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. BFA-induced ADP-ribosylation of p38. Postnuclear supernatants Labeling of p38 was detectable also in control samples and from RBL cells were incubated with [32P]NAD in the presence of was markedly (7-fold) enhanced by BFA. A 38-kDa protein various concentrations of BFA for 60 min at 37°C. The proteins were identified as GAPDH has been reported to covalently bind analyzed by SDS/PAGE, autoradiography, and densitometry. Sim- [32P]NAD in the presence ofNO (30-32). We tested the effect ilar results were obtained in three different experiments run in of NO in RBL cells. Sodium nitroprusside, a NO-generating duplicate, with SD < 5%. (D) Dose dependence ofthe BFA-induced agent, induced the appearance of a 38-kDa labeled band on inhibition of ARF binding to Golgi membranes in vitro. Golgi SDS/PAGE and of three labeled spots with sizes of 38 kDa membranes and cytosol from RBL cells were incubated at 37°C for and pI values of 7.2, 7.4, and 7.6 on IEF-SDS/PAGE (Fig. 30 min in the presence of the indicated concentrations of BFA and of 20 jLM guanosine 5'-[-thio]triphosphate (a nonhydrolyzable an- 2). The substrate at pI 7.4 comigrated precisely in IEF-SDS/ alogue of GTP) during the last 15 min of incubation. The amount of PAGE with the single protein spot labeled in the presence of membrane-bound ARF (at 20 kDa) was assessed by immunoblotting. BFA, indicating that the latter is a GAPDH isoform (Fig. 2B). Furthermore, commercial purified GAPDH from rabbit skel- shown), consistent with mono(ADP-ribosyl)ation. The cova- etal muscle became labeled in a [32P]NAD-dependent manner lent binding ofNAD to GAPDH, as induced by NO (32), was by cellular extracts containing BFA. Its migration in SDS/ excluded in the case ofthe BFA-induced reaction, since BFA PAGE was identical to that of the 38-kDa substrate of the stimulated the incorporation of only adenine from NAD BFA-stimulated reaction from RBL cells (Fig. 3). To examine (Table 1). The chemical reactivity of the BFA-stimulated whether NO could interfere with the BFA-stimulated reac- linkage of radiolabel from [32P]NAD with GAPDH was tion, a cell extract was pretreated with NO and nonradioac- assessed. tive NAD. This pretreatment markedly decreased the ability The incorporated radioactivity was stable to treat- of NO to stimulate radioactive labeling of GAPDH in a ment with NH2OH or HgCl2 (Fig. 4A) but was sensitive to subsequent incubation, whereas it had no effect on the acid and base (Table 2). The product released by HCl from modification stimulated by BFA (Fig. 4B). [32P]GAPDH comigrated with standard ADP-ribose on an- p50 has a size in SDS/PAGE in the same range as that of ion-exchange HPLC (32), again consistent with ADP- tubulin and of the larger form of the G, a subunit, both ribosylation. The background labeling of GAPDH that was GTP-binding proteins that can be ADP-ribosylated by cellu- observed in the absence of BFA had different properties. lar mono(ADP-ribosyl)transferases (11, 33, 34). However, This linkage was sensitive to HgCl2 or base but was stable in neither protein comigrated in urea/SDS/PAGE with the acid and so was attributed to nonenzymatic ADP-ribosylation labeled p50 band (data not shown). (see below) of the reactive cysteine residue in GAPDH, as Characterization of the BFA-Stimulated Modification as has been described (35). As ADP-ribosylation can be due to Enzymatic ADP-ribosylation. p38 radiolabeled in the presence nonenzymic formation of adducts of acceptor proteins with of BFA and [32P]NAD was separated by SDS/PAGE, trans- ADP-ribose generated by NADases, we used free radioactive ferred to nitrocellulose, and incubated with snake venom ADP-ribose as a substrate instead ofNAD (see Materials and phosphodiesterase. A large fraction (70%) of the protein- Methods). No labeling ofp50, and a very slight labeling ofp38 bound radioactivity from BFA-treated samples became sol- by [32P]ADP-ribose, was observed with or without BFA, uble and was identified as AMP by TLC and HPLC (data not under conditions where [32P]NAD fully supported the BFA- Downloaded by guest on September 27, 2021 1116 Cell Biology: De Matteis et al. Proc. Natl. Acad. Sci. USA 91 (1994)

