scholarly journals A role for calmodulin in organelle membrane tubulation.

1995 ◽  
Vol 6 (7) ◽  
pp. 871-887 ◽  
Author(s):  
P de Figueiredo ◽  
W J Brown
Keyword(s):  
Golgi Complex ◽  
Brefeldin A ◽  
Retrograde Transport ◽  
Coated Vesicle ◽  
Calmodulin Antagonists ◽  
Membrane Tubulation ◽  
Membrane Tubules ◽  
Potent Drug ◽  
Uniform Diameter ◽  
Golgi Network

Membrane tubules of uniform diameter (60-80 nm) and variable lengths have been seen to extend from the main bodies of the Golgi complex, trans Golgi network (TGN), and endosomes. In the case of endosomes, these tubules appear to mediate membrane and receptor recycling events. Brefeldin A (BFA) is a potent drug that completely blocks coated vesicle formation from the Golgi complex and TGN, but at the same time causes the enhanced formation of membrane tubules from these same organelles. Recently, experiments have shown that calmodulin antagonists inhibit the transport of receptors out of endosomes, perhaps by inhibiting the formation of recycling tubules. Using the potent calmodulin-specific antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide (W-13), and N-(4-aminobutyl)-5-chloro-1-naphthalenesulfonamide (C-1), we found that the recycling of transferrin from endosomes to the cell surface was significantly inhibited, resulting in the formation of enlarged endosomal vacuoles. In addition, these same calmodulin antagonists also potently inhibited the formation of BFA-stimulated membrane tubules from the Golgi complex, TGN, and endosomes. In the case of the Golgi complex, failure to form tubules resulted in the inhibition of BFA-stimulated retrograde transport to the endoplasmic reticulum. These results suggest that calmodulin is a general regulator of membrane tubulation and is capable of influencing the morphology of several organelles.

Retrograde trafficking of both Golgi complex and TGN markers to the ER induced by nordihydroguaiaretic acid and cyclofenil diphenol

1998 ◽  
Vol 111 (7) ◽  
pp. 951-965 ◽  
Author(s):  
D. Drecktrah ◽  
P. de Figueiredo ◽  
R.M. Mason ◽  
W.J. Brown
Keyword(s):  
Golgi Complex ◽  
Membrane Trafficking ◽  
Molecular Mechanisms ◽  
Nordihydroguaiaretic Acid ◽  
Retrograde Transport ◽  
Lipoxygenase Inhibitor ◽  
Golgi Stack ◽  
Golgi Network ◽  
In Vivo Effects

Previous studies have shown that the Golgi stack and the trans-Golgi network (TGN) may play a role in capturing escaped resident endoplasmic reticulum (ER) proteins, and directing their retrograde transport back to that organelle. Whether this retrograde movement represents a highly specific or more generalized membrane trafficking pathway is unclear. To better understand both the retrograde and anterograde trafficking pathways of the secretory apparatus, we examined more closely the in vivo effects of two structurally unrelated compounds, the potent lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA), and the non-steroidal estrogen cyclofenil diphenol (CFD), both of which are known to inhibit secretion. In the presence of these compounds, transport of vesicular stomatitis virus G membrane glycoprotein from the ER to the Golgi complex, and from the TGN to the cell surface, was inhibited potently and rapidly. Surprisingly, we found that NDGA and CFD stimulated the rapid, but not concomitant, retrograde movement of both Golgi stack and TGN membrane proteins back to the ER until both organelles were morphologically absent from cells. Both NDGA- and CFD-stimulated TGN and Golgi retrograde membrane trafficking were inhibited by microtubule depolymerizing agents and energy poisons. Removal of NDGA and CFD resulted in the complete, but not concomitant, reformation of both Golgi stacks and their closely associated TGN compartments. These studies suggest that NDGA and CFD unmask a generalized bulk recycling pathway to the ER for both Golgi and TGN membranes and, further, that NDGA and CFD are useful for investigating the molecular mechanisms that control the formation and maintenance of both the Golgi stack proper and the TGN.

scholarly journals Influenza virus hemagglutinin trimers and monomers maintain distinct biochemical modifications and intracellular distribution in brefeldin A-treated cells.

