The small GTP-binding protein rab6p is redistributed in the cytosol by brefeldin A

1993 ◽  
Vol 106 (3) ◽  
pp. 789-802 ◽  
Author(s):  
M. Roa ◽  
V. Cornet ◽  
C.Z. Yang ◽  
B. Goud
Keyword(s):  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Brefeldin A ◽  
Atp Depletion ◽  
Cell Fractionation ◽  
Microtubule Organizing Center ◽  
Trans Golgi Network ◽  
Gtp Binding ◽  
Organizing Center ◽  
Golgi Network

Rab6 protein belongs to the Sec4/Ypt/rab subfamily of small GTP-binding proteins involved in intracellular membrane trafficking in yeast and mammalian cells. Its localization both in medial and trans-Golgi network prompted us to study the effects of brefeldin A (BFA) on rab6p redistribution. By two techniques, indirect immunofluorescence and cell fractionation, we investigated the fate of rab6p and compared it to other Golgi or trans-Golgi network markers in BHK-21 and NIH-3T3 cells. BFA, at 5 micrograms/ml, induced redistribution of rab6p according to a biphasic process: during the first 10–15 minutes, tubulo-vesicular structures--colabelled with a bona fide medial Golgi marker called CTR 433--were observed; these structures were then replaced by punctate diffuse staining, which was stable for up to 3 hours. The 110 kDa peripheral membrane protein beta-COP was released much more rapidly from the Golgi membranes, whereas the trans-Golgi network marker TGN 38 relocated to the microtubule organizing center. The kinetics of reversion of BFA action on these antigens was also followed by immunofluorescence. Consistent with these results, rab6 antigen, originally found as 40% in the cytosolic versus 60% in the particulate (P 150,000 g) fraction, became almost entirely cytosolic; moreover, it partitioned in the aqueous phase of Triton X-114 whereas the membrane fraction was detergent-soluble. Rab6p did not become part of the coatomers after its BFA-induced release from Golgi structures. Three requirements seemed to be necessary for such a release: integrity of the microtubules, presence of energy, and a hypothetical trimeric G protein, as revealed by the respective roles of nocodazole, ATP depletion, and sensitivity to aluminium fluoride. Finally, we have shown that BFA does not prevent attachment of newly synthesized rab6p to membranes.

scholarly journals Perturbation of the morphology of the trans-Golgi network following Brefeldin A treatment: redistribution of a TGN-specific integral membrane protein, TGN38.

1992 ◽  
Vol 116 (1) ◽  
pp. 85-94 ◽  
Author(s):  
B Reaves ◽  
G Banting
Keyword(s):  
Membrane Protein ◽  
Morphological Changes ◽  
Brefeldin A ◽  
Integral Membrane Protein ◽  
Polyclonal Antiserum ◽  
Atp Depletion ◽  
Microtubule Organizing Center ◽  
Trans Golgi Network ◽  
Organizing Center ◽  
Golgi Network

Brefeldin A (BFA) has a dramatic effect on the morphology of the Golgi apparatus and induces a rapid redistribution of Golgi proteins into the ER (Lippincott-Schwartz, J., L. C. Yuan, J. S. Bonifacino, and R. D. Klausner. 1989. Cell. 56:801-813). To date, no evidence that BFA affects the morphology of the trans-Golgi network (TGN) has been presented. We describe the results of experiments, using a polyclonal antiserum to a TGN specific integral membrane protein (TGN38) (Luzio, J.P., B. Brake, G. Banting, K. E. Howell, P. Braghetta, and K. K. Stanley. 1990. Biochem. J. 270:97-102), which demonstrate that incubation of cells with BFA does induce morphological changes to the TGN. However, rather than redistributing to the ER, the majority of the TGN collapses around the microtubule organizing center (MTOC). The effect of BFA upon the TGN is (a) independent of protein synthesis, (b) fully reversible (c) microtubule dependent (as shown in nocodazole-treated cells), and (d) relies upon the hydrolysis of GTP (as shown by performing experiments in the presence of GTP gamma S). ATP depletion reduces the ability of BFA to induce a redistribution of Golgi proteins into the ER; however, it has no effect upon the BFA-induced relocalizations of the TGN. These data confirm that the TGN is an organelle which is independent of the Golgi, and suggest a dynamic interaction between the TGN and microtubules which is centered around the MTOC.

