Use of brefeldin A to define sites of glycosphingolipid synthesis: GA2/GM2/GD2 synthase is trans to the brefeldin A block.

Young, William W.; Lutz, Mallory S.; Mills, S. E.; Lechler-Osborn, Susanne

doi:10.1073/pnas.87.17.6838

Cited by 88 publications

(52 citation statements)

References 25 publications

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“…The simplest interpretation of these results is that in these cell clones GalNAc-T was concentrated in the distal (TGN) part of the Golgi complex. Consistent with this, in the same clones BFA produced a complete block in the synthesis of the complex gangliosides GM2, GM1 and GD1a, as occurs in other cell systems that naturally express GalNAc-T activity [6][7][8].…”

Section: Discussionsupporting

confidence: 56%

“…Drugs affecting intra-Golgi transport [6][7][8] or distal Golgi function [9][10][11] block the synthesis of GalNAc-bearing gangliosides [12] but not that of simple gangliosides (GM3,GD3), in both neural and non-neural cells. These results indicate that GalNAc-T does not act in proximal (cis-, medial-and trans-) Golgi compartments that sustain the synthesis of simple gangliosides.…”

Section: Introductionmentioning

confidence: 99%

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GA2/GM2/GD2 synthase localizes to the trans-Golgi network of CHO-K1 cells

Giraudo

Fritz

Maccioni

1999

Biochemical Journal

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U D P - G a l N A c: l a c t o s y l c e r a m i d e / G M 3 / G D 3 β-1,4-N-a c e t y lgalactosaminyltransferase (GalNAc-T) transforms its acceptors into the gangliosides GA2, GM2 and GD2. It is well established that it is a Golgi-located glycosyltransferase, but its sub-Golgi localization is still unclear. We addressed this question in Chinese hamster ovary K1 cell clones stably transfected with a c-myc-tagged version of GalNAc-T which express the enzyme at different levels of activity. In these cell clones we examined the effect of brefeldin A (BFA) on the synthesis of glycolipids (in metabolic-labelling experiments) and on the sub-Golgi localization of the GalNAc-T (by immunocytochemistry). We found that in cell clones expressing moderate levels of activity, GalNAc-T immunoreactivity behaved as thetrans-Golgi network (TGN) marker mannose-6-P receptor (M6PR) both in BFA-treated and untreated cells, and that BFA completely blocked the synthesis of GM2, GM1 and GD1a. On the other hand, in cell clones expressing high levels of activity and treated with BFA, most GalNAc-T immunoreactivity redistributed to the endoplasmic reticulum, as did the medial-Golgi marker mannosidase II, and the synthesis of GM2, GM1 and GD1a was not completely blocked. These results indicate that GalNAc-T is a TGN-located enzyme and that the mechanism that localizes it to this compartment involves steps that, when saturated, lead to its mislocalization to the cis-, medial- or trans-Golgi. Changes of Golgi membrane properties by modification of local glycolipid composition due to the activity of the expressed enzyme were not the main cause of mislocalization, since it persists when glycolipid synthesis is inhibited with D,L-threo-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol-HCl.

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Section: Discussionsupporting

confidence: 56%

Section: Introductionmentioning

confidence: 99%

GA2/GM2/GD2 synthase localizes to the trans-Golgi network of CHO-K1 cells

Giraudo

Fritz

Maccioni

1999

Biochemical Journal

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“…These studies generally con¢rmed the presence of early acting and the absence of late acting transferases in the proximal Golgi. A tagged GalNAc transferase was not limited to the trans-Golgi [174] in contrast to predictions from brefeldin studies [145,165]. Whether or not this represents a transfection artefact has been critically discussed [173,175].…”

Section: Glycosphingolipidscontrasting

confidence: 41%

Sphingolipid transport in eukaryotic cells

Meer

Holthuis

2000

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biolog

153

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Sphingolipids constitute a sizeable fraction of the membrane lipids in all eukaryotes and are indispensable for eukaryotic life. First of all, the involvement of sphingolipids in organizing the lateral domain structure of membranes appears essential for processes like protein sorting and membrane signaling. In addition, recognition events between complex glycosphingolipids and glycoproteins are thought to be required for tissue differentiation in higher eukaryotes and for other specific cell interactions. Finally, upon certain stimuli like stress or receptor activation, sphingolipids give rise to a variety of second messengers with effects on cellular homeostasis. All sphingolipid actions are governed by their local concentration. The intricate control of their intracellular topology by the proteins responsible for their synthesis, hydrolysis and intracellular transport is the topic of this review. ß

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“…Interestingly, this change in the localization of NBD-ceramide was very similar to the change induced by BFA (Fig. 1), which has been shown to disrupt protein transport between the ER and Golgi (8)(9)(10)(11)(12)(13)(14)(15). One of the earliest events after BFA treatment is the dissociation of the 110-kDa Golgi-associated peripheral membrane protein from the Golgi membrane to the cytosol (16).…”

Section: Controlmentioning

confidence: 64%

“…Brefeldin A (BFA) is a fungal antibiotic that has been found to affect intracellular protein transport in mammalian cells (3,4). BFA inhibits protein secretion (5-7) and dramatically disintegrates the Golgi apparatus, resulting in the redistribution of the Golgi proteins into the ER (8)(9)(10)(11)(12)(13)(14)(15). It has been suggested that BFA inhibits the anterograde membrane transport from the ER to the Golgi without affecting the retrograde transport from the Golgi to the ER (5,8,10,13).…”

mentioning

confidence: 99%

Bacterial ADP-ribosyltransferase with a substrate specificity of the rho protein disassembles the Golgi apparatus in Vero cells and mimics the action of brefeldin A.

Sugai

Chen

1992

Proc. Natl. Acad. Sci. U.S.A.

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Epidermal-cell differentiation inhibitor (EDIN) is an exoenzyme produced by Staphylococcus aureus that catalyzes the ADP-ribosylation of rho proteins, members of the small GTP-binding protein family. In this study we demonstrate that EDIN induces a rapid morphological change in the Golgi structure of monkey kidney Vero cells that is similar to the changes elicited by brefeldin A (BFA). Treatment of Vero cells with EDIN resulted in a rapid disappearance of N-7-(4-nitrobenzo-2-oxa-1,3-diazole)-6-aminocaproylsphingosine, a 110-kDa protein (13-COP, coat protein), and mannoidase II from the Golgi structure. Lower doses of EDIN and BFA had a synergistic effect on the redistribution ofthe Golgi markers. The similities in the effects of EDIN and BFA in Vero cells also include the EDIN-or BFA-mediated protection of Vero cells from ricin cytotoxicity and prevention of the effects of EDIN or

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Use of brefeldin A to define sites of glycosphingolipid synthesis: GA2/GM2/GD2 synthase is trans to the brefeldin A block.

Cited by 88 publications

References 25 publications

GA2/GM2/GD2 synthase localizes to the trans-Golgi network of CHO-K1 cells

GA2/GM2/GD2 synthase localizes to the trans-Golgi network of CHO-K1 cells

Sphingolipid transport in eukaryotic cells

Bacterial ADP-ribosyltransferase with a substrate specificity of the rho protein disassembles the Golgi apparatus in Vero cells and mimics the action of brefeldin A.

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