1991 ◽  
Vol 2 (7) ◽  
pp. 549-563 ◽  
Author(s):  
G Russ ◽  
J R Bennink ◽  
T Bächi ◽  
J W Yewdell
Keyword(s):  
Endoplasmic Reticulum ◽  
Influenza Virus ◽  
Monoclonal Antibodies ◽  
Golgi Complex ◽  
Brefeldin A ◽  
Retrograde Transport ◽  
Cell Fractionation ◽  
Influenza Virus Hemagglutinin ◽  
Infected Cells ◽  
Vesicular Compartment

Brefeldin A (BFA) induces the retrograde transport of proteins from the Golgi complex (GC) to the endoplasmic reticulum (ER). It is uncertain, however, whether the drug completely merges the ER with post-ER compartments, or whether some of their elements remain physically and functionally distinct. We investigated this question by the use of monoclonal antibodies specific for monomers and trimers of the influenza virus hemagglutinin (HA). In untreated influenza virus-infected cells, monomers and trimers almost exclusively partition into the ER and GC, respectively. In BFA-treated cells, both monomers and trimers are detected in the ER by immunofluorescence. Cell fractionation experiments indicate, however, that whereas HA monomers synthesized in the presence of BFA reside predominantly in vesicles with a characteristic density of the ER, HA trimers are primarily located in lighter vesicles characteristic of post-ER compartments. Biochemical experiments confirm that in BFA-treated cells, trimers are more extensively modified than monomers by GC-associated enzymes. Additional immunofluorescence experiments reveal that in BFA-treated cells, HA monomers can exist in an ER subcompartment less accessible to trimers and, conversely, that trimers are present in a vesicular compartment less accessible to monomers. These findings favor the existence of a post-ER compartment for which communication with the ER is maintained in the presence of BFA and suggest that trimers cycle between this compartment and the ER, but have access to only a portion of the ER.

scholarly journals Syntaxin 5-Dependent Retrograde Transport to thetrans-Golgi Network Is Required for Adeno-Associated Virus Transduction

2014 ◽  
Vol 89 (3) ◽  
pp. 1673-1687 ◽  
Author(s):  
Mathieu E. Nonnenmacher ◽  
Jean-Christophe Cintrat ◽  
Daniel Gillet ◽  
Thomas Weber
Keyword(s):  
Gene Delivery ◽  
Golgi Apparatus ◽  
Intracellular Transport ◽  
Brefeldin A ◽  
Retrograde Transport ◽  
Transport Pathway ◽  
Adeno Associated Virus ◽  
Viral Attachment ◽  
Golgi Network ◽  
Endosomal System

ABSTRACTIntracellular transport of recombinant adeno-associated virus (AAV) is still incompletely understood. In particular, the trafficking steps preceding the release of incoming AAV particles from the endosomal system into the cytoplasm, allowing subsequent nuclear import and the initiation of gene expression, remain to be elucidated fully. Others and we previously showed that a significant proportion of viral particles are transported to the Golgi apparatus and that Golgi apparatus disruption caused by the drug brefeldin A efficiently blocks AAV serotype 2 (AAV2) transduction. However, because brefeldin A is known to exert pleiotropic effects on the entire endosomal system, the functional relevance of transport to the Golgi apparatus for AAV transduction remains to be established definitively. Here, we show that AAV2 trafficking toward thetrans-Golgi network (TGN) and the Golgi apparatus correlates with transduction efficiency and relies on a nonclassical retrograde transport pathway that is independent of the retromer complex, late endosomes, and recycling endosomes. AAV2 transduction is unaffected by the knockdown of syntaxins 6 and 16, which are two major effectors in the retrograde transport of both exogenous and endogenous cargo. On the other hand, inhibition of syntaxin 5 function by small interfering RNA silencing or treatment with cyclized Retro-2 strongly decreases AAV2 transduction and transport to the Golgi apparatus. This inhibition of transduction is observed with several AAV serotypes and a number of primary and immortalized cells. Together, our data strongly suggest that syntaxin 5-mediated retrograde transport to the Golgi apparatus is a broadly conserved feature of AAV trafficking that appears to be independent of the identity of the receptors used for viral attachment.IMPORTANCEGene therapy constitutes a promising approach for the treatment of life-threatening conditions refractory to any other form of remedy. Adeno-associated virus (AAV) vectors are currently being evaluated for the treatment of diseases such as Duchenne muscular dystrophy, hemophilia, heart failure, Parkinson's disease, and others. Despite their promise as gene delivery vehicles, a better understanding of the biology of AAV-based vectors is necessary to improve further their efficacy. AAV vectors must reach the nucleus in order to deliver their genome, and their intracellular transport is not fully understood. Here, we dissect an important step of the intracellular journey of AAV by showing that retrograde transport of capsids to thetrans-Golgi network is necessary for gene delivery. We show that the AAV trafficking route differs from that of known Golgi apparatus-targeted cargos, and we raise the possibility that this nonclassical pathway is shared by most AAV variants, regardless of their attachment receptors.

scholarly journals Organization of the Yeast Golgi Complex into at Least Four Funtionally Distinct Compartments

2000 ◽  
Vol 11 (1) ◽  
pp. 171-182 ◽  
Author(s):  
William T. Brigance ◽  
Charles Barlowe ◽  
Todd R. Graham
Keyword(s):  
Golgi Complex ◽  
Brefeldin A ◽  
Proteolytic Processing ◽  
Golgi Compartment ◽  
Specific Antisera ◽  
Sensitive Factor ◽  
Processing Event ◽  
Golgi Network

Pro-α-factor (pro-αf) is posttranslationally modified in the yeast Golgi complex by the addition of α1,6-, α1,2-, and α1,3-linked mannose to N-linked oligosaccharides and by a Kex2p-initiated proteolytic processing event. Previous work has indicated that the α1,6- and α1,3-mannosylation and Kex2p-dependent processing of pro-αf are initiated in three distinct compartments of the Golgi complex. Here, we present evidence that α1,2-mannosylation of pro-αf is also initiated in a distinct Golgi compartment. Linkage-specific antisera and an endo-α1,6-d-mannanase (endoM) were used to quantitate the amount of each pro-αf intermediate during transport through the Golgi complex. We found that α1,6-, α1,2-, and α1,3-mannose were sequentially added to pro-αf in a temporally ordered manner, and that the intercompartmental transport factor Sec18p/N-ethylmaleimide-sensitive factor was required for each step. The Sec18p dependence implies that a transport event was required between each modification event. In addition, most of the Golgi-modified pro-αf that accumulated in brefeldin A-treated cells received only α1,6-mannosylation as did ∼50% of pro-αf transported to the Golgi in vitro. This further supports the presence of an early Golgi compartment that houses an α1,6-mannosyltransferase but lacks α1,2-mannosyltransferase activity in vivo. We propose that the α1,6-, α1,2-, and α1,3-mannosylation and Kex2p-dependent processing events mark the cis, medial,trans, and trans-Golgi network of the yeast Golgi complex, respectively.

scholarly journals Dynamic measurement of the pH of the Golgi complex in living cells using retrograde transport of the verotoxin receptor.

1996 ◽  
Vol 134 (6) ◽  
pp. 1387-1399 ◽  
Author(s):  
J H Kim ◽  
C A Lingwood ◽  
D B Williams ◽  
W Furuya ◽  
M F Manolson ◽  
...  
Keyword(s):  
Golgi Complex ◽  
Brefeldin A ◽  
Retrograde Transport ◽  
Vero Cells ◽  
Ionic Species ◽  
Cytosolic Calcium ◽  
Enterohemorrhagic Escherichia Coli ◽  
Live Cells ◽  
B Subunit ◽  
Acute Changes