A A a b c d B + a b c de f g h kDa kDa kDa 38- 40- a a 50 -- 38-a MP Q

38 - b B a b c d e f Ca b c d e f

38- 38 FIG. 4. Comparison between the ADP-ribosylation reactions induced by NO and BFA. (A) Sensitivity of the ADP-ribosylated FiG. 2. ADP-ribosylation ofp38 in the presence of BFA and NO. products to HgCl2 and NH2OH. Postnuclear supernatants of RBL (A) Postnuclear supernatants of RBL cells were incubated with cells were incubated in the presence of P2P]NAD plus BFA at 30 [32P]NAD for the ADP-ribosylation assay in the presence of buffer jug/ml (lanes a-c), 100 pM sodium nitroprusside (lanes d-), or 10 nM (lane a), BFA at 30 pg/ml (lane b), or sodium nitroprusside at 100 ,M pertussis toxin (lanes g-i) for 60 min at 370C. The reaction was (lane c) or 200 iLM (lane d) at 37C for 60 min. Proteins were then stopped by the addition ofcold 1o (wt/vol) trichloroacetic acid and analyzed by SDS/PAGE and autoradiography. Similar results were then suspended in a solution containing 1 mM NaCl (lanes a, d, and obtained in at least five experiments run in duplicate. (B) Postnuclear g), 1 mM HgCI2 (lanes b, e, and h), or 500 mM NH2OH at pH 7 (lanes supernatants of RBL cells were incubated with (32P]NAD plus BFA c, f, and i). The incubation was stopped after 60 iin by the addition at 30 Mg/ml (gel a), 100 pM sodium nitroprusside (gel b), or BFA and of Laemmli sample buffer and then proteins were analyzed by sodium nitroprusside (gel c). Proteins were then analyzed by IEF- SDS/PAGE and autoradiography. Similarresults were obtained in at SDS/PAGE and autoradiography. Each experiment was repeated at least four experiments run in duplicate. (B) Effect of pretreatment least four times (in duplicate), with similar results. with NO on the ADP-ribosylation induced by NO and BFA. Post- nuclear supernatants ofRBL cells (50 Mg ofprotein per sample) were stimulated reaction (Fig. 1A). Moreover, neither 10 mM preincubated with buffer (lanes a-c) or 200 pM sodium nitroprusside isoniazide, an inhibitor of some NADases (36), nor 1 mM (lanes d- for60 min at 3rC in the presence of10 pM nonradioactive nonradioactive ADP-ribose affected the reaction with NAD. At the end of the pretreatment cell extracts were incubated [32P]NAD (data not shown). BFA did not affect endogenous with [32P]NAD for 60 min at 37-C in the presence of buffer (lanes a NADases (data not shown). and d), BFA at 30 pg/ml (lanes b and e), or 100 pAM sodium Requirement for Cellular Factors for the BFA-Stimulated nitroprusside (lanes c and f). Proteins were then analyzed by SDS/ PAGE and autoradiography. Similar results were obtained in four Reacton. BFA, unlike NO, depended on the presence of separate experiments run in duplicate. (C) Requirement for cellular postnuclear supernatant to stimulate [32P]NAD-dependent factors for BFA-induced, but not for NO-induced, ADP-ribosylation labeling of p38 and p50 (Fig. 4C). Postnuclear supernatant of GAPDH. Purified rabbit muscle GAPDH (10 pg per sample) was was separated into membranes and cytosol by ultracentrifu- incubated with (32P]NAD and buffer (anes a and d), 100 pM sodium gation (100,000 x g). Both fractions supported the BFA- nitroprusside (lanes b and e), or BFA at 30 pug/ml (lanes c and 0f in induced ADP-ribosylation of purified GAPDH, but the mem- the absence (lanes a-c) or presence (lanes d-) of cell extract (50 pug branes had a much higher specific activity (Fig. 3 D-f). The of protein per sample) under the conditions described of the ADP- ribosylation assay. Proteins were then analyzed by SDS/PAGE and results are consistent with the BFA-dependent enzyme being autoradiography. Each experiment was repeated at least three times mostly (but not exclusively) membrane-bound and with the (in duplicate), with similar results. substrates (GAPDH and p50) being mostly cytosolic (Fig. 3 A-C). Membranes largely retained their BFA-dependent result argues against a role for the reactive C-3 atom of BFA ADP-ribosyltransferase activity after salt washing (2 M KCl) (38), which is present in B5, in stimulating ADP-ribosylation. but not after boiling. Treatment with 1% (wt/vol) Triton The concentrations of BFA that stimulate ADP-ribosylation X-100 or 