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

Alexander Mironov, Antonino Colanzi, Maria Giuseppina Silletta, Giusy Fiucci, Silvio Flati, Aurora Fusella, Roman Polishchuk, Alexander Mironov, Giuseppe Di Tullio, Roberto Weigert, Vivek Malhotra, Daniela Corda, Maria Antonietta De Matteis, Alberto Luini

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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.

Original languageEnglish
Pages (from-to)1109-1118
Number of pages10
JournalJournal of Cell Biology
Volume139
Issue number5
DOIs
Publication statusPublished - Dec 1 1997

ASJC Scopus subject areas

  • Cell Biology

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    Mironov, A., Colanzi, A., Silletta, M. G., Fiucci, G., Flati, S., Fusella, A., Polishchuk, R., Mironov, A., Di Tullio, G., Weigert, R., Malhotra, V., Corda, D., De Matteis, M. A., & Luini, A. (1997). Role of NAD+ and ADP-ribosylation in the maintenance of the Golgi structure. Journal of Cell Biology, 139(5), 1109-1118. https://doi.org/10.1083/jcb.139.5.1109