The B subunit of verotoxin (VT1B) from enterohemorrhagic Escherichia coli is responsible for the attachment of the holotoxin to the cell surface, by binding to the glycolipid, globotriaosyl ceramide. After receptor-mediated endocytosis, the toxin is targeted to the Golgi complex by a process of retrograde transport. We took advantage of this unique property of VT1B to measure the pH of the Golgi complex in intact live cells. Purified recombinant VT1B was labeled with either rhodamine or fluorescein for subcellular localization by confocal microscopy. After 1 h at 37 degrees C, VT1B accumulated in a juxtanuclear structure that colocalized with several Golgi markers, including alpha-mannosidase II, beta-COP, and NBD-ceramide. Moreover, colchicine and brefeldin A induced dispersal of the juxtanuclear staining, consistent with accumulation of VT1B in the Golgi complex. Imaging of the emission of fluorescein-labeled VT1B was used to measure intra-Golgi pH (pHG), which was calibrated in situ with ionophores. In intact Vero cells, pHG averaged 6.45 +/- 0.03 (standard error). The acidity of the Golgi lumen dissipated rapidly upon addition of bafilomycin A1, a blocker of vacuolar-type ATPases, pHG remained constant despite acidification of the cytosol by reversal of the plasmalemmal Na+/H+ antiport. Similarly, pHG was unaffected by acute changes in cytosolic calcium. Furthermore, pHG recovered quickly toward the basal level after departures imposed with weak bases. These findings suggest that pHG is actively regulated, despite the presence of a sizable H+ "leak" pathway. The ability of VT1B to target the Golgi complex should facilitate not only studies of acid-base regulation, but also analysis of other ionic species.

scholarly journals Inhibition of Membrane Tubule Formation and Trafficking by Isotetrandrine, an Antagonist of G-protein-regulated Phospholipase A2 Enzymes

2004 ◽  
Vol 15 (4) ◽  
pp. 1871-1880 ◽  
Author(s):  
Diane Chan ◽  
Marian Strang ◽  
Bret Judson ◽  
William J. Brown
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Golgi Complex ◽  
Brefeldin A ◽  
Enzyme Activation ◽  
Heterotrimeric G Proteins ◽  
Tubule Formation ◽  
Retrograde Trafficking ◽  
Er Exit Sites ◽  
Golgi Network

Previous studies have established a role for cytoplasmic phospholipase A2 (PLA2) activity in tubule-mediated retrograde trafficking between the Golgi complex and the endoplasmic reticulum (ER). However, little else is known about how membrane tubule formation is regulated. This study demonstrates that isotetrandrine (ITD), a biscoclaurine alkaloid known to inhibit PLA2 enzyme activation by heterotrimeric G-proteins, effectively prevented brefeldin A (BFA)-induced tubule formation from the Golgi complex and retrograde trafficking to the ER. In addition, ITD inhibited BFA-stimulated tubule formation from the trans-Golgi network and endosomes. ITD inhibition of the BFA response was potent (IC50 ∼10-20 μM) and rapid (complete inhibition with a 10-15-min preincubation). ITD also inhibited normal retrograde trafficking as revealed by the formation of nocodazole-induced Golgi mini-stacks at ER exit sites. Treatment of cells with ITD alone caused the normally interconnected Golgi ribbons to become fragmented and dilated, but cisternae were still stacked and located in a juxtanuclear position. These results suggest that a G-protein-binding PLA2 enzyme plays a pivotal role in tubule mediated trafficking between the Golgi and the ER, the maintenance of the interconnected ribbons of Golgi stacks, and tubule formation from endosomes.

GLUT4 in cultured skeletal myotubes is segregated from the transferrin receptor and stored in vesicles associated with TGN

1996 ◽  
Vol 109 (13) ◽  
pp. 2967-2978 ◽  
Author(s):  
E. Ralston ◽  
T. Ploug
Keyword(s):  
Immunoelectron Microscopy ◽  
Golgi Complex ◽  
Transferrin Receptor ◽  
Glucose Transporter ◽  
Brefeldin A ◽  
Immunogold Electron Microscopy ◽  
Trans Golgi Network ◽  
Storage Vesicles ◽  
Skeletal Myotubes ◽  
Golgi Network

