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Stimulation of Endogenous ADP-Ribosylation by Brefeldin A

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Stimulation of Endogenous ADP-Ribosylation by Brefeldin A Proc. Nadl. Acad. Sci. USA Vol. 91, pp. 1114-1118, February 1994 Cell Biology Stimulation of endogenous ADP-ribosylation by brefeldin A MARIA ANTONIETTA DE MATTEIS*, MARIA Di GIROLAMOt, ANTONINO COLANZI*, MERCE PALLASt, GIUSEPPE Di TULLIOt, LEE J. MCDONALD§, JOEL Moss§, GIOVANNA SANTINI*, SERGEI BANNYKHt, DANIELA CORDAt, AND ALBERTO LUINIt *Unit of Physiopathology of Secretion, tLaboratory of Molecular and Cellular Endocrinology, and tLaboratory of Molecular Neurobiology, Istituto di Ricerche Farmacologiche "Mario Negri," Consorzio Mario Negri Sud, 66030 S. Maria Imbaro (Chieti), Italy; and kLaboratory of Cellular Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892 Communicated by Martin Rodbell, October 4, 1993 ABSTRACT Brefeldin A (BFA) is a fungal metabolite that ribosyl)transferase (11). Indeed, a family of brain exerts profound and general Inhibitor actions on membrane mono(ADP-ribosyl)transferases has been reported to be sen- transport. At least some ofthe BFA effects are due to inhibition sitive to ARF (14). These considerations prompted us to of the GDP-GTP exchange on the ADP-ribosylation factor determine whether BFA, possibly by perturbing ARF bind- (ARF) catalyzed by membrane protein(s). ARF activation is ing, might affect cellular ADP-ribosylations. Here we report likely to be a key event In the of non-dathrln coat that BFA markedly stimulates the ADP-ribosylation of two components, including ARF Itself, onto tranp organelles. cytosolic proteins of38 and 50 kDa (p38 and p50). p38 appears ARF, In addition to partidpating In membrane trnsport, is to be identical with an isoform of glyceraldehyde-3- known to function as a cofactor in the enzymatic activit of phosphate dehydrogenase (GAPDH), a glycolytic enzyme cholera toxin, a bacterial ADP-ribosyltansferase. In this study and a multifunctional protein that has been implicated in we have examined whether BFA, in addition to inhibiting several cellular processes (15-22). The binding components membrane transport, might affect endogenous ADP- mediating the effects of BFA on ADP-ribosylation appear to ribosylation in eukaryotic cells. Two cytosolic proteins of 38 possess the same ligand specificity as that of the BFA and 50 kDa were enzymatically ADP-ribosylated In the pres- receptor involved in inhibiting membrane traffic and ARF ence of BFA in cellular extracts. The 38-kDa sbre was binding to the Golgi complex. These results demonstrate the tentatively identified as the glycolytic enzyme glyceraldehyde- existence of a BFA-sensitive ADP-ribosyltransferase that 3-phosphate dehydrogenase. The BFA-binding nts may play a role in membrane traffic. mediating inhibition of membrane traffic and stimulaton of ADP-ribosylatlon appear to have the same lgand icit. These data demonstrate the existence of a BFA-seisltive MATERIALS AND METHODS mono(ADP-rlbosyl)transferase that may play a role in mem- Materials. NAD, GAPDH, nucleotides, BFA, sodium ni- brane movements. troprusside, nicotinamide, isoniazide, phenylmethylsulfonyl fluoride,'leupeptin, Coon's modified Ham's F-12 medium, The fungal toxin brefeldin A (BFA) has been widely used to ADP-ribose, and AMP were from Sigma. Crotalus adaman- analyze the mechanisms of membrane transport. The effects teus venom phosphodiesterase I was from Worthington. of BFA include the disappearance of non-clathrin-coated Tissue culture materials were from GIBCO. GTP and ATP buds and transport vesicles, the inhibition of constitutive were from Boebringer Mannheim. [32PJNAD was from Du- secretion, and a series of changes in'shape, location, and pont/NEN, and [adenine-14C]NAD and [nicotinamide- function of the organelles of the exocytic and endocytic 14C]NAD were from Amersham. Pertussis toxin was kindly pathways (1-4). These changes are preceded and probably, provided by Rino Rappuoli (Istituto Ricerche Immunobio- at least in part, caused by the release ofa set ofproteins from logiche, Siena, Italy); BFA analogues B36 and B5 were a gift the Golgi complex including two major non-clathrin coat from J. Donaldson (National Institutes of Health, Bethesda, proteins, the ADP-ribosylation factor (ARF, a small Ras-like MD). The monoclonal antibody against ARRl (1D9) was a GTPase) and 8COP, a component of the cytosolic protein gift from R. Kahn (National Institutes of Health, Bethesda, complex "coatomer" (5-7). Moreover, BFA inhibits the MD). GDP-GTP exchage on ARF catalyzed by a Gojgi protein Preparation of Postnudear Supernatant. Rat basophilic and the binding ofARF to Golgi membranes, suggesting that leukemia (RBL) and Fischer rat thyroid line 5 (FRTL5) cells the components involved in ARF association to transport were grown as described (23, 24). Postnuclear supernatant, organelles may be the primary targets of BFA and a key site cytosol, and membranes from both cell lines were prepared of regulation of vesicular transport pathways (8, 9). (24, 25). ARF, in addition to being involved in membrane transport, ADP-ribosylatio Assay. Samples (50 pg of protein) were has long been known as a cofactor in the ADP-ribosylation of incubated at 370C for 60 min (unless otherwise specified) in the a subunit ofthe GTP-binding protein Gs by cholera toxin 50 A4 of either 100 mM potassium phosphate buffer, pH and to be able to interact directly with cholera toxin (10-12). 