Characterization of Chemical Inhibitors of Brefeldin A-activated Mono-ADP-ribosylation
1997; Elsevier BV; Volume: 272; Issue: 22 Linguagem: Inglês
10.1074/jbc.272.22.14200
ISSN1083-351X
AutoresRoberto Weigert, Antonino Colanzi, Mironov Aa, Roberto Buccione, Claudia Cericola, Maria G. Sciulli, Giovanna Santini, Silvio Flati, Aurora Fusella, Julie G. Donaldson, Maria Di Girolamo, Daniela Corda, Maria Antonietta De Matteis, Alberto Luini,
Tópico(s)PARP inhibition in cancer therapy
ResumoBrefeldin A, a toxin inhibitor of vesicular traffic, induces the selective mono-ADP-ribosylation of two cytosolic proteins, glyceraldehyde-3-phosphate dehydrogenase and the novel GTP-binding protein BARS-50. Here, we have used a new quantitative assay for the characterization of this reaction and the development of specific pharmacological inhibitors. Mono-ADP-ribosylation is activated by brefeldin A with an EC50 of 17.0 ± 3.1 μg/ml, but not by biologically inactive analogs including a brefeldin A stereoisomer. Brefeldin A acts by increasing theVmax of the reaction, whereas it does not influence the Km of the enzyme for NAD+(154 ± 13 μm). The enzyme is an integral membrane protein present in most tissues and is modulated by Zn2+, Cu2+, ATP (but not by other nucleotides), pH, temperature, and ionic strength. To identify inhibitors of the reaction, a large number of drugs previously tested as blockers of bacterial ADP-ribosyltransferases were screened. Two classes of molecules, one belonging to the coumarin group (dicumarol, coumermycin A1, and novobiocin) and the other to the quinone group (ilimaquinone, benzoquinone, and naphthoquinone), rather potently and specifically inhibited brefeldin A-dependent mono-ADP-ribosylation. When tested in living cells, these molecules antagonized the tubular reticular redistribution of the Golgi complex caused by brefeldin A at concentrations similar to those active in the mono-ADP-ribosylation assay in vitro, suggesting a role for mono-ADP-ribosylation in the cellular actions of brefeldin A. Brefeldin A, a toxin inhibitor of vesicular traffic, induces the selective mono-ADP-ribosylation of two cytosolic proteins, glyceraldehyde-3-phosphate dehydrogenase and the novel GTP-binding protein BARS-50. Here, we have used a new quantitative assay for the characterization of this reaction and the development of specific pharmacological inhibitors. Mono-ADP-ribosylation is activated by brefeldin A with an EC50 of 17.0 ± 3.1 μg/ml, but not by biologically inactive analogs including a brefeldin A stereoisomer. Brefeldin A acts by increasing theVmax of the reaction, whereas it does not influence the Km of the enzyme for NAD+(154 ± 13 μm). The enzyme is an integral membrane protein present in most tissues and is modulated by Zn2+, Cu2+, ATP (but not by other nucleotides), pH, temperature, and ionic strength. To identify inhibitors of the reaction, a large number of drugs previously tested as blockers of bacterial ADP-ribosyltransferases were screened. Two classes of molecules, one belonging to the coumarin group (dicumarol, coumermycin A1, and novobiocin) and the other to the quinone group (ilimaquinone, benzoquinone, and naphthoquinone), rather potently and specifically inhibited brefeldin A-dependent mono-ADP-ribosylation. When tested in living cells, these molecules antagonized the tubular reticular redistribution of the Golgi complex caused by brefeldin A at concentrations similar to those active in the mono-ADP-ribosylation assay in vitro, suggesting a role for mono-ADP-ribosylation in the cellular actions of brefeldin A. Mono-ADP-ribosylation is a post-translational modification of proteins whereby the adenosine 5′-diphosphoribose moiety of NAD+ is transferred to one of a number of amino acid residues (1Ueda K. Annu. Rev. Biochem. 1985; 54: 73-100Crossref PubMed Scopus (659) Google Scholar, 2Moss J. Vaughan M. Adv. Enzymol. Relat. Areas Mol. Biol. 1988; 61: 303-379PubMed Google Scholar). Several well characterized mono-ADP-ribosylation reactions are catalyzed by bacterial toxins and result in the permanent modification of GTPases with key regulatory functions in cellular processes (1Ueda K. Annu. Rev. Biochem. 1985; 54: 73-100Crossref PubMed Scopus (659) Google Scholar, 2Moss J. Vaughan M. Adv. Enzymol. Relat. Areas Mol. Biol. 1988; 61: 303-379PubMed Google Scholar). Similar reactions are also catalyzed by eukaryotic enzymes, but their cellular significance, despite recent advances (3Okazaki I.J. H. J. Kim McElvaney N.G. Lesma E. Moss J. Blood. 1996; 86Google Scholar, 4Koch-Nolte F. Petersen D. Balasubramanian S. Haag F. