Characterization of a cis-Golgi matrix protein, GM130.

Antisera raised to a detergent- and salt-resistant matrix fraction from rat liver Golgi stacks were used to screen an expression library from rat liver cDNA. A full-length clone was obtained encoding a protein of 130 kD (termed GM130), the COOH-terminal domain of which was highly homologous to a Golgi human auto-antigen, golgin-95 (Fritzler et al., 1993). Biochemical data showed that GM130 is a peripheral cytoplasmic protein that is tightly bound to Golgi membranes and part of a larger oligomeric complex. Predictions from the protein sequence suggest that GM130 is an extended rod-like protein with coiled-coil domains. Immunofluorescence microscopy showed partial overlap with medial- and trans-Golgi markers but almost complete overlap with the cis-Golgi network (CGN) marker, syntaxin5. Immunoelectron microscopy confirmed this location showing that most of the GM130 was located in the CGN and in one or two cisternae on the cis-side of the Golgi stack. GM130 was not re-distributed to the ER in the presence of brefeldin A but maintained its overlap with syntaxin5 and a partial overlap with the ER-Golgi intermediate compartment marker, p53. Together these results suggest that GM130 is part of a cis-Golgi matrix and has a role in maintaining cis-Golgi structure.

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A GRASP55-rab2 effector complex linking Golgi structure to membrane traffic

The Journal of Cell Biology ◽

10.1083/jcb.200108079 ◽

2001 ◽

Vol 155 (6) ◽

pp. 877-884 ◽

Cited By ~ 157

Author(s):

Benjamin Short ◽

Christian Preisinger ◽

Roman Körner ◽

Robert Kopajtich ◽

Olwyn Byron ◽

...

Keyword(s):

Golgi Apparatus ◽

Matrix Protein ◽

Protein Transport ◽

Coiled Coil ◽

Membrane Traffic ◽

Secretory Protein ◽

Rab Gtpase ◽

Rab Proteins ◽

Golgi Structure ◽

Golgi Matrix

Membrane traffic between the endoplasmic reticulum (ER) and Golgi apparatus and through the Golgi apparatus is a highly regulated process controlled by members of the rab GTPase family. The GTP form of rab1 regulates ER to Golgi transport by interaction with the vesicle tethering factor p115 and the cis-Golgi matrix protein GM130, also part of a complex with GRASP65 important for the organization of cis-Golgi cisternae. Here, we find that a novel coiled-coil protein golgin-45 interacts with the medial-Golgi matrix protein GRASP55 and the GTP form of rab2 but not other Golgi rab proteins. Depletion of golgin-45 disrupts the Golgi apparatus and causes a block in secretory protein transport. These results demonstrate that GRASP55 and golgin-45 form a rab2 effector complex on medial-Golgi essential for normal protein transport and Golgi structure.

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Golgi-bound Rab34 is a novel member of the secretory pathways

Clinical & Investigative Medicine ◽

10.25011/cim.v30i4.2854 ◽

2007 ◽

Vol 30 (4) ◽

pp. 82

Author(s):

Neil M. Goldenberg ◽

Sergio Grinstein ◽

Mel Silverman

Keyword(s):

Hela Cells ◽

Secretory Pathway ◽

Brefeldin A ◽

Dominant Negative ◽

Temperature Sensitive ◽

Class I Mhc ◽

Golgi Stack ◽

Vesicular Stomatitis ◽

Golgi Network ◽

Intracellular Vesicle Transport

Background: Golgi-localized Rab34 has been implicated in repositioning of lysosomes and activation of macropinocytosis. Methods: Using HeLa cells we undertook a detailed investigation of Rab34 involvement in intracellular vesicle transport. Results: Immunoelectron microscopy and immunocytochemistry confirmed that Rab34 is localized to the Golgi stack and that active Rab34 shifts lysosomes to the cell centre. Contrary to a previous report, we found that Rab34 is not concentrated at membrane ruffles and is not involved in macropinocytosis. Also, Rab34 induced repositioning of lysosomes does not affect transport of the mannose 6-phosphate receptor to endosomes. Most strikingly, HeLa cells depleted of Rab34 by transfection with dominant-negative Rab34, or following RNA interference, failed to transport the temperature-sensitive Vesicular Stomatitis Virus G-protein fused to GFP (VSVG-GFP) from the Golgi to the plasma membrane. Transfection with mouse Rab34 rescued this defect. Using endogenous MHC class I (MHC) as a marker, an endoglycosidase H resistance assay showed that ER to medial Golgi traffic remains intact in knock-down cells indicating that Rab34 specifically functions in post-Golgi transport. Further, brefeldin A treatment revealed that Rab34 acts at the Golgi, not the trans-Golgi network. Conclusion: Collectively, these results define Rab34 as a novel member of the secretory pathway acting at the Golgi.

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

Journal of Cell Science ◽

10.1242/jcs.107.4.933 ◽

1994 ◽

Vol 107 (4) ◽

pp. 933-943 ◽

Cited By ~ 3

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.