3-[(3-cholamidopropyl)dimethylammonio]-2- hydroxy-l-propanesulfonate resulted in nearly complete sol- were similar to those that inhibit ARF binding to Golgi ubilization of the enzyme without loss of activity or sensi- membranes in vitro, under identical experimental conditions, tivity to BFA (data not shown). the EC50 values being 15-30 and -15 ug/ml in the ARF Posinble Relationships Between ADP-ribosylation and Mem- binding and ADP-ribosylation assays, respectively (refs. 9 brane Transport. We examined whether the BFA effects on and 39 and Fig. 1 C and D). These data are consistent with the ADP-ribosylation and those on membrane transport might be possibility that the same binding sites mediate the BFA mediated by the same binding components. The methyl ester effects on ARF binding and ADP-ribosylation. Of note, the and epoxide derivatives of BFA (B5 and B36, respectively; potency ofBFA in both ofthese in vitro assays (ARE binding see refs. 8 and 37), which are inactive on membrane traffic and ARF binding, were also inactive in stimulating ADP- Table 1. Incorporation of radiolabel into GAPDH from ribosylation of GAPDH and p5O (Table 3). Incidentally, this [nicotinamide-14C]NAD or [adenine-14C]NAD NAD, nmol/mol of GAPDH A B C D E F [nicotinamide-14C]/ Incubation [nicotinamide-14C] [adenine-14C] [adenine-14C] Control 0.03 ± 0.02 0.2 ± 0.07 0.15 ± 0.16 kDa- 38 i Nitroprusside 11.3 + 0.44 10.8 ± 0.91 1.05 ± 0.13 4mo.00'.~ ~ ~ _Vw- 4 :X aa; BFA 0.09 ± 0.04 1.45 ± 0.07 0.06 ± 0.03

BFA - + - + - + - + - + - + GAPDH (200 Mig) was incubated with 0.1 mM [adenine-14C]NAD or [nicotinanaide-14C]NAD (0.44 ACi/ml) in 0.1 mM potassium FIG. 3. BFA-induced ADP-ribosylation of authentic purified phosphate, pH 7.5/10 mM thymidine/2.5 mM MgCl2/5 mM dithio- GAPDH. Postnuclear supernatants (A and D), membranes (B and E), threitol (total volume, 0.1 ml). When present, BFA was at 100 pg/ml and cytosol (C and F) from RBL cells (50 Mg of protein per sample) and added along with 5 Mug of rat brain membrane protein; sodium were incubated with [32P]NAD in the absence (A-C) or presence nitroprusside was used at 1 mM. After a 60-min incubation at 37"C, (D-F) of GAPDH (10 pg/ml) from rabbit muscle, with BFA (30 proteins were denatured with 2% SDS, and components bound to jMg/ml) where indicated. Proteins were then analyzed by SDS/PAGE GAPDH were separated from free nucleotide over PD-10 desalting and autoradiography. Similar results were obtained in four separate columns (Bio-Rad) as described (32). The data are means ± SD of experiments run in duplicate. triplicate measurements from one of three equivalent experiments. Downloaded by guest on September 27, 2021 Cell Biology: De Matteis et al. Proc. Natl. Acad. Sci. USA 91 (1994) 1117 Table 2. Chemical sensitivity of the BFA-stimulated, (41-43) was excluded since (i) free radioactive ADP-ribose [32P]NAD-dependent modification of GAPDH did not support p50 labeling in either the presence or the Radiolabel Major released absence of BFA and supported only a very low (and BFA- Treatment released, % product(s) insensitive) GAPDH labeling which had different chemical properties than those ofthe modification stimulated by BFA, HCl (0.1 M) 94 ADP-ribose (ii) nonradioactive ADP-ribose did not affect the NAD- NaOH (0.1 M) 100 5'-AMP and ADP-ribose supported GAPDH and p50 labeling induced by BFA, and HgCl2 (0.01 M) <5 (iii) BFA had no effect on the rate of NAD degradation by NH2OH (1 M) <5 endogenous NADases. Rabbit muscle GAPDH (0.3 mg) was incubated with [adenylate- Together, these results demonstrate that BFA stimulates a 32P1NAD (0.1 mM, 20 uCi) in 0.1 M potassium phosphate, pH 7.5/10 mono(ADP-ribosyl)transferase. These enzymes can be dis- mM thymidine/2.5 mM MgCl2/5 mM dithiothreitol with S pg of rat tinguished depending on the amino acid that they modify. brain membrane protein, without or with BFA (50 pg/ml), for 30 min Only arginine-, cysteine-, and asparagine-specific enzymes at 370C (total volume, 0.1 ml). GAPDH was denatured by boiling in have been so far demonstrated in animal cells SDS and separated from unincorporated NAD by chromatography (44-46). over a desalting column (32). Samples were treated with additions as Interestingly, the stability of the BFA-induced