Role of NAD 1 and ADP - Ribosylation in the Maintenance of the Golgi Structure

We have investigated the role of the ADPribosylation 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 1 (the ADP-ribose donor) or by using selective pharmacological blockers in permeabilized cells. In NAD 1 -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 1 triggered Golgi disassembly by BFA. This effect of NAD 1 was mimicked by the use of pre–ADPribosylated 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. T he Golgi apparatus is a complex structure that can be schematically viewed as composed of two basic elements: flat disc-shaped cisternae and tubularreticular networks. Groups of three to eight cisternae piled in stacks are in continuity with cisternae of adjacent stacks through tubular-reticular elements. The overall tridimensional appearance of the Golgi complex is therefore ribbon-like, with alternating compact (stacked cisternae) and noncompact (tubular-reticular) zones; the cis and trans poles of the complex are made mostly of tubular networks (Tanaka et al., 1986; Rambourg and Clermont, 1990; Clermont et al., 1994). A notable feature of these structures is that despite their complexity they are highly dynamic: stacks can rapidly change shape and tubules can be seen to emanate from, or retract to, the cisternae under a variety of conditions (Lippincott-Schwartz et al., 1989; Cole et al., 1996). Given the central role of the Golgi complex in the secretory process, there is much interest in understanding the molecular mechanisms responsible for generating and maintaining the organelle’s structure as well as the relationships existing between such structure and the organelle’s functions. However, although recent significant progress mainly based on studies of Golgi reassembly after fragmentation induced by the toxin ilimaquinone or durAddress 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: [email protected] and luini@cmns. mnegri.it on M ay 4, 2017 D ow nladed fom Published December 1, 1997