scholarly journals The secretion inhibitor Exo2 perturbs trafficking of Shiga toxin between endosomes and the trans-Golgi network

2008 ◽  
Vol 414 (3) ◽  
pp. 471-484 ◽  
Author(s):  
Robert A. Spooner ◽  
Peter Watson ◽  
Daniel C. Smith ◽  
Frédéric Boal ◽  
Mohammed Amessou ◽  
...  
Keyword(s):  
Cholera Toxin ◽  
Membrane Trafficking ◽  
Shiga Toxin ◽  
Mammalian Cells ◽  
Brefeldin A ◽  
Anterograde Transport ◽  
Trans Golgi Network ◽  
Molecule Inhibitor ◽  
Early Endosomes ◽  
Golgi Network

The small-molecule inhibitor Exo2 {4-hydroxy-3-methoxy-(5,6,7,8-tetrahydrol[1]benzothieno[2,3-d]pyrimidin-4-yl)hydraz-one benzaldehyde} has been reported to disrupt the Golgi apparatus completely and to stimulate Golgi–ER (endoplasmic reticulum) fusion in mammalian cells, akin to the well-characterized fungal toxin BFA (brefeldin A). It has also been reported that Exo2 does not affect the integrity of the TGN (trans-Golgi network), or the direct retrograde trafficking of the glycolipid-binding cholera toxin from the TGN to the ER lumen. We have examined the effects of BFA and Exo2, and found that both compounds are indistinguishable in their inhibition of anterograde transport and that both reagents significantly disrupt the morphology of the TGN in HeLa and in BS-C-1 cells. However, Exo2, unlike BFA, does not induce tubulation and merging of the TGN and endosomal compartments. Furthermore, and in contrast with its effects on cholera toxin, Exo2 significantly perturbs the delivery of Shiga toxin to the ER. Together, these results suggest that the likely target(s) of Exo2 operate at the level of the TGN, the Golgi and a subset of early endosomes, and thus Exo2 provides a more selective tool than BFA for examining membrane trafficking in mammalian cells.

Polarization-dependent apical membrane CFTR targeting underlies cAMP-stimulated Cl- secretion in epithelial cells

1994 ◽  
Vol 266 (1) ◽  
pp. C254-C268 ◽  
Author(s):  
A. P. Morris ◽  
S. A. Cunningham ◽  
A. Tousson ◽  
D. J. Benos ◽  
R. A. Frizzell
Keyword(s):  
Epithelial Cells ◽  
Apical Membrane ◽  
Brefeldin A ◽  
Microtubule Organizing Center ◽  
Cl Secretion ◽  
Organizing Center ◽  
Reversible Time ◽  
Golgi Network ◽  
Ht 29 ◽  
Cl Conductance