GAPDH- and indicated (2 hr, 370C). Radioactivity released was quantified in p50-ADP-ribose bonds to mercuric ions, hydroxylamine, ultrafiltrates (Centri-free, Amicon) ofthe reactions, and the released acid, and base is different from that of the known bonds radioactive products were analyzed by HPLC, as described (32). between the above amino acids and ADP-ribose, suggesting that the BFA-induced modification is of a novel kind. and ADP-ribosylation) was lower than its potency in causing Certain ADP-ribosyltransferases have been shown to be rapid (5 min) in vivo cytosolic redistribution of ARF and activated by polylysine, histones, and other cationic com- pCOP in the two cell lines used in this study (EC50, 1-3 pounds (47-49) but the action of these agents has been ,ug/ml, as assessed by ARF immunostaining; data not attributed to their physical-chemical properties. In contrast, shown). This kind of discrepancy between the potencies of the activation of ADP-ribosyltransferase by BFA appears to BFA in in vitro and in vivo assays has been previously be mediated by a specific binding site (a novel mechanism, to observed (9, 37). Its cause is unclear. our knowledge, for a member of this enzyme family). More- over, the BFA sensitivity of the enzyme is retained after Triton or CHAPS solubilization, suggesting that the BFA- DISCUSSION binding component(s) may be on the ADP-ribosyltransferase This paper reports that BFA, which disrupts membrane itself or on a solubilized complex (this, incidentally, rules out traffic via a specific intracellular "receptor," stimulates, via indirect BFA effects on ADP-ribosylation secondary to BFA- a similar or identical binding component(s), the mono(ADP- induced alterations of membrane traffic). ribosyl)ation of the glycolytic enzyme GAPDH and of a Substrates of the BFA-Sensitive Mono(ADP-ribosyl)trans- protein of 50 kDa. The significance of these observations is ferase. The identity of the 38-kDa protein as a GAPDH twofold: (i) they demonstrate the regulation ofan endogenous isoform is made very likely by the identity of its electropho- mono(ADP-ribosyl)transferase through a specific BFA re- retic properties in two-dimensional gels to those of GAPDH ceptor and (ii) they suggest that ADP-ribosylation may play and by the fact that authentic purified GAPDH is ADP- a regulatory role in membrane traffic. ribosylated in the presence of BFA. GAPDH has been Nature of the BFA-Stimulated Reaction. Several reactions characterized as a glycolytic enzyme. Thus, the possibility of could theoretically explain the observed labeling of GAPDH a link between GAPDH and the process of membrane traffic and in might seem surprising. However, this enzyme is known to be p50 the presence of [32P]NAD. They include, in a multifunctional protein and has been implicated in several addition to mono(ADP-ribosyl)ation, poly(ADP-ribosyl)a- cellular activities, including the nuclear export of tRNA (15), tion (a nuclear reaction involved in DNA repair), phospho- phosphorylation (it binds ATP) (16), regulation of actin adenylylation (40), and the covalent binding of NAD to filaments and microtubules (17, 18), DNA repair (19), and the GAPDH (32). The observations that (i) labeled GAPDH regulation of calcium released only radioactive AMP when exposed to snake release from the endoplasmic reticulum venom phosphodiesterase, (ii) BFA stimulated the incorpo- (20). It has also been reported to induce membrane fusion and ration of the adenine but not of the nicotinamide portion of to bind small GTPases, two properties consistent with a NAD into GAPDH (thereby excluding the binding of NAD), possible role in membrane traffic (21, 22). The identity of the and (iii) labeled GAPDH released ADP-ribose when exposed 50-kDa substrate is unknown. An attractive hypothesis is that to acid this protein might be an a subunit of a G protein, since G provide very strong evidence that the reaction is proteins have been implicated in coat assembly on transport indeed ADP-ribosylation. Nonenzymic ADP-ribosylation organelles and in the formation of transport vesicles (6). Table 3. Effect of BFA analogues on ADP-ribosylation Possible Role of the BFA-Sensitive Mono(ADP-ribosyl)trans- of GAPDH