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 Intracellular movement of two mannose 6-phosphate receptors: return to the Golgi apparatus.

1988 ◽  
Vol 106 (3) ◽  
pp. 617-628 ◽  
Author(s):  
J R Duncan ◽  
S Kornfeld
Keyword(s):  
Sialic Acid ◽  
Golgi Apparatus ◽  
Chinese Hamster ◽  
Murine Lymphoma ◽  
Trans Golgi Network ◽  
Intracellular Movement ◽  
Golgi Region ◽  
Golgi Compartment ◽  
Mannose 6 Phosphate ◽  
Golgi Network

We have used Chinese hamster ovary (CHO) cells and a murine lymphoma cell line to study the recycling of the 215-kD and the 46-kD mannose 6-phosphate receptors to various regions of the Golgi to determine the site where the receptors first encounter newly synthesized lysosomal enzymes. For assessing return to the trans-most Golgi compartments containing sialyltransferase (trans-cisternae and trans-Golgi network), the oligosaccharides of receptor molecules on the cell surface were labeled with [3H]galactose at 4 degrees C. Upon warming to 37 degrees C, the [3H]galactose residues on both receptors were substituted with sialic acid with a t1/2 approximately 3 hrs. Other glycoproteins acquired sialic acid at least 8-10 times slower. Return of the receptors to the trans-Golgi cisternae containing galactosyltransferase could not be detected. Return to the cis/middle Golgi cisternae containing alpha-mannosidase I was measured by adding deoxymannojirimycin, a mannosidase I inhibitor, during the initial posttranslational passage of [3H]mannose-labeled glycoproteins through the Golgi, thereby preserving oligosaccharides which would be substrates for alpha-mannosidase I. After removal of the inhibitor, return to the early Golgi with subsequent passage through the Golgi complex was measured by determining the conversion of the oligosaccharides from high mannose to complex-type units. This conversion was very slow for the receptors and other glycoproteins (t1/2 approximately 20 h). Exposure of the receptors and other glycoproteins to the dMM-sensitive alpha-mannosidase without movement through the Golgi apparatus was determined by measuring the loss of mannose residues from these proteins. This loss was also slow. These results indicate that both Man-6-P receptors routinely return to the Golgi compartment which contains sialyltransferase and recycle through other regions of the Golgi region less frequently. We infer that the trans-Golgi network is the major site for lysosomal enzyme sorting in CHO and murine lymphoma cells.

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.

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 Lysosomal Hydrolase Mannose 6-Phosphate Uncovering Enzyme Resides in the trans-Golgi Network

2001 ◽  
Vol 12 (6) ◽  
pp. 1623-1631 ◽  
Author(s):  
Jack Rohrer ◽  
Rosalind Kornfeld
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Lysosomal Hydrolases ◽  
Intracellular Location ◽  
Trans Golgi Network ◽  
Cytosolic Domain ◽  
Cytosolic Domains ◽  
Mannose 6 Phosphate ◽  
Green Fluorescent ◽  
Golgi Network

A crucial step in lysosomal biogenesis is catalyzed by “uncovering” enzyme (UCE), which removes a coveringN-acetylglucosamine from the mannose 6-phosphate (Man-6-P) recognition marker on lysosomal hydrolases. This study shows that UCE resides in the trans-Golgi network (TGN) and cycles between the TGN and plasma membrane. The cytosolic domain of UCE contains two potential endocytosis motifs: 488YHPL and C-terminal 511NPFKD. YHPL is shown to be the more potent of the two in retrieval of UCE from the plasma membrane. A green-fluorescent protein-UCE transmembrane-cytosolic domain fusion protein colocalizes with TGN 46, as does endogenous UCE in HeLa cells, showing that the transmembrane and cytosolic domains determine intracellular location. These data imply that the Man-6-P recognition marker is formed in the TGN, the compartment where Man-6-P receptors bind cargo and are packaged into clathrin-coated vesicles.

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 Oligomerization of atrans-Golgi/trans-Golgi Network Retained Protein Occurs in the Golgi Complex and May Be Part of Its Retention

1995 ◽  
Vol 270 (15) ◽  
pp. 8815-8821 ◽  
Author(s):  
Jacomine Krijnse Locker ◽  
Dirk-Jan E. Opstelten ◽  
Maria Ericsson ◽  
Marian C. Horzinek ◽  
Peter J. M. Rottier
Keyword(s):  
Golgi Complex ◽  
Trans Golgi Network ◽  
Golgi Network

Nucleotide depletion increases trafficking of gentamicin to the Golgi complex in LLC-PK1 cells

2002 ◽  
Vol 283 (6) ◽  
pp. F1422-F1429 ◽  
Author(s):  
Ruben M. Sandoval ◽  
Robert L. Bacallao ◽  
Kenneth W. Dunn ◽  
Jeffrey D. Leiser ◽  
Bruce A. Molitoris
Keyword(s):  
Cell Culture ◽  
Golgi Complex ◽  
Lens Culinaris ◽  
Ricinus Communis ◽  
Physiological State ◽  
Complex Mechanism ◽  
Trans Golgi Network ◽  
Golgi Network ◽  
Texas Red ◽  
Number Of Cells

Having shown rapid trafficking of aminoglycosides to the Golgi complex in cell culture, we focused on the injurious interaction that occurs when gentamicin administration is preceded by renal ischemia. Using Texas red-labeled gentamicin as a tracer, we determined that 15 min of cellular nucleotide depletion did not significantly increase subsequent uptake. However, cells previously depleted of nucleotides accumulated significantly more Texas red-labeled gentamicin within a dispersed Golgi complex. Using Ricinus communis and Lens culinaris lectins, which label specific compartments of the Golgi complex ( trans-Golgi network/ trans and medial/ cis compartments, respectively), we determined that the medial/ cis compartment dispersed after 15 min of nucleotide depletion but the trans-Golgi network/ trans compartment remained unaffected. An increase in the number of cells exhibiting disrupted medial/ cis-Golgi morphology after repletion in physiological media containing gentamicin was also seen. In summary, the increase in nephrotoxicity seen when ischemia precedes aminoglycoside uptake may be part of a complex mechanism initially involving increased Golgi accumulation and prolonged Golgi dispersion. The Golgi complex must then endure the effects of gentamicin accumulated in larger quantities in an aberrant physiological state.

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.

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