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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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BFA induces the ADP-ribosylation of BARS-50 and  GAPDH in permeabilized cells.  (A) RBL cells were permeabilized  with 3 U/ml SLO and exposed to  10 μg/ml BFA in the presence of  32P-NAD+ for 20 or 60 min at  37°C. At the end of the incubation, proteins were separated on  SDS-PAGE, and the radioactivity bound to BARS-50 and  GAPDH in the presence of BFA  was evaluated by fluorography.  Similar results were obtained in  four experiments. (B) Cytosol was  ADP-ribosylated exactly as described in Materials and Methods.  Proteins were separated on SDS-PAGE and the radioactive bands  revealed by fluorography. Only  BARS-50 and GAPDH were  detectably ADP-ribosylated by  BFA. GAPDH was also weakly  modified in the absence of the  toxin, due to a nonenzymatic  ADP-ribosylation different from  that induced by BFA (De Matteis  et al., 1994).
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Figure 3: BFA induces the ADP-ribosylation of BARS-50 and GAPDH in permeabilized cells. (A) RBL cells were permeabilized with 3 U/ml SLO and exposed to 10 μg/ml BFA in the presence of 32P-NAD+ for 20 or 60 min at 37°C. At the end of the incubation, proteins were separated on SDS-PAGE, and the radioactivity bound to BARS-50 and GAPDH in the presence of BFA was evaluated by fluorography. Similar results were obtained in four experiments. (B) Cytosol was ADP-ribosylated exactly as described in Materials and Methods. Proteins were separated on SDS-PAGE and the radioactive bands revealed by fluorography. Only BARS-50 and GAPDH were detectably ADP-ribosylated by BFA. GAPDH was also weakly modified in the absence of the toxin, due to a nonenzymatic ADP-ribosylation different from that induced by BFA (De Matteis et al., 1994).

Mentions: Cytosol and membranes were prepared from rat brain as described (De Matteis et al., 1994). Cytosol (10 mg/ml) and salt-washed membranes (2 mg/ml) were incubated in the presence or absence of 200 μM NAD+ or 100 μM BFA or both for 60 min at 37°C. Under these experimental conditions the ADP-ribosylation of BARS-50 (evaluated in parallel experiments run in the presence of 32P-NAD+) was maximal (>90%), whereas that of GAPDH was only partial (3–4%). No other proteins were detectably ADP-ribosylated by BFA (see Fig. 3). At the end of the incubation the samples were centrifuged at 100,000 g for 60 min and then the supernatants (cytosol) were dialyzed for 16 h at 4°C and used in immunofluorescence experiments in permeabilized cells as described below.


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)

BFA induces the ADP-ribosylation of BARS-50 and  GAPDH in permeabilized cells.  (A) RBL cells were permeabilized  with 3 U/ml SLO and exposed to  10 μg/ml BFA in the presence of  32P-NAD+ for 20 or 60 min at  37°C. At the end of the incubation, proteins were separated on  SDS-PAGE, and the radioactivity bound to BARS-50 and  GAPDH in the presence of BFA  was evaluated by fluorography.  Similar results were obtained in  four experiments. (B) Cytosol was  ADP-ribosylated exactly as described in Materials and Methods.  Proteins were separated on SDS-PAGE and the radioactive bands  revealed by fluorography. Only  BARS-50 and GAPDH were  detectably ADP-ribosylated by  BFA. GAPDH was also weakly  modified in the absence of the  toxin, due to a nonenzymatic  ADP-ribosylation different from  that induced by BFA (De Matteis  et al., 1994).
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Related In: Results  -  Collection

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

Figure 3: BFA induces the ADP-ribosylation of BARS-50 and GAPDH in permeabilized cells. (A) RBL cells were permeabilized with 3 U/ml SLO and exposed to 10 μg/ml BFA in the presence of 32P-NAD+ for 20 or 60 min at 37°C. At the end of the incubation, proteins were separated on SDS-PAGE, and the radioactivity bound to BARS-50 and GAPDH in the presence of BFA was evaluated by fluorography. Similar results were obtained in four experiments. (B) Cytosol was ADP-ribosylated exactly as described in Materials and Methods. Proteins were separated on SDS-PAGE and the radioactive bands revealed by fluorography. Only BARS-50 and GAPDH were detectably ADP-ribosylated by BFA. GAPDH was also weakly modified in the absence of the toxin, due to a nonenzymatic ADP-ribosylation different from that induced by BFA (De Matteis et al., 1994).
Mentions: Cytosol and membranes were prepared from rat brain as described (De Matteis et al., 1994). Cytosol (10 mg/ml) and salt-washed membranes (2 mg/ml) were incubated in the presence or absence of 200 μM NAD+ or 100 μM BFA or both for 60 min at 37°C. Under these experimental conditions the ADP-ribosylation of BARS-50 (evaluated in parallel experiments run in the presence of 32P-NAD+) was maximal (>90%), whereas that of GAPDH was only partial (3–4%). No other proteins were detectably ADP-ribosylated by BFA (see Fig. 3). At the end of the incubation the samples were centrifuged at 100,000 g for 60 min and then the supernatants (cytosol) were dialyzed for 16 h at 4°C and used in immunofluorescence experiments in permeabilized cells as described below.

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