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Role of NAD+ and ADP-ribosylation in the maintenance of the Golgi structure.

Mironov A, Colanzi A, Silletta MG, Fiucci G, Flati S, Fusella A, Polishchuk R, Mironov A, Di Tullio G, Weigert R, Malhotra V, Corda D, De Matteis MA, Luini A - J. Cell Biol. (1997)

Bottom Line: This effect of NAD+ was mimicked by the use of pre-ADP- ribosylated cytosol.Biol.These results indicate that ADP-ribosylation plays a role in the Golgi disassembling activity of BFA, and suggest that the ADP-ribosylated substrates are components of the machinery controlling the structure of the Golgi apparatus.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Oncology, Istituto di Ricerche Farmacologiche Mario Negri, Consorzio Mario Negri Sud, 66030 Santa Maria Imbaro (Chieti), Italy. mironov@cmns.mnegri.it

ABSTRACT
We have investigated the role of the ADP- ribosylation induced by brefeldin A (BFA) in the mechanisms controlling the architecture of the Golgi complex. BFA causes the rapid disassembly of this organelle into a network of tubules, prevents the association of coatomer and other proteins to Golgi membranes, and stimulates the ADP-ribosylation of two cytosolic proteins of 38 and 50 kD (GAPDH and BARS-50; De Matteis, M.A., M. DiGirolamo, A. Colanzi, M. Pallas, G. Di Tullio, L.J. McDonald, J. Moss, G. Santini, S. Bannykh, D. Corda, and A. Luini. 1994. Proc. Natl. Acad. Sci. USA. 91:1114-1118; Di Girolamo, M., M.G. Silletta, M.A. De Matteis, A. Braca, A. Colanzi, D. Pawlak, M.M. Rasenick, A. Luini, and D. Corda. 1995. Proc. Natl. Acad. Sci. USA. 92:7065-7069). To study the role of ADP-ribosylation, this reaction was inhibited by depletion of NAD+ (the ADP-ribose donor) or by using selective pharmacological blockers in permeabilized cells. In NAD+-depleted cells and in the presence of dialized cytosol, BFA detached coat proteins from Golgi membranes with normal potency but failed to alter the organelle's structure. Readdition of NAD+ triggered Golgi disassembly by BFA. This effect of NAD+ was mimicked by the use of pre-ADP- ribosylated cytosol. The further addition of extracts enriched in native BARS-50 abolished the ability of ADP-ribosylated cytosol to support the effect of BFA. Pharmacological blockers of the BFA-dependent ADP-ribosylation (Weigert, R., A. Colanzi, A. Mironov, R. Buccione, C. Cericola, M.G. Sciulli, G. Santini, S. Flati, A. Fusella, J. Donaldson, M. DiGirolamo, D. Corda, M.A. De Matteis, and A. Luini. 1997. J. Biol. Chem. 272:14200-14207) prevented Golgi disassembly by BFA in permeabilized cells. These inhibitors became inactive in the presence of pre-ADP-ribosylated cytosol, and their activity was rescued by supplementing the cytosol with a native BARS-50-enriched fraction. These results indicate that ADP-ribosylation plays a role in the Golgi disassembling activity of BFA, and suggest that the ADP-ribosylated substrates are components of the machinery controlling the structure of the Golgi apparatus.

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Ultrastructure of the Golgi complex in permeabilized  cells: NAD+ is required for the effect of BFA. Intact RBL cells (a  and b) were treated with 3 μg/ml BFA for 15 min (b), or were  permeabilized with 3 U/ml SLO (c–f), and incubated for 20 min  at 37°C in the absence (c) or in the presence of BFA alone (d), or  of 150 μM NAD+ (e), or of BFA in combination with 150 μM  NAD+ (f). The cells were then processed for electron microscopy. Experiments were repeated at least three times in duplicate  with similar results. Bar, 0.5 μm.
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Figure 2: Ultrastructure of the Golgi complex in permeabilized cells: NAD+ is required for the effect of BFA. Intact RBL cells (a and b) were treated with 3 μg/ml BFA for 15 min (b), or were permeabilized with 3 U/ml SLO (c–f), and incubated for 20 min at 37°C in the absence (c) or in the presence of BFA alone (d), or of 150 μM NAD+ (e), or of BFA in combination with 150 μM NAD+ (f). The cells were then processed for electron microscopy. Experiments were repeated at least three times in duplicate with similar results. Bar, 0.5 μm.

Mentions: To examine the effects of NAD+ on the Golgi structure, cells were depleted of the nucleotide by a permeabilization protocol designed to selectively porate the plasma membrane. The resulting membrane damage and cell rounding caused a less than perfect resolution of the Golgi complex at the immunofluorescence level; however, the redistribution of Golgi markers into the ER by BFA remained easily detectable. Moreover, the fine structure of the organelle was very well preserved (see Figs. 1 and 2). It is known that the permeabilization with SLO results in the rapid loss of most low molecular weight soluble molecules, including NAD+, from the cell interior (Bhakdi et al., 1993). In addition, the brain cytosol normally used in experiments with permeabilized cells was extensively dialyzed. Fig. 1 shows that whereas in intact cells BFA caused the expected rapid and complete diffuse redistribution of Golgi markers (with ∼50% effective concentration [EC50] of 0.3 μg/ml), in NAD+-depleted cells (permeabilized and exposed to dialyzed cytosol), the toxin dramatically lost activity (Fig. 1 d) even when used at concentrations up to 100-fold higher than those active in intact cells (not shown). The loss of BFA activity was not due to ATP depletion or microtubule depolymerization (both conditions are known to impede Golgi redistribution), because these experiments were routinely conducted in the presence of an ATP-regenerating system and the microtubule stabilizer taxol.