There is little consensus on the nature of the storage compartment of the glucose transporter GLUT4, in non-stimulated cells of muscle and fat. More specifically, it is not known whether GLUT4 is localized to unique, specialized intracellular storage vesicles, or to vesicles that are part of the constitutive endosomal-lysosomal pathway. To address this question, we have investigated the localization of the endogenous GLUT4 in non-stimulated skeletal myotubes from the cell line C2, by immunofluorescence and immunoelectron microscopy. We have used a panel of antibodies to markers of the Golgi complex (alpha mannosidase II and giantin), of the trans-Golgi network (TGN38), of lysosomes (lgp110), and of early and late endosomes (transferrin receptor and mannose-6-phosphate receptor, respectively), to define the position of their subcellular compartments. By immunofluorescence, GLUT4 appears concentrated in the core of the myotubes. It is primarily found around the nuclei, in a pattern suggesting an association with the Golgi complex, which is further supported by colocalization with giantin and by immunogold electron microscopy. GLUT4 appears to be in the trans-most cisternae of the Golgi complex and in vesicles just beyond, i.e. in the structures that constitute the trans-Golgi network (TGN). In myotubes treated with brefeldin A, the immunofluorescence pattern of GLUT4 is modified, but it differs from both Golgi complex markers and TGN38. Instead, it resembles the pattern of the transferrin receptor, which forms long tubules. In untreated cells, double staining for GLUT4 and transferrin receptor by immunofluorescence shows similar but distinct patterns. Immunoelectron microscopy localizes transferrin receptor, detected by immunoperoxidase, to large vesicles, presumably endosomes, very close to the GLUT4-containing tubulo-vesicular elements. In brefeldin A-treated cells, a network of tubules of approximately 70 nm diameter, studded with varicosities, stains for both GLUT4 and transferrin receptor, suggesting that brefeldin A has caused fusion of the transferrin receptor and GLUT4-containing compartments. The results suggest that GLUT4 storage vesicles constitute a specialized compartment that is either a subset of the TGN, or is very closely linked to it. The link between GLUT4 vesicles and transferrin receptor containing endosomes, as revealed by brefeldin A, may be important for GLUT4 translocation in response to muscle stimulation.

A hypothesis on the traffic of MG160, a medial Golgi sialoglycoprotein, from the trans-Golgi network to the Golgi cisternae

1994 ◽  
Vol 107 (3) ◽  
pp. 529-537 ◽  
Author(s):  
P.A. Johnston ◽  
A. Stieber ◽  
N.K. Gonatas
Keyword(s):  
Sialic Acid ◽  
Golgi Apparatus ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Brefeldin A ◽  
Retrograde Transport ◽  
Transport Pathway ◽  
Trans Golgi Network ◽  
Covalently Linked ◽  
Golgi Network

We have reported that MG160, an intrinsic membrane sialoglycoprotein of the Golgi apparatus (GA), resides in the medial cisternae of the organelle (Gonatas et al. (1989) J. Biol. Chem. 264, 646–653). In order to resolve the question whether MG160 acquires sialic acid residues in the trans cisternae or trans-Golgi network (TGN) prior to its retrograde transport, we have examined the effects of brefeldin A (BFA) on the post-translational processing of MG160, and the distribution of internalized wheat germ agglutinin covalently linked with HRP (WGA-HRP), which labels the TGN (Gonatas et al. (1977) J. Cell Biol. 73, 1–13). In BFA-treated PC12 cells, MG160 acquires resistance to endo H, but fails to be sialylated. This effect occurs in parallel with the redistribution of MG160 into an ER compartment dispersed throughout the cytoplasm including the nuclear envelope, and the collapse of the WGA-HRP-labelled TGN into vesicles and tubules surrounding the centriole. These results suggest that MG160 is not sialylated in BFA-treated cells because it is sequestered from the sialyltransferase enzyme(s), presumably located in the TGN, and provide evidence supporting the hypothesis for a retrograde transport pathway that recycles resident GA proteins, including MG160, between the Golgi cisternae and the TGN. To examine further the above hypothesis we studied cells treated with BFA and then allowed to recover from the effect of the drug for various lengths of time. After 15 minutes of recovery, cisternae of the Golgi apparatus, typically found in the pericentriolar region, are labeled by both MG160 and WGA-HRP.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Compartmentation of the Golgi complex: brefeldin-A distinguishes trans-Golgi cisternae from the trans-Golgi network.

