Brefeldin A causes structural and functional alterations of the trans-Golgi network of MDCK cells

1994 ◽  
Vol 107 (4) ◽  
pp. 933-943 ◽  
Author(s):  
M. Wagner ◽  
A.K. Rajasekaran ◽  
D.K. Hanzel ◽  
S. Mayor ◽  
E. Rodriguez-Boulan
Keyword(s):  
Mdck Cells ◽  
Brefeldin A ◽  
Adaptor Proteins ◽  
Fungal Metabolite ◽  
Surface Transport ◽  
Structural Alterations ◽  
Golgi Stack ◽  
Trans Golgi Network ◽  
Influenza Hemagglutinin ◽  
Golgi Network

The trans-Golgi network (TGN) of MDCK cells is exquisitely sensitive to the fungal metabolite brefeldin A (BFA), in contrast to the refractory Golgi stack of these cells. At a concentration of 1 microgram/ml, BFA promoted extensive tubulation of the TGN while the medical Golgi marker alpha-mannosidase II was not affected. Tubules emerging minutes after addition of the drug contained both the apical marker influenza hemagglutinin (HA), previously accumulated at 20 degrees C, and the fusion protein interleukin receptor/TGN38 (TGG), a TGN marker that recycles basolaterally, indicating that, in contrast to TGN vesicles, TGN-derived tubules cannot sort apical and basolateral proteins. After 60 minutes treatment with BFA, HA and TGG tubules formed extensive networks widely spread throughout the cell, different from the focused centrosomal localization previously described in non-polarized cells. The TGG network partially codistributed with an early endosomal tubular network loaded with transferrin, suggesting that the TGG and endosomal networks had fused or that TGG had entered the endosomal network via surface recycling and endocytosis. The extensive structural alterations of the TGN were accompanied by functional disruptions, such as the extensive mis-sorting of influenza HA, and by the release of the TGN marker gamma-adaptin. Our results suggest the involvement of BFA-sensitive adaptor proteins in TGN-->surface transport.

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.

Effect of brefeldin A on heparan sulphate biosynthesis in Madin–Darby canine kidney cells

2002 ◽  
Vol 362 (2) ◽  
pp. 359-366 ◽  
Author(s):  
Svein Olav KOLSET ◽  
Kristian PRYDZ ◽  
Katja FJELDSTAD ◽  
Fariba SAFAIYAN ◽  
Tram Thu VUONG ◽  
...  
Keyword(s):  
Mdck Cells ◽  
Heparan Sulphate ◽  
Brefeldin A ◽  
Sodium Chlorate ◽  
Cell Types ◽  
Madin Darby Canine Kidney ◽  
Golgi Stack ◽  
Golgi Network ◽  
Darby Canine Kidney ◽  
Canine Kidney

Brefeldin A (BFA) perturbs the organization of the Golgi apparatus, such that Golgi stack components are fused with the endoplasmic reticulum (ER) and separated from the trans-Golgi network. In many cell types, BFA blocks the secretion of macromolecules but still allows the action of Golgi enzymes in the ER. Treatment of cells with BFA has been reported to inhibit the secretion of heparan sulphate (HS) proteoglycans and alter the structure of their HS components, but the nature of such structural alterations has not been characterized in detail. We analysed the effect of BFA on HS biosynthesis in Madin—Darby canine kidney (MDCK) cells, in which the Golgi complex is more resistant towards BFA than in most other cell types. We found that MDCK cells were able to secrete HS proteoglycans in spite of BFA treatment. However, the secretion of HS was reduced and the secreted HS differed from that produced by untreated cells. In BFA-treated cells, two structurally distinct pools of HS were generated. One pool was similar to HS from control cells, with the exception that the 6-O-sulphation of glucosamine (GlcN) residues was reduced. In contrast, the other pool consisted of largely unmodified N-acetylheparosan polymers with a low (<20%) proportion of N-sulphated GlcN residues but a substantial proportion of N-unsubstituted GlcN units, indicating that it had been acted upon by N-deacetylases and partly by the N-sulphotransferases, but not by O-sulphotransferases. Together, these findings represent a previously unrecognized alteration in HS biosynthesis caused by BFA, and differ dramatically from our previous findings in MDCK cells pertaining to the undersulphation of HS caused by sodium chlorate treatment.

scholarly journals SMAP2, a Novel ARF GTPase-activating Protein, Interacts with Clathrin and Clathrin Assembly Protein and Functions on the AP-1–positive Early Endosome/Trans-Golgi Network

2006 ◽  
Vol 17 (6) ◽  
pp. 2592-2603 ◽  
Author(s):  
Waka Natsume ◽  
Kenji Tanabe ◽  
Shunsuke Kon ◽  
Naomi Yoshida ◽  
Toshio Watanabe ◽  
...  
Keyword(s):  
Brefeldin A ◽  
Adaptor Proteins ◽  
Early Endosome ◽  
Dependent Manner ◽  
Transferrin Receptors ◽  
Gtpase Activating Protein ◽  
Trans Golgi Network ◽  
Early Endosomes ◽  
Golgi Network