Role of NAD+ and ADP-ribosylation in the maintenance of the Golgi structure.

Mironov A, Colanzi A, Silletta MG, Fiucci G, Flati S, Fusella A, Polishchuk R, Mironov A, Di Tullio G, Weigert R, Malhotra V, Corda D, De Matteis MA, Luini A - J. Cell Biol. (1997)

Ultrastructure of the Golgi complex in permeabilized  cells: NAD+ is required for the effect of BFA. Intact RBL cells (a  and b) were treated with 3 μg/ml BFA for 15 min (b), or were  permeabilized with 3 U/ml SLO (c–f), and incubated for 20 min  at 37°C in the absence (c) or in the presence of BFA alone (d), or  of 150 μM NAD+ (e), or of BFA in combination with 150 μM  NAD+ (f). The cells were then processed for electron microscopy. Experiments were repeated at least three times in duplicate  with similar results. Bar, 0.5 μm.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2140198&req=5

Figure 2: Ultrastructure of the Golgi complex in permeabilized cells: NAD+ is required for the effect of BFA. Intact RBL cells (a and b) were treated with 3 μg/ml BFA for 15 min (b), or were permeabilized with 3 U/ml SLO (c–f), and incubated for 20 min at 37°C in the absence (c) or in the presence of BFA alone (d), or of 150 μM NAD+ (e), or of BFA in combination with 150 μM NAD+ (f). The cells were then processed for electron microscopy. Experiments were repeated at least three times in duplicate with similar results. Bar, 0.5 μm.
Mentions: To examine the effects of NAD+ on the Golgi structure, cells were depleted of the nucleotide by a permeabilization protocol designed to selectively porate the plasma membrane. The resulting membrane damage and cell rounding caused a less than perfect resolution of the Golgi complex at the immunofluorescence level; however, the redistribution of Golgi markers into the ER by BFA remained easily detectable. Moreover, the fine structure of the organelle was very well preserved (see Figs. 1 and 2). It is known that the permeabilization with SLO results in the rapid loss of most low molecular weight soluble molecules, including NAD+, from the cell interior (Bhakdi et al., 1993). In addition, the brain cytosol normally used in experiments with permeabilized cells was extensively dialyzed. Fig. 1 shows that whereas in intact cells BFA caused the expected rapid and complete diffuse redistribution of Golgi markers (with ∼50% effective concentration [EC50] of 0.3 μg/ml), in NAD+-depleted cells (permeabilized and exposed to dialyzed cytosol), the toxin dramatically lost activity (Fig. 1 d) even when used at concentrations up to 100-fold higher than those active in intact cells (not shown). The loss of BFA activity was not due to ATP depletion or microtubule depolymerization (both conditions are known to impede Golgi redistribution), because these experiments were routinely conducted in the presence of an ATP-regenerating system and the microtubule stabilizer taxol.

Bottom Line: This effect of NAD+ was mimicked by the use of pre-ADP- ribosylated cytosol.Biol.These results indicate that ADP-ribosylation plays a role in the Golgi disassembling activity of BFA, and suggest that the ADP-ribosylated substrates are components of the machinery controlling the structure of the Golgi apparatus.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Oncology, Istituto di Ricerche Farmacologiche Mario Negri, Consorzio Mario Negri Sud, 66030 Santa Maria Imbaro (Chieti), Italy. mironov@cmns.mnegri.it

ABSTRACT
We have investigated the role of the ADP- ribosylation induced by brefeldin A (BFA) in the mechanisms controlling the architecture of the Golgi complex. BFA causes the rapid disassembly of this organelle into a network of tubules, prevents the association of coatomer and other proteins to Golgi membranes, and stimulates the ADP-ribosylation of two cytosolic proteins of 38 and 50 kD (GAPDH and BARS-50; De Matteis, M.A., M. DiGirolamo, A. Colanzi, M. Pallas, G. Di Tullio, L.J. McDonald, J. Moss, G. Santini, S. Bannykh, D. Corda, and A. Luini. 1994. Proc. Natl. Acad. Sci. USA. 91:1114-1118; Di Girolamo, M., M.G. Silletta, M.A. De Matteis, A. Braca, A. Colanzi, D. Pawlak, M.M. Rasenick, A. Luini, and D. Corda. 1995. Proc. Natl. Acad. Sci. USA. 92:7065-7069). To study the role of ADP-ribosylation, this reaction was inhibited by depletion of NAD+ (the ADP-ribose donor) or by using selective pharmacological blockers in permeabilized cells. In NAD+-depleted cells and in the presence of dialized cytosol, BFA detached coat proteins from Golgi membranes with normal potency but failed to alter the organelle's structure. Readdition of NAD+ triggered Golgi disassembly by BFA. This effect of NAD+ was mimicked by the use of pre-ADP- ribosylated cytosol. The further addition of extracts enriched in native BARS-50 abolished the ability of ADP-ribosylated cytosol to support the effect of BFA. Pharmacological blockers of the BFA-dependent ADP-ribosylation (Weigert, R., A. Colanzi, A. Mironov, R. Buccione, C. Cericola, M.G. Sciulli, G. Santini, S. Flati, A. Fusella, J. Donaldson, M. DiGirolamo, D. Corda, M.A. De Matteis, and A. Luini. 1997. J. Biol. Chem. 272:14200-14207) prevented Golgi disassembly by BFA in permeabilized cells. These inhibitors became inactive in the presence of pre-ADP-ribosylated cytosol, and their activity was rescued by supplementing the cytosol with a native BARS-50-enriched fraction. These results indicate that ADP-ribosylation plays a role in the Golgi disassembling activity of BFA, and suggest that the ADP-ribosylated substrates are components of the machinery controlling the structure of the Golgi apparatus.

Show MeSH
Related in: MedlinePlus