The relationship between adenosine 3',5'-cyclic monophosphate (cAMP)-mediated Cl- secretion and the cellular location of the cystic fibrosis transmembrane conductance regulator (CFTR) was determined in both polarized (Cl.19A) and unpolarized (parental) HT-29 colonocytes expressing similar levels of CFTR mRNA and protein. CFTR immunolocalized to the apical membrane domain of polarized colonocytes exhibiting cAMP-responsive Cl- secretion. In contrast, CFTR staining was perinuclear in unpolarized colonocytes, which gave little or no cAMP-stimulated Cl- conductance responses. Thus cAMP-stimulated Cl- secretion coincided with an apical localization of CFTR. Brefeldin A (BFA) was used to perturb glycoprotein targeting in these cells. In polarized colonocytes, BFA caused a reversible, time-dependent decrease in the Cl-conductance response to cAMP but not Ca2+. Apical CFTR redistributed into large coalesced intracellular vesicles, located within the same plane as the microtubule organizing center, a marker for the trans-Golgi network (TGN). In preconfluent monolayers or unpolarized HT-29 cells, BFA had no effect on CFTR staining, which remained perinuclear. Mature, Golgi-processed CFTR protein was isolated from both polarized and unpolarized colonocytes. Thus the mechanism for polarization-dependent apical membrane CFTR targeting and the acquisition of cAMP-dependent Cl- secretion lies at or beyond the late Golgi-TGN in epithelial cells.

GGA proteins: new players in the sorting game

2001 ◽  
Vol 114 (19) ◽  
pp. 3413-3418 ◽  
Author(s):  
Annette L. Boman
Keyword(s):  
Membrane Trafficking ◽  
Sequence Homology ◽  
Mammalian Cells ◽  
Gene Knockout ◽  
Protein Domains ◽  
Vesicle Formation ◽  
Trans Golgi Network ◽  
And Function ◽  
Golgi Network ◽  
Cargo Sorting

The GGA proteins are a novel family of proteins that were discovered nearly simultaneously by several labs studying very different aspects of membrane trafficking. Since then, several studies have described the GGA proteins and their functions in yeast and mammalian cells. Four protein domains are present in all GGA proteins, as defined by sequence homology and function. These different domains interact directly with ARF proteins, cargo and clathrin. Alteration of the levels of GGA proteins by gene knockout or overexpression affects specific trafficking events between the trans-Golgi network and endosomes. These data suggest that GGAs function as ARF-dependent, monomeric clathrin adaptors to facilitate cargo sorting and vesicle formation at the trans-Golgi network.

scholarly journals A novel GTP-binding protein–adaptor protein complex responsible for export of Vangl2 from the trans Golgi network

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Yusong Guo ◽  
Giulia Zanetti ◽  
Randy Schekman
Keyword(s):  
Mammalian Cells ◽  
Planar Cell Polarity ◽  
Binding Protein ◽  
Adaptor Protein ◽  
Gtp Binding Protein ◽  
Epithelial Surface ◽  
Trans Golgi Network ◽  
Gtp Binding ◽  
Signaling Receptors ◽  
Golgi Network

Planar cell polarity (PCP) requires the asymmetric sorting of distinct signaling receptors to distal and proximal surfaces of polarized epithelial cells. We have examined the transport of one PCP signaling protein, Vangl2, from the trans Golgi network (TGN) in mammalian cells. Using siRNA knockdown experiments, we find that the GTP-binding protein, Arfrp1, and the clathrin adaptor complex 1 (AP-1) are required for Vangl2 transport from the TGN. In contrast, TGN export of Frizzled 6, which localizes to the opposing epithelial surface from Vangl2, does not depend on Arfrp1 or AP-1. Mutagenesis studies identified a YYXXF sorting signal in the C-terminal cytosolic domain of Vangl2 that is required for Vangl2 traffic and interaction with the μ subunit of AP-1. We propose that Arfrp1 exposes a binding site on AP-1 that recognizes the Vangl2 sorting motif for capture into a transport vesicle destined for the proximal surface of a polarized epithelial cell.

scholarly journals Sec6/8 complexes on trans-Golgi network and plasma membrane regulate late stages of exocytosis in mammalian cells

2001 ◽  
Vol 155 (4) ◽  
pp. 593-604 ◽  
Author(s):  
Charles Yeaman ◽  
Kent K. Grindstaff ◽  
Jessica R. Wright ◽  
W. James Nelson
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Rat Kidney ◽  
Brefeldin A ◽  
Intercellular Junctions ◽  
Trans Golgi Network ◽  
Cargo Protein ◽  
Normal Rat ◽  
Golgi Network ◽  
Exocytic Pathway