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The Golgi matrix protein giantin is required for normal cilia function in zebrafish

10.1101/117507 ◽

2017 ◽

Author(s):

Dylan J.M. Bergen ◽

Nicola L. Stevenson ◽

Roderick E.H. Skinner ◽

David J. Stephens ◽

Chrissy L. Hammond

Keyword(s):

Neural Tube ◽

Matrix Protein ◽

Intraflagellar Transport ◽

Loss Of Function ◽

Golgi Stack ◽

Summary Statement ◽

Almost All ◽

Golgi Organization ◽

Golgi Matrix

AbstractThe Golgi is essential for glycosylation of newly synthesised proteins including almost all cell-surface and extracellular matrix proteoglycans. Giantin, encoded by the golgb1 gene, is a member of the golgin family of proteins that reside within the Golgi stack but its function remains elusive. Loss-of-function of giantin in rats causes osteochondrodysplasia; knockout mice show milder defects, notably a cleft palate. In vitro, giantin has been implicated in Golgi organization, biosynthetic trafficking, and ciliogenesis. Here we show that loss-of-function of giantin in zebrafish, using either morpholino or knockout techniques, causes defects in cilia function. Giantin morphants have fewer cilia in the neural tube and those remaining are longer. Mutants have the same number of cilia in the neural tube but these cilia are also elongated. Scanning electron microscopy shows that loss of giantin results in an accumulation of material at the ciliary tip, consistent with a loss-of-function of retrograde intraflagellar transport. Mutants show milder defects than morphants consistent with adaptation to loss of giantin.Summary statementLoss of giantin following either morpholino injection or genome editing in zebrafish results in defects in ciliogenesis.

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

Biochemical Journal ◽

10.1042/bj3620359 ◽

2002 ◽

Vol 362 (2) ◽

pp. 359-366 ◽

Cited By ~ 1

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.

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Association of coagulation factor XIII-A with Golgi proteins within monocyte-macrophages: implications for subcellular trafficking and secretion

Blood ◽

10.1182/blood-2009-08-231316 ◽

2010 ◽

Vol 115 (13) ◽

pp. 2674-2681 ◽

Cited By ~ 38

Author(s):

Paul A. Cordell ◽

Benjamin T. Kile ◽

Kristina F. Standeven ◽

Emma C. Josefsson ◽

Richard J. Pease ◽

...

Keyword(s):

Plasma Membrane ◽

Factor Xiii ◽

Matrix Protein ◽

Interleukin 1 ◽

Coagulation Factor ◽

Human Monocyte ◽

Metabolic Labeling ◽

Intracellular Targeting ◽

Golgi Network ◽

Golgi Matrix

AbstractFactor XIII-A (FXIII-A) is present in the cytosol of platelets, megakaryocytes, monocytes, osteoblasts, and macrophages and may be released from cells by a nonclassical pathway. We observed that plasma FXIII-A levels were unchanged in thrombocytopenic mice (Bcl-xPlt20/Plt20 and Mpl−/−), which implicates nonclassical secretion from nucleated cells as the source of plasma FXIII-A. We, therefore, examined the intracellular targeting of FXIII-A in the THP-1 (monocyte/macrophage) cell line and in human monocyte–derived macrophages. Metabolic labeling of THP-1 cells did not show release of 35S-FXIII-A either under basal conditions or when interleukin 1-β was released in response to cell stress. However, immunofluorescence of THP-1 cells and primary macrophages showed that FXIII-A associated with podosomes and other structures adjacent to the plasma membrane, which also contain trans-Golgi network protein-46 and Golgi matrix protein-130 (GM130) but not the endoplasmic reticulum luminal protein, protein disulphide isomerase. Further, FXIII-A was present in GM130-positive intracellular vesicles that could mediate its transport, and in other contexts GM130 and its binding partner GRASP have been implicated in the delivery of nonclassically secreted proteins to the plasma membrane. Hence, this mechanism may precede FXIII-A release into the extracellular matrix from macrophages and its release into plasma from the cell type of origin.

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A Family of Proteins with γ-Adaptin and Vhs Domains That Facilitate Trafficking between the Trans-Golgi Network and the Vacuole/Lysosome

The Journal of Cell Biology ◽

10.1083/jcb.149.1.67 ◽

2000 ◽

Vol 149 (1) ◽

pp. 67-80 ◽

Cited By ~ 231

Author(s):

Jennifer Hirst ◽

Winnie W.Y. Lui ◽

Nicholas A. Bright ◽

Nicholas Totty ◽

Matthew N.J. Seaman ◽

...

Keyword(s):

Coiled Coil ◽

Coated Vesicles ◽

Carboxypeptidase Y ◽

Golgi Stack ◽

Trans Golgi Network ◽

Terminal Domain ◽

Vhs Domain ◽

Golgi Network ◽

Terminal Domains

We have cloned and characterized members of a novel family of proteins, the GGAs. These proteins contain an NH2-terminal VHS domain, one or two coiled-coil domains, and a COOH-terminal domain homologous to the COOH-terminal “ear” domain of γ-adaptin. However, unlike γ-adaptin, the GGAs are not associated with clathrin-coated vesicles or with any of the components of the AP-1 complex. GGA1 and GGA2 are also not associated with each other, although they colocalize on perinuclear membranes. Immunogold EM shows that these membranes correspond to trans elements of the Golgi stack and the TGN. GST pulldown experiments indicate that the GGA COOH-terminal domains bind to a subset of the proteins that bind to the γ-adaptin COOH-terminal domain. In yeast there are two GGA genes. Deleting both of these genes results in missorting of the vacuolar enzyme carboxypeptidase Y, and the cells also have a defective vacuolar morphology phenotype. These results indicate that the function of the GGAs is to facilitate the trafficking of proteins between the TGN and the vacuole, or its mammalian equivalent, the lysosome.

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

Biochemical Journal ◽

10.1042/bj3010069 ◽

1994 ◽

Vol 301 (1) ◽

pp. 69-73 ◽

Cited By ~ 46

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.

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