7.5/2.5 mM MgCl2/1 mM ATP/1 mM GTP/10 mM thymi- ADP-ribosylation is a posttranslational modification of pro- dine/5 mM dithiothreitol (ADP-ribosylation buffer) or (when teins produced by the transfer of ADP-ribose from NAD to specified) ARF binding buffer (26) containing 0.5 POCi of specific amino acid residues, which can be catalyzed by both [32P]NAD (specific activity, 800 Ci/mmol; 1 Ci = 37 GBq). bacterial toxins and eukaryotic enzymes (13). Analogous to BFA was stored (10 mg/ml) in dimethyl sulfoxide at -20oC. its role in the activation of the exogenous ADP-ribosyltrans- Appropriate control samples with dimethyl sulfoxide were ferase cholera toxin, one of the physiological functions of present in all the experiments. Samples were analyzed by ARF may be to activate an endogenous cellular mono(ADP- Abbreviations: BFA, brefeldin A; ARF, ADP-ribosylation factor; The publication costs ofthis article were defrayed in part by page charge GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RBL, rat ba- payment. This article must therefore be hereby marked "advertisement" sophilic leukemia; FRTL5, Fischer rat thyroid line 5; IEF, isoelectric in accordance with 18 U.S.C. §1734 solely to indicate this fact. focusing; NADase, NAD glycohydrolase. 1114 Downloaded by guest on September 27, 2021 Cell Biology: De Matteis et al. Proc. Natl. Acad. Sci. USA 91 (1994) 1115 SDS/PAGE (8% SDS with 4 M urea) (24) followed by A B autoradiography and densitometry (LKB ultroscan-XL). kDa a b cd e f Ch ARF-Binding Assay. The assay was performed as described 50- c (26). Membrane-bound ARF was assessed by immunoblot U with the monoclonal anti-ARF antibody 1D9. ._ Production of [32P]ADP-ribose. [32P]NAD (0.5 uCi per Q w sample) was incubated in 15 Al with 25 ,ug of FRTL5 cell 38- m_ d membranes, possessing high NAD glycohydrolase (NADase) activity (24), for 20 min at 370C and centrifuged. The super- natant was used as a source of ADP-ribose. After this C Time, min incubation 90o of the radioactivity was in ADP-ribose and co 10% in ADP, and NAD was undetectable (data not shown). D Isoelectric Focusing (IEF)-SDS/PAGE. For IEF the O'Far- (_ rell procedure (27) with the following modifications was used: 20- Ampholines (pH 3.5-10) were used at a concentration of 3% - 3 10 30 100 300 a-0 in the gel but not added to the sample buffer, and the sample 100 200 BFA, gg/ml was loaded at the acidic end of the gel rod. After IEF (16 hr 10 20 at 400 V), SDS/PAGE was performed in an 8-15% gradient BFA, rig/ml gel. Other Methods. Assay of the sensitivity of the ADP- FIG. 1. BFA-induced ADP-ribosylation ofa 38-kDa and a 50-kDa ribosylated protein to NH2OH and HgCl2 (28), TLC analysis protein in RBL and FRTL5 cells. (A) NAD, but not ADP-ribose, of NAD breakdown products (29), and snake venom phos- supports the BFA-induced ADP-ribosylation. Postnuclear superna- phodiesterase digestion ofADP-ribosylated GAPDH (29, 30) tants (50 ttg of protein per sample) from RBL cells (lanes a and b) or were performed as described. FRTL5 cells (lanes c-f were incubated with [32P]NAD under the conditions for the ADP-ribosylation assay in the absence (lanes a, c, and e) or presence (lanes b, d, and f) of BFA (30 ,ug/ml) for 60 min RESULTS at 37C. For lanes e and f, [32P]ADP-ribose (prepared as described in Materials and Methods) was added to the reaction mixture. Similar BFA Induces Labeling of Two Cellular Proteins in the results were obtained in at least five different experiments run in Presence of [32P]NAD. The effect of BFA on protein labeling duplicate. (B) Time course of basal and BFA-induced ADP- in the presence of [32P]NAD was examined in postnuclear ribosylation of p38. Postnuclear supernatants from RBL cells were supernatant from RBL and FRTL5 cells. In the presence of incubated with [32P]NAD in the absence (n) or presence (o) of BFA [32PJNAD, BFA induced a dose- and time-dependent labeling (30 ug/ml) for the indicated times at 37C. The proteins were oftwo proteins of38 and 50 kDa (Fig. 1). The lowest effective analyzed by SDS/PAGE followed by autoradiography and densi- concentration of BFA varied 2 and tometry. Similar results were obtained in three different experiments between 5 Mg/ml and the run in duplicate, with SD < 5%. (C) Dose dependence of the EC50 between 15 and 30 ,g/ml in different experiments.
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