Steady-state localization of a medial-Golgi glycosyltransferase involves transit through the trans-Golgi network

2001 ◽  
Vol 358 (1) ◽  
pp. 33-40 ◽  
Author(s):  
Andrew S. OPAT ◽  
Fiona HOUGHTON ◽  
Paul A. GLEESON
Keyword(s):  
Steady State ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Retrograde Transport ◽  
Asymmetric Distribution ◽  
Transport Pathways ◽  
Golgi Stack ◽  
Trans Golgi Network ◽  
Golgi Network

The steady-state localization of medial-Golgi enzymes is likely to involve retrograde transport pathways; however, the trafficking of these resident enzymes through the Golgi stack is unclear. To investigate if the medial-Golgi enzyme β-1,2-N-acetylglucosaminyltransferase I (GlcNAc-TI) is transported to the late Golgi, a modified GlcNAc-TI bearing an N-glycan site on the C-terminus was constructed. The modified GlcNAc-TI was demonstrated to be functionally active in vivo, and was localized to the Golgi stack of transfected cells. In stable Chinese-hamster ovary (CHO) cell clones, the N-glycosylated GlcNAc-TI carried sialylated complex N-glycan chains. Pulse-chase studies showed that the majority of GlcNAc-TI was sialylated within 60min of synthesis. Treatment of transfected CHO cells with Brefeldin A resulted in the glycosylated GlcNAc-TI bearing endo-β-N-acetylglucosaminidase H resistant chains; however, the sialylation of glycosylated GlcNAc-TI was dramatically reduced. These data imply that, in CHO cells, newly synthesized GlcNAc-TI is transported rapidly through the Golgi stack to the trans-Golgi network, suggesting that GlcNAc-TI continuously recycles from the late Golgi. Furthermore, this data suggests that retrograde transport pathways play an important role in establishing the asymmetric distribution of GlcNAc-TI within the Golgi stack.

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 FIP1/RCP Binding to Golgin-97 Regulates Retrograde Transport from Recycling Endosomes to the trans-Golgi Network

2010 ◽  
Vol 21 (17) ◽  
pp. 3041-3053 ◽  
Author(s):  
Jian Jing ◽  
Jagath R. Junutula ◽  
Christine Wu ◽  
Jemima Burden ◽  
Hugo Matern ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Binding Protein ◽  
Retrograde Transport ◽  
Recycling Endosome ◽  
Transport Pathways ◽  
Recycling Endosomes ◽  
Trans Golgi Network ◽  
C Terminus ◽  
Transport Vesicles ◽  
Golgi Network

Many proteins are retrieved to the trans-Golgi Network (TGN) from the endosomal system through several retrograde transport pathways to maintain the composition and function of the TGN. However, the molecular mechanisms involved in these distinct retrograde pathways remain to be fully understood. Here we have used fluorescence and electron microscopy as well as various functional transport assays to show that Rab11a/b and its binding protein FIP1/RCP are both required for the retrograde delivery of TGN38 and Shiga toxin from early/recycling endosomes to the TGN, but not for the retrieval of mannose-6-phosphate receptor from late endosomes. Furthermore, by proteomic analysis we identified Golgin-97 as a FIP1/RCP-binding protein. The FIP1/RCP-binding domain maps to the C-terminus of Golgin-97, adjacent to its GRIP domain. Binding of FIP1/RCP to Golgin-97 does not affect Golgin-97 recruitment to the TGN, but appears to regulate the targeting of retrograde transport vesicles to the TGN. Thus, we propose that FIP1/RCP binding to Golgin-97 is required for tethering and fusion of recycling endosome-derived retrograde transport vesicles to the TGN.

scholarly journals The Golgin GCC88 Is Required for Efficient Retrograde Transport of Cargo from the Early Endosomes to the Trans-Golgi Network

2007 ◽  
Vol 18 (12) ◽  
pp. 4979-4991 ◽  
Author(s):  
Zi Zhao Lieu ◽  
Merran C. Derby ◽  
Rohan D. Teasdale ◽  
Charles Hart ◽  
Priscilla Gunn ◽  
...  
Keyword(s):  
Shiga Toxin ◽  
Retrograde Transport ◽  
Transport Pathway ◽  
Transport Pathways ◽  
Recycling Endosomes ◽  
Trans Golgi Network ◽  
Early Endosomes ◽  
Cargo Proteins ◽  
Mannose 6 Phosphate ◽  
Golgi Network

