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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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Dicumarol prevents the tubular-reticular transformation of the Golgi apparatus induced by BFA. RBL cells were  treated with the indicated BFA concentrations for 15 min after a  30-min pretreatment with 200 μM dicumarol. They were then processed for electron microscopy. Dicumarol (and ilimaquinone,  not shown) prevents the tubular-reticular transformation and disappearance of the Golgi stacks induced by moderate (a), but not  by high concentrations of BFA (b). Similar results were obtained  in three independent experiments run in duplicate. Bar, 0.5 μm.
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Figure 6: Dicumarol prevents the tubular-reticular transformation of the Golgi apparatus induced by BFA. RBL cells were treated with the indicated BFA concentrations for 15 min after a 30-min pretreatment with 200 μM dicumarol. They were then processed for electron microscopy. Dicumarol (and ilimaquinone, not shown) prevents the tubular-reticular transformation and disappearance of the Golgi stacks induced by moderate (a), but not by high concentrations of BFA (b). Similar results were obtained in three independent experiments run in duplicate. Bar, 0.5 μm.

Mentions: As recently reported, several compounds belonging to two different chemical groups, one containing a coumarin, and the other a quinone ring, act as inhibitors of the BFA- dependent ADP-ribosylation in vitro (Weigert et al., 1997). Dicumarol and ilimaquinone, a marine sponge metabolite that causes the gradual and reversible breakdown of the Golgi complex (Takizawa et al., 1993), are relatively potent and nontoxic representatives of the two classes of compounds. Remarkably, ADP-ribosylation inhibitors are able to antagonize the BFA-induced redistribution of Golgi enzymes into the ER in intact cells (Weigert et al., 1997), with potencies similar to those observed in assays of BFA-dependent ADP-ribosylation in vitro. This suggests that they inhibit the BFA-induced Golgi disassembly by inhibiting ADP-ribosylation. We wanted to directly test this possibility by assessing whether ADP-ribosylation inhibitors would lose their effect in permeabilized cells exposed to pre–ADP-ribosylated cytosol. The effect of these agents on the fine structure of the Golgi complex, both in intact and permeabilized cells, was first characterized. In intact cells, as expected, a prominent early effect of BFA was the disorganization and tubular-vesicular transformation of the Golgi complex (Fig. 2 b); dicumarol (Fig. 6 a) or ilimaquinone (not shown) strongly inhibited these alterations and, in fact, afforded a remarkable preservation of the stack structure. The effects of the inhibitors, in line with previous data (Weigert et al., 1997), were dose-dependent and dependent on the dose of BFA, since higher concentrations of the toxin overcame the inhibition (Fig. 6 b). In permeabilized cells, dicumarol had very similar effects to those seen in vivo in that it inhibited the BFA and NAD+-induced Golgi disassembly in the presence of control cytosol (Fig. 7 b). It was then tested whether dicumarol would maintain its ability to antagonize BFA in the presence of pre–ADP-ribosylated cytosol. Remarkably, under these conditions, the effect of dicumarol was largely prevented (Fig. 7 d). Furthermore, when the pre–ADP-ribosylated cytosol was complemented with enriched native (nonADP-ribosylated) BARS-50 and NAD+ (Fig. 7 f), dicumarol regained its property to prevent the BFA-induced redistribution of the Golgi apparatus. GAPDH had no effect (not shown). The addition of pre–ADP-ribosylated BARS-50 under the same conditions was unable to restore dicumarol activity (Fig. 7 h). These experiments indicate that dicumarol either acts by preventing the ADP-ribosylation of BARS-50 or that it requires unmodified BARS-50 to exert its effects.


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)

Dicumarol prevents the tubular-reticular transformation of the Golgi apparatus induced by BFA. RBL cells were  treated with the indicated BFA concentrations for 15 min after a  30-min pretreatment with 200 μM dicumarol. They were then processed for electron microscopy. Dicumarol (and ilimaquinone,  not shown) prevents the tubular-reticular transformation and disappearance of the Golgi stacks induced by moderate (a), but not  by high concentrations of BFA (b). Similar results were obtained  in three independent experiments run in duplicate. Bar, 0.5 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 6: Dicumarol prevents the tubular-reticular transformation of the Golgi apparatus induced by BFA. RBL cells were treated with the indicated BFA concentrations for 15 min after a 30-min pretreatment with 200 μM dicumarol. They were then processed for electron microscopy. Dicumarol (and ilimaquinone, not shown) prevents the tubular-reticular transformation and disappearance of the Golgi stacks induced by moderate (a), but not by high concentrations of BFA (b). Similar results were obtained in three independent experiments run in duplicate. Bar, 0.5 μm.
Mentions: As recently reported, several compounds belonging to two different chemical groups, one containing a coumarin, and the other a quinone ring, act as inhibitors of the BFA- dependent ADP-ribosylation in vitro (Weigert et al., 1997). Dicumarol and ilimaquinone, a marine sponge metabolite that causes the gradual and reversible breakdown of the Golgi complex (Takizawa et al., 1993), are relatively potent and nontoxic representatives of the two classes of compounds. Remarkably, ADP-ribosylation inhibitors are able to antagonize the BFA-induced redistribution of Golgi enzymes into the ER in intact cells (Weigert et al., 1997), with potencies similar to those observed in assays of BFA-dependent ADP-ribosylation in vitro. This suggests that they inhibit the BFA-induced Golgi disassembly by inhibiting ADP-ribosylation. We wanted to directly test this possibility by assessing whether ADP-ribosylation inhibitors would lose their effect in permeabilized cells exposed to pre–ADP-ribosylated cytosol. The effect of these agents on the fine structure of the Golgi complex, both in intact and permeabilized cells, was first characterized. In intact cells, as expected, a prominent early effect of BFA was the disorganization and tubular-vesicular transformation of the Golgi complex (Fig. 2 b); dicumarol (Fig. 6 a) or ilimaquinone (not shown) strongly inhibited these alterations and, in fact, afforded a remarkable preservation of the stack structure. The effects of the inhibitors, in line with previous data (Weigert et al., 1997), were dose-dependent and dependent on the dose of BFA, since higher concentrations of the toxin overcame the inhibition (Fig. 6 b). In permeabilized cells, dicumarol had very similar effects to those seen in vivo in that it inhibited the BFA and NAD+-induced Golgi disassembly in the presence of control cytosol (Fig. 7 b). It was then tested whether dicumarol would maintain its ability to antagonize BFA in the presence of pre–ADP-ribosylated cytosol. Remarkably, under these conditions, the effect of dicumarol was largely prevented (Fig. 7 d). Furthermore, when the pre–ADP-ribosylated cytosol was complemented with enriched native (nonADP-ribosylated) BARS-50 and NAD+ (Fig. 7 f), dicumarol regained its property to prevent the BFA-induced redistribution of the Golgi apparatus. GAPDH had no effect (not shown). The addition of pre–ADP-ribosylated BARS-50 under the same conditions was unable to restore dicumarol activity (Fig. 7 h). These experiments indicate that dicumarol either acts by preventing the ADP-ribosylation of BARS-50 or that it requires unmodified BARS-50 to exert its effects.

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