Sec6/8 complex regulates delivery of exocytic vesicles to plasma membrane docking sites, but how it is recruited to specific sites in the exocytic pathway is poorly understood. We identified an Sec6/8 complex on trans-Golgi network (TGN) and plasma membrane in normal rat kidney (NRK) cells that formed either fibroblast- (NRK-49F) or epithelial-like (NRK-52E) intercellular junctions. At both TGN and plasma membrane, Sec6/8 complex colocalizes with exocytic cargo protein, vesicular stomatitis virus G protein (VSVG)-tsO45. Newly synthesized Sec6/8 complex is simultaneously recruited from the cytosol to both sites. However, brefeldin A treatment inhibits recruitment to the plasma membrane and other treatments that block exocytosis (e.g., expression of kinase-inactive protein kinase D and low temperature incubation) cause accumulation of Sec6/8 on the TGN, indicating that steady-state distribution of Sec6/8 complex depends on continuous exocytic vesicle trafficking. Addition of antibodies specific for TGN- or plasma membrane–bound Sec6/8 complexes to semiintact NRK cells results in cargo accumulation in a perinuclear region or near the plasma membrane, respectively. These results indicate that Sec6/8 complex is required for several steps in exocytic transport of vesicles between TGN and plasma membrane.

scholarly journals Intracellular Localization of Phospholipase D1 in Mammalian Cells

2001 ◽  
Vol 12 (4) ◽  
pp. 943-955 ◽  
Author(s):  
Zachary Freyberg ◽  
David Sweeney ◽  
Anirban Siddhanta ◽  
Sylvain Bourgoin ◽  
Michael Frohman ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
Mammalian Cells ◽  
Intracellular Localization ◽  
Brefeldin A ◽  
Cell Types ◽  
Immunogold Electron Microscopy ◽  
Cell Fractionation ◽  
Cell Nuclei ◽  
Phospholipase D1 ◽  
Golgi Network

Phospholipase D (PLD) hydrolyzes phosphatidylcholine to generate phosphatidic acid. In mammalian cells this reaction has been implicated in the recruitment of coatomer to Golgi membranes and release of nascent secretory vesicles from the trans-Golgi network. These observations suggest that PLD is associated with the Golgi complex; however, to date, because of its low abundance, the intracellular localization of PLD has been characterized only indirectly through overexpression of chimeric proteins. We have used highly sensitive antibodies to PLD1 together with immunofluorescence and immunogold electron microscopy as well as cell fractionation to identify the intracellular localization of endogenous PLD1 in several cell types. Although PLD1 had a diffuse staining pattern, it was enriched significantly in the Golgi apparatus and was also present in cell nuclei. On fragmentation of the Golgi apparatus by treatment with nocodazole, PLD1 closely associated with membrane fragments, whereas after inhibition of PA synthesis, PLD1 dissociated from the membranes. Overexpression of an hemagglutinin-tagged form of PLD1 resulted in displacement of the endogenous enzyme from its perinuclear localization to large vesicular structures. Surprisingly, when the Golgi apparatus collapsed in response to brefeldin A, the nuclear localization of PLD1 was enhanced significantly. Our data show that the intracellular localization of PLD1 is consistent with a role in vesicle trafficking from the Golgi apparatus and suggest that it also functions in the cell nucleus.