1990 ◽  
Vol 111 (3) ◽  
pp. 893-899 ◽  
Author(s):  
N W Chege ◽  
S R Pfeffer
Keyword(s):  
Sialic Acid ◽  
Golgi Complex ◽  
Brefeldin A ◽  
Fungal Metabolite ◽  
Trans Golgi Network ◽  
Lectin Affinity ◽  
Mannose 6 Phosphate ◽  
Lectin Chromatography ◽  
Golgi Network ◽  
Lines Of Evidence

The Golgi complex is composed of at least four distinct compartments, termed the cis-, medial, and trans-Golgi cisternae and the trans-Golgi network (TGN). It has recently been reported that the organization of the Golgi complex is disrupted in cells treated with the fungal metabolite, brefeldin-A. Under these conditions, it was shown that resident enzymes of the cis-, medial, and trans-Golgi return to the ER. We report here that 300-kD mannose 6-phosphate receptors, when pulse-labeled within the ER of brefeldin-A-treated cells, acquired numerous N-linked galactose residues with a half time of approximately 2 h, as measured by their ability to bind to RCA-I lectin affinity columns. In contrast, Limax flavus lectin chromatography revealed that less than 10% of these receptors acquired sialic acid after 8 h in brefeldin-A. Two lines of evidence suggested that proteins within and beyond the TGN did not return to the ER in the presence of brefeldin-A. First, the majority of 300-kD mannose 6-phosphate receptors present in the TGN and endosomes did not return to the ER after up to 6 h in brefeldin-A, as determined by their failure to contact galactosyltransferase that had relocated there. Moreover, although mannose 6-phosphate receptors did not acquire sialic acid when present in the ER of brefeldin-A-treated cells, they were readily sialylated when labeled at the cell surface and transported to the TGN. These experiments indicate that galactosyltransferase, a trans-Golgi enzyme, returns to the endoplasmic reticulum in the presence of brefeldin-A, while the bulk of sialyltransferase, a resident of the TGN, does not. Our findings support the proposal that the TGN is a distinct, fourth compartment of the Golgi apparatus that is insensitive to brefeldin-A.

scholarly journals Laa1p, a Conserved AP-1 Accessory Protein Important for AP-1 Localization in Yeast

2006 ◽  
Vol 17 (7) ◽  
pp. 3304-3317 ◽  
Author(s):  
G. Esteban Fernández ◽  
Gregory S. Payne
Keyword(s):  
Protein Transport ◽  
Brefeldin A ◽  
Coated Vesicle ◽  
Carboxypeptidase Y ◽  
Protein Distribution ◽  
Glutathione S Transferase ◽  
Growth Defects ◽  
Highly Sensitive ◽  
Stable Association ◽  
Golgi Network

AP-1 and Gga adaptors participate in clathrin-mediated protein transport between the trans-Golgi network and endosomes. Both adaptors contain homologous domains that act to recruit accessory proteins involved in clathrin-coated vesicle formation, but the spectrum of known adaptor-binding partners is limited. This study describes an evolutionarily conserved protein of Saccharomyces cerevisiae, Laa1p (Yjl207cp), that interacts and functions specifically with AP-1. Deletion of LAA1, when combined with a conditional mutation in clathrin heavy chain or deletion of GGA genes, accentuated growth defects and increased disruption of clathrin-dependent α-factor maturation and transport of carboxypeptidase Y to the vacuole. In contrast, such genetic interactions were not observed between deletions of LAA1 and AP-1 subunit genes. Laa1p preferentially interacted with AP-1 compared with Gga proteins by glutathione S-transferase-fusion affinity binding and coimmunoprecipitations. Localization of AP-1 and Laa1p, but not Gga proteins, was highly sensitive to brefeldin A, an inhibitor of ADP-ribosylation factor (Arf) activation. Importantly, deletion of LAA1 caused mislocalization of AP-1, especially in cells at high density (postdiauxic shift), but it did not affect Gga protein distribution. Our results identify Laa1p as a new determinant of AP-1 localization, suggesting a model in which Laa1p and Arf cooperate to direct stable association of AP-1 with appropriate intracellular membranes.

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