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© The Rockefeller University Press,
0021-9525/1997//1109 $5.00
The Journal of Cell Biology, Volume 139, Number 5,
, 1997 1109-1118
Article |
Role of NAD+ and ADP-Ribosylation in the Maintenance of the Golgi Structure
Department of Biology, University of California at San Diego, La Jolla, California 92093
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.
Abbreviations used in this paper: BARS-50, BFA-dependent ADP-ribosylation substrate of 50 kD; BFA, brefeldin A; COP, coat proteins; EC, effective concentration; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Man, mannosidase; PB, permeabilization buffer; RBL, rat basophilic leukemia cells; SLO, streptolycin O.
This research was supported in part by a grant from the Italian National Research Council (Convenzione Consiglio Nazionale delle Ricerche-Consorzio Mario Negri Sud and Progetto Finalizzato contract #96.00755.PF39) and the Italian Association for Cancer Research. R. Weigert is the recipient of a fellowship from the Centro di Formazione e Studi per il Mezzogiorno.
Address all correspondence to Alexander Mironov and Alberto Luini, Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Via Nazionale, 66030 S. Maria Imbaro (Chieti), Italy. Tel.: 39.872.570.334. Fax: 39.872.578.240. E-mail: mironov{at}cmns.mnegri.it and luini{at}cmns.mnegri.it
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