Retrograde transport pathways from early/recycling endosomes to the trans-Golgi network (TGN) are poorly defined. We have investigated the role of TGN golgins in retrograde trafficking. Of the four TGN golgins, p230/golgin-245, golgin-97, GCC185, and GCC88, we show that GCC88 defines a retrograde transport pathway from early endosomes to the TGN. Depletion of GCC88 in HeLa cells by interference RNA resulted in a block in plasma membrane–TGN recycling of two cargo proteins, TGN38 and a CD8 mannose-6-phosphate receptor cytoplasmic tail fusion protein. In GCC88-depleted cells, cargo recycling was blocked in the early endosome. Depletion of GCC88 dramatically altered the TGN localization of the t-SNARE syntaxin 6, a syntaxin required for endosome to TGN transport. Furthermore, the transport block in GCC88-depleted cells was rescued by syntaxin 6 overexpression. Internalized Shiga toxin was efficiently transported from endosomes to the Golgi of GCC88-depleted cells, indicating that Shiga toxin and TGN38 are internalized by distinct retrograde transport pathways. These findings have identified an essential role for GCC88 in the localization of TGN fusion machinery for transport from early endosomes to the TGN, and they have allowed the identification of a retrograde pathway which differentially selects TGN38 and mannose-6-phosphate receptor from Shiga toxin.

GGA proteins regulate retrograde transport of BACE1 from endosomes to the trans-Golgi network

2005 ◽  
Vol 29 (3) ◽  
pp. 453-461 ◽  
Author(s):  
Tina Wahle ◽  
Kai Prager ◽  
Nikolai Raffler ◽  
Christian Haass ◽  
Michael Famulok ◽  
...  
Keyword(s):  
Retrograde Transport ◽  
Trans Golgi Network ◽  
Golgi Network

The steady state distribution of humTGN46 is not significantly altered in cells defective in clathrin-mediated endocytosis

1998 ◽  
Vol 111 (23) ◽  
pp. 3451-3458 ◽  
Author(s):  
G. Banting ◽  
R. Maile ◽  
E.P. Roquemore
Keyword(s):  
Steady State ◽  
Cell Surface ◽  
Dominant Negative ◽  
Type I ◽  
Steady State Distribution ◽  
State Distribution ◽  
Trans Golgi Network ◽  
Defective Mutant ◽  
Dynamin I ◽  
Golgi Network

It has been shown previously that whilst the rat type I integral membrane protein TGN38 (ratTGN38) is predominantly localised to the trans-Golgi network this protein does reach the cell surface from where it is internalised and delivered back to the trans-Golgi network. This protein thus provides a suitable tool for the investigation of trafficking pathways between the trans-Golgi network and the cell surface and back again. The human orthologue of ratTGN38, humTGN46, behaves in a similar fashion. These proteins are internalised from the cell surface via clathrin mediated endocytosis, a process which is dependent upon the GTPase activity of dynamin. We thus reasoned that humTGN46 would accumulate at the surface of cells rendered defective in clathrin mediated endocytosis by virtue of the fact that they express a GTPase defective mutant of dynamin I. It did not. In fact, expression of a dominant negative GTPase defective mutant of dynamin I had no detectable effect on the steady state distribution of humTGN46. One explanation for this observation is that humTGN46 does not travel directly to the cell surface from the trans-Golgi network. Further studies on cells expressing the dominant negative GTPase defective mutant of dynamin I indicate that the major recycling pathway for humTGN46 is in fact between the trans-Golgi network and the early endosome.