ferase in Vesicular Traffic. BFA inhibits the activation (GDP- GTP exchange) and the subsequent binding of ARF to Golgi ADP-ribosylated membranes, most probably an essential event for non- GAPDH, clathrin coat assembly and formation of transport vesicles. Treatment (arbitrary OD units) Thus, the inhibition ofARF binding is likely to explain at least Control 0.15 ± 0.07 an important part of the cellular effects of BFA. However, BFA (30 ,g/ml) 1.40 ± 0.30 there is no clear evidence that all of the complex changes B36 (30 Mg/ml) 0.20 ± 0.05 induced by BFA (see Introduction) can be attributed solely to B36 (60 pg/ml) 0.18 ± 0.08 inhibition of ARF binding. The stimulation of the mono(ADP- B5 (30 pg/ml) 0.15 + 0.06 ribosyl)transferase described in this study is an additional Postnuclear supernatants from RBL cells were incubated with mechanism of action of BFA that may play a role in the [32P]NAD in the presence of buffer (control) or the indicated con- toxin's effects on membrane traffic. Support for this hypoth- centrations of BFA or BFA analogues for 60 min at 37°C. Proteins esis is provided by the fact that the enzyme appears to be were then analyzed by SDS/PAGE, autoradiography, and densitom- activated by BFA via the same (or a similar) binding com- etry. The data are means t SD ofduplicate measurements from three ponent(s) through which BFA perturbs ARF binding and equivalent experiments. membrane movements. Moreover, a recent series of exper- Downloaded by guest on September 27, 2021 1118 Cell Biology: De Matteis et al. Proc. Nad. Acad. Sci. USA 91 (1994) iments with inhibitors of ADP-ribosylation has shown that 20. Kim, K. C., Caswell, A. H., Talvenheimo, J. A. & Brandt, these agents inhibit some cellular effects of BFA and affect N. R. (1990) Biochemistry 29, 9281-9289. the morphology ofthe secretory pathway without any sign of 21. L6pez-Vinals, A. E., Farias, R. N. & Morero, R. D. (1987) Biochem. Biophys. Res. Commun. 143, 403-409. general toxicity (unpublished work). 22. Doucet, J.-P. & Tuana, B. S. (1991) J. Biol. Chem. 266, A simple model to explain our results might be that the 17613-17620. protein (or protein complex) responsible for ARF activation 23. De Matteis, M. A., Di Tullio, G., Buccione, R. & Luini, A. and binding also possesses ADP-ribosyltransferase activity (1991) J. Biol. Chem. 266, 10452-10460. and that both these properties are affected by BFA. ARF 24. Di Girolamo, M., D'Arcangelo, D., Cacciamani, T., Gierschik, itself might be the endogenous stimulator of this ADP- P. & Corda, D. (1992) J. Biol. Chem. 267, 17397-17403. 25. Kiley, S., Schaap, D., Parker, P., Hsieh, L.-L. & Jaken, S. ribosyltransferase. (1990) J. Biol. Chem. 265, 15704-15712. 26. De Matteis, M. A., Santini, G., Kahn, R. A., Di Tullio, G. & The first two authors contributed equally to this paper. We thank Luini, A. (1993) Nature (London) 364, 818-820. M. Vaughan for useful discussions and critical reading of the 27. O'Farrell, P. H. (1975) J. Biol. Chem. 250, 4007-4021. manuscript, J. Donaldson for the generous gift ofthe BFA analogues 28. Aktories, K., Just, I. & Rosenthal, W. (1988) Biochem. Bio- B5 and B36, R. Kahn for the generous gift of monoclonal anti-ARF phys. Res. Commun. 1, 361-367. antibody 1D9, and M. Falasca for help with the phosphodiesterase 29. Cassel, D. & Pfeuffer, T. (1979) Proc. Natl. Acad. Sci. USA 76, experiments. We are grateful to A. Valentini for excellent technical 2669-2673. assistance and to R. Bertazzi for preparation of the figures. This 30. Kots, A. Y., Skurat, A. V., Sergienko, E. A., Bulargina, T. V. study was supported in part by the Italian Association for Cancer & Severin, E. S. (1992) FEBS Lett. 360, 9-12. Research, the Agenzia per la Promozione e lo Sviluppo del Mezzo- 31. Zhang, J. & Snyder, S. H. (1992) Proc. Natl. Acad. Sci. USA giorno (PR-2 and PR-3), and the Italian National Research Council 89, 9382-9385. (92.01296.PF70, BTBS, and N092.02378.PF39, ACRO). A.C. is the 32. McDonald, L. J. & Moss, J. (1993) Proc. Natl. Acad. Sci. USA recipient of a fellowship from the Centro di Formazione e Studi per 90, 6238-6241. il Mezzogiorno. 33. Scaife, R. M., Wilson, L. & Purich, D. L. 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