We recently reported that SMAP1, a GTPase-activating protein (GAP) for Arf6, directly interacts with clathrin and regulates the clathrin-dependent endocytosis of transferrin receptors from the plasma membrane. Here, we identified a SMAP1 homologue that we named SMAP2. Like SMAP1, SMAP2 exhibits GAP activity and interacts with clathrin heavy chain (CHC). Furthermore, we show that SMAP2 interacts with the clathrin assembly protein CALM. Unlike SMAP1, however, SMAP2 appears to be a regulator of Arf1 in vivo, because cells transfected with a GAP-negative SMAP2 mutant were resistant to brefeldin A. SMAP2 colocalized with the adaptor proteins for clathrin AP-1 and EpsinR on the early endosomes/trans-Golgi-network (TGN). Moreover, overexpression of SMAP2 delayed the accumulation of TGN38/46 molecule on the TGN. This suggests that SMAP2 functions in the retrograde, early endosome-to-TGN pathway in a clathrin- and AP-1–dependent manner. Thus, the SMAP gene family constitutes an important ArfGAP subfamily, with each SMAP member exerting both common and distinct functions in vesicle trafficking.

scholarly journals Okadaic acid treatment leads to a fragmentation of the trans-Golgi network and an increase in expression of TGN38 at the cell surface

1994 ◽  
Vol 301 (1) ◽  
pp. 69-73 ◽  
Author(s):  
M Horn ◽  
G Banting
Keyword(s):  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Acid Treatment ◽  
Brefeldin A ◽  
Fold Increase ◽  
Integral Membrane Protein ◽  
Golgi Stack ◽  
Trans Golgi Network ◽  
Marker Proteins ◽  
Golgi Network

Okadaic acid (OA) is a protein phosphatase inhibitor which has, among other properties, previously been shown to induce a fragmentation of the cisternae of the Golgi stack [for review, see Lucocq (1992) J. Cell Sci. 103, 875-880]. The effects of OA an reversible and mimic intracellular events which occur during mitosis. To date, due to a lack of endogenous marker proteins, the effects of OA on the trans-Golgi network (TGN) has not been studied. Certain drugs, e.g. Brefeldin A (BFA), have different effects on the morphology of the Golgi stack and the TGN; it is therefore relevant to ask what effect(s) OA has on the TGN. We now present data from a study in which we have used antibodies to TGN38, an integral membrane protein predominantly localized to the TGN of rat NRK cells [Luzio, Brake, Banting, Howell, Braghetta and Stanley (1990) Biochem. J. 270, 97-102], to investigate the effects of OA on this organelle. OA induces a reversible fragmentation of the TGN. This fragmentation occurs with similar kinetics to that observed within the Golgi stack, and is independent of protein synthesis. The sensitivity of the TGN to OA is similar to that of the Golgi stack. The fragmentation of the TGN induced by OA also leads to a 10-fold increase in the level of TGN38 expressed at the plasma membrane.

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.

scholarly journals Structural disruption of the trans-Golgi network does not interfere with the acute stimulation of glucose and amino acid uptake by insulin-like growth factor I in muscle cells

1994 ◽  
Vol 297 (2) ◽  
pp. 289-295 ◽  
Author(s):  
H S Hundal ◽  
P J Bilan ◽  
T Tsakiridis ◽  
A Marette ◽  
A Klip
Keyword(s):  
Amino Acid ◽  
Brefeldin A ◽  
Glucose Transporters ◽  
Amino Acid Uptake ◽  
Acid Uptake ◽  
Trans Golgi Network ◽  
Maximal Activation ◽  
Igf I ◽  
Golgi Network ◽  
Stimulation Of

The effects of insulin-like growth factor I (IGF-I) on glucose and amino acid uptake were investigated in fully differentiated L6 muscle cells, in order to determine whether the two processes are functionally related. Transport of both glucose and amino acid (methylaminoisobutyric acid, MeAIB) was activated rapidly in response to IGF-I. Stimulation reached a peak within 30 min and was sustained for up to 90 min. Maximal activation of either glucose or MeAIB transport was achieved at 3 nM IGF-I; the half-maximal activation (ED50) of glucose transport was at 107 pM and that of MeAIB transport was at 36 pM. Stimulation of amino acid uptake occurred in the absence or presence of glucose, suggesting that this response is not secondary to increased glucose intake. Incubation of cells for 1 h with Brefeldin A (5 micrograms/ml), which disassembles the Golgi apparatus and inhibits the secretory pathway in eukaryotic cells, had no effect on the acute IGF-I activation of glucose and MeAIB transport. Moreover, Brefeldin A caused wide redistribution of the trans-Golgi antigen TGN38, as assessed by subcellular fractionation, without affecting the distribution of glucose transporters. The finding that the degree of activation, time response and sensitivity to IGF-I and Brefeldin A were similar for both glucose and MeAIB transport suggests commonalities in the IGF-I mechanism of recruitment of glucose transporters and stimulation of amino acid transport through System A. An integral trans-Golgi network does not appear to be required for the acute IGF-I stimulation of glucose or amino acid transport, even though stimulation of glucose transport occurs through recruitment of glucose transporters from intracellular stores in these cells. We propose that the donor site of glucose transporters (and perhaps of amino acid transporters) involved in the acute response to IGF-I lies beyond the trans-Golgi network, perhaps in an endosomal compartment in close proximity to the plasma membrane.

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.

Sign in / Sign up

Export Citation Format

Share Document