Suppression of tumor formation in vivo by expression of the JE gene in malignant cells

1991 ◽  
Vol 11 (6) ◽  
pp. 3125-3131
Author(s):  
B J Rollins ◽  
M E Sunday
Keyword(s):  
Cho Cells ◽  
Chinese Hamster ◽  
Suppressive Effect ◽  
Tumor Formation ◽  
Host Animal ◽  
Early Growth Response ◽  
Discernible Effect ◽  
Early Growth Response Gene

The early growth response gene JE encodes a monocyte chemoattractant, MCP-1. The JE/MCP-1 protein attracts and stimulates human monocytes and induces monocyte-mediated inhibition of tumor cell growth in vitro. Expression of human or murine JE/MCP-1 in Chinese hamster ovary (CHO) cells completely suppressed their ability to form tumors in nude mice. Coinjection of JE/MCP-1-expressing cells with nonexpressing CHO cells or with HeLa cells also prevented tumor formation. Since JE/MCP-1 expression had no discernible effect on the tranformed phenotype of these cells in vitro, the suppressive effect depends on host animal factors. These factors are likely to be components of the inflammatory response, because JE/MCP-1-expressing cells elicited a predominantly monocytic infiltrate at the site of injection. Our results suggest that JE/MCP-1 protein may be useful in cancer therapy.

scholarly journals Hydrophobic glycosides of N-acetylglucosamine can act as primers for polylactosamine synthesis and can affect glycolipid synthesis in vivo

1995 ◽  
Vol 307 (3) ◽  
pp. 791-797 ◽  
Author(s):  
D C A Neville ◽  
R A Field ◽  
M A J Ferguson
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Culture Medium ◽  
Linear Array ◽  
Chinese Hamster ◽  
Living Cells ◽  
Anomeric Configuration ◽  
Glycolipid Synthesis ◽  
Synthetic Capacity

Several hydrophobic glycosides of N-acetylglucosamine (GlcNAc) served as primers for polylactosamine synthesis when added to Chinese hamster ovary (CHO) cells. The modified glycosides, containing one to six lactosamine repeats in linear array, were sialylated and secreted into the culture medium. The relative efficiencies of the glycosides to serve as primers were dependent on the nature of the aglycone and on the anomeric configuration of the GlcNAc residue. The same compounds were tested for their effects on glycolipid synthesis in CHO cells. All of the beta-glycosides significantly inhibited the synthesis of the lactoseries glycolipid GM3 whereas the alpha-glycoside was inactive. The compound GlcNAc alpha 1-O-benzyl- was the most efficient primer of polylactosamine synthesis and had no effect on glycolipid synthesis. This compound may have potential for the assay of the polylactosamine synthetic capacity of living cells.

scholarly journals A versatile nanobody-based toolkit to analyze retrograde transport from the cell surface

2018 ◽  
Vol 115 (27) ◽  
pp. E6227-E6236 ◽  
Author(s):  
Dominik P. Buser ◽  
Kai D. Schleicher ◽  
Cristina Prescianotto-Baschong ◽  
Martin Spiess
Keyword(s):  
Electron Microscopy ◽  
Cell Surface ◽  
Adaptor Protein ◽  
Retrograde Transport ◽  
Transport Pathways ◽  
Consensus Sequences ◽  
Retrograde Traffic ◽  
Biochemical Detection ◽  
Golgi Network ◽  
Kinetics Of

Retrograde transport of membranes and proteins from the cell surface to the Golgi and beyond is essential to maintain homeostasis, compartment identity, and physiological functions. To study retrograde traffic biochemically, by live-cell imaging or by electron microscopy, we engineered functionalized anti-GFP nanobodies (camelid VHH antibody domains) to be bacterially expressed and purified. Tyrosine sulfation consensus sequences were fused to the nanobody for biochemical detection of trans-Golgi arrival, fluorophores for fluorescence microscopy and live imaging, and APEX2 (ascorbate peroxidase 2) for electron microscopy and compartment ablation. These functionalized nanobodies are specifically captured by GFP-modified reporter proteins at the cell surface and transported piggyback to the reporters’ homing compartments. As an application of this tool, we have used it to determine the contribution of adaptor protein-1/clathrin in retrograde transport kinetics of the mannose-6-phosphate receptors from endosomes back to the trans-Golgi network. Our experiments establish functionalized nanobodies as a powerful tool to demonstrate and quantify retrograde transport pathways.

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.

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