Multimeric connexin interactions prior to the trans-Golgi network

2001 ◽  
Vol 114 (22) ◽  
pp. 4013-4024
Author(s):  
Jayasri Das Sarma ◽  
Rita A. Meyer ◽  
Fushan Wang ◽  
Valsamma Abraham ◽  
Cecilia W. Lo ◽  
...  
Keyword(s):  
Brefeldin A ◽  
Dominant Negative ◽  
Alveolar Epithelial Cells ◽  
Trans Golgi Network ◽  
Alveolar Epithelial ◽  
C Terminus ◽  
Gradient Fractionation ◽  
Golgi Network ◽  
Nih 3T3 ◽  
Gap Junction Hemichannels

Cells that express multiple connexins have the capacity to form heteromeric (mixed) gap junction hemichannels. We used a dominant negative connexin construct, consisting of bacterial β-galactosidase fused to the C terminus of connexin43 (Cx43/β-gal), to examine connexin compatibility in NIH 3T3 cells. Cx43/β-gal is retained in a perinuclear compartment and inhibits Cx43 transport to the cell surface. The intracellular connexin pool induced by Cx43/β-gal colocalized with a medial Golgi apparatus marker and was readily disassembled by treatment with brefeldin A. This was unexpected, since previous studies indicated that Cx43 assembly into hexameric hemichannels occurs in the trans-Golgi network (TGN) and is sensitive to brefeldin A. Further analysis by sucrose gradient fractionation showed that Cx43 and Cx43/β-gal were assembled into a subhexameric complex. Cx43/β-gal also specifically interacted with Cx46, but not Cx32, consistent with the ability of Cx43/β-gal to simultaneously inhibit multiple connexins. We confirmed that interactions between Cx43/β-gal and Cx46 reflect the ability of Cx43 and Cx46 to form heteromeric complexes, using HeLa and alveolar epithelial cells, which express both connexins. In contrast, ROS osteoblastic cells, which differentially sort Cx43 and Cx46, did not form Cx43/Cx46 heteromers. Thus, cells have the capacity to regulate whether or not compatible connexins intermix.

scholarly journals p230 is associated with vesicles budding from the trans-Golgi network

1996 ◽  
Vol 109 (12) ◽  
pp. 2811-2821 ◽  
Author(s):  
P.A. Gleeson ◽  
T.J. Anderson ◽  
J.L. Stow ◽  
G. Griffiths ◽  
B.H. Toh ◽  
...  
Keyword(s):  
Brefeldin A ◽  
Immunogold Labeling ◽  
Coated Vesicles ◽  
Specific Marker ◽  
Very High Frequency ◽  
Trans Golgi Network ◽  
Golgi Membranes ◽  
Vesicle Biogenesis ◽  
Heptad Repeats ◽  
Golgi Network

Transport vesicle formation requires the association of cytosolic proteins with the membrane. We have previously described a brefeldin-A sensitive, hydrophilic protein (p230), containing a very high frequency of heptad repeats, found in the cytosol and associated with Golgi membranes. We show here that p230 is localised on the trans-Golgi network, by immunogold labeling of HeLa cell cryosections using alpha 2,6 sialyltransferase as a compartment-specific marker. The role of G protein activators on the binding of p230 to Golgi membranes and in vesicle biogenesis has been investigated. Treatment of streptolysin-O permeabilised HeLa cells with either GTP gamma S or AlF4- resulted in accumulation of p230 on Golgi membranes. Furthermore, immunolabeling of isolated Golgi membranes treated with AlF4-, to induce the accumulation of vesicles, showed that p230 is predominantly localised to the cytoplasmic surface of trans-Golgi network-derived budding structures and small coated vesicles. p230-labeled vesicles have a thin (approximately 10 nm) electron dense cytoplasmic coat and could be readily distinguished from clathrin-coated vesicles. Dual immunogold labeling of perforated cells, or of cryosections of treated Golgi membranes, revealed that p230 and the trans-Golgi network-associated p200, which we show here to be distinct molecules, appear to be localised on separate populations of vesicles budding from the trans-Golgi network. These results strongly suggest the presence of distinct populations of non-clathrin coated vesicles derived from the trans-Golgi network. As p230 recycles between the cytosol and buds/vesicles of TGN membranes, a process regulated by G proteins, we propose that p230 is involved in the biogenesis of a specific population of non-clathrin coated vesicles.

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