that conducts to an abortive pentameric Arf1-Mg(2+)-GDP-BFA-Sec7 complex. With BFA, the association rate of the abortive complex is drastically reduced by a factor of 42, and by contrast, the 15-fold decrease of the dissociation rate concurs to stabilize the pentameric complex. These specific kinetic signatures have allowed distinguishing the level and nature as well as the fate in real time of formed complexes according to experimental conditions. Thus, we showed that in the presence of GDP, the BFA-resistant Sec7 domain of Arno can also associate to form a pentameric complex, which suggests that the uncompetitive inhibition by BFA and the nucleotide allosteric effect combine to stabilize such abortive complex.
Topics: ADP-Ribosylation Factor 1; Allosteric Site; Binding, Competitive; Biotinylation; Brefeldin A; Catalysis; Escherichia coli; GTPase-Activating Proteins; Guanine Nucleotide Exchange Factors; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Kinetics; Plasmids; Protein Binding; Surface Plasmon Resonance
PubMed: 23255605
DOI: 10.1074/jbc.M112.391748 -
The Journal of Biological Chemistry Jun 2001Many animal cells release ATP into the extracellular medium, and often this release is mechanosensitive. However, the mechanisms underlying this release are not well...
Many animal cells release ATP into the extracellular medium, and often this release is mechanosensitive. However, the mechanisms underlying this release are not well understood. Using the luciferin-luciferase bioluminescent assay we demonstrate that a Xenopus oocyte releases ATP at a basal rate approximately 0.01 fmol/s, and gentle mechanical stimulation can increase this to 50 fmol/s. Brefeldin A, nocodazole, and progesterone-induced- maturation block basal and mechanosensitive ATP release. These treatments share the common feature of disrupting the Golgi complex and vesicle trafficking to the cell surface and thereby block protein secretion and membrane protein insertion. We propose that ATP release occurs when protein transport vesicles enriched in ATP fuse with the plasma membrane. Collagenase, integrin-binding peptides, and cytochalasin D also block ATP release, indicating that extracellular, membrane and cytoskeletal elements are involved in the release process. Elevation of intracellular Ca(2+) does not evoke ATP release but potentiates mechanosensitive ATP release. Our study indicates a novel mechanism of mechanotransduction that would allow cells to regulate membrane trafficking and protein transport/secretion in response to mechanical loading.
Topics: Adenosine Triphosphate; Animals; Brefeldin A; Calcium; Cells, Cultured; Collagenases; Cytochalasin D; Integrins; Luminescent Measurements; Nocodazole; Oligopeptides; Oocytes; Peptides, Cyclic; Progesterone; Protein Transport; Stress, Mechanical; Xenopus
PubMed: 11320093
DOI: 10.1074/jbc.M101500200 -
Cells Dec 2019The Golgi apparatus undergoes disorganization in response to stress, but it is able to restore compact and perinuclear structure under recovery. This self-organization...
BACKGROUND
The Golgi apparatus undergoes disorganization in response to stress, but it is able to restore compact and perinuclear structure under recovery. This self-organization mechanism is significant for cellular homeostasis, but remains mostly elusive, as does the role of giantin, the largest Golgi matrix dimeric protein.
METHODS
In HeLa and different prostate cancer cells, we used the model of cellular stress induced by Brefeldin A (BFA). The conformational structure of giantin was assessed by proximity ligation assay and atomic force microscopy. The post-BFA distribution of Golgi resident enzymes was examined by 3D SIM high-resolution microscopy.
RESULTS
We detected that giantin is rather flexible than an extended coiled-coil dimer and BFA-induced Golgi disassembly was associated with giantin monomerization. A fusion of the nascent Golgi membranes after BFA washout is forced by giantin re-dimerization via disulfide bond in its luminal domain and assisted by Rab6a GTPase. GM130-GRASP65-dependent enzymes are able to reach the nascent Golgi membranes, while giantin-sensitive enzymes appeared at the Golgi after its complete recovery via direct interaction of their cytoplasmic tail with N-terminus of giantin.
CONCLUSION
Post-stress recovery of Golgi is conducted by giantin dimer and Golgi proteins refill membranes according to their docking affiliation rather than their intra-Golgi location.
Topics: Brefeldin A; Endoplasmic Reticulum Stress; Golgi Apparatus; Golgi Matrix Proteins; HeLa Cells; Humans; Immunoprecipitation; Male; Membrane Proteins; Microscopy, Atomic Force; Microscopy, Confocal; Prostatic Neoplasms; Protein Binding
PubMed: 31847122
DOI: 10.3390/cells8121631 -
PloS One 2013Members of the glycolipid transfer protein superfamily (GLTP) are found from animals and fungi to plants and red micro-alga. Eukaryotes that encode the glucosylceramide...
Members of the glycolipid transfer protein superfamily (GLTP) are found from animals and fungi to plants and red micro-alga. Eukaryotes that encode the glucosylceramide synthase responsible for the synthesis of glucosylceramide, the precursor for most glycosphingolipids, also produce GLTPs. Cells that does not synthesize glucosylceramide neither express GLTPs. Based on this genetic relationship there must be a strong correlation between the synthesis of glucosylceramide and GLTPs. To regulate the levels of glycolipids we have used inhibitors of intracellular trafficking, glycosphingolipid synthesis and degradation, and small interfering RNA to down-regulate the activity of glucosylceramide synthase activity. We found that GLTP expression, both at the mRNA and protein levels, is elevated in cells that accumulate glucosylceramide. Monensin and brefeldin A block intracellular vesicular transport mechanisms. Brefeldin A treatment leads to accumulation of newly synthesized glucosylceramide, galactosylceramide and lactosylceramide in a fused endoplasmic reticulum-Golgi complex. On the other hand, inhibiting glycosphingolipid degradation with conduritol-B-epoxide, that generates glucosylceramide accumulation in the lysosomes, did not affect the levels of GLTP. However, glycosphingolipid synthesis inhibitors like PDMP, NB-DNJ and myriocin, all decreased glucosylceramide and GLTP below normal levels. We also found that an 80% loss of glucosylceramide due to glucosylceramide synthase knockdown resulted in a significant reduction in the expression of GLTP. We show here that interfering with membrane trafficking events and simple neutral glycosphingolipid synthesis will affect the expression of GLTP. We postulate that a change in the glucosylceramide balance causes a response in the GLTP expression, and put forward that GLTP might play a role in lipid directing and sensing of glucosylceramide at the ER-Golgi interface.
Topics: Biological Transport; Blotting, Western; Brefeldin A; Carrier Proteins; Cells, Cultured; Ceramides; Galactosylceramides; Glucosylceramides; Glycosphingolipids; Humans; Monensin; RNA Interference; Real-Time Polymerase Chain Reaction; Sphingomyelins
PubMed: 23894633
DOI: 10.1371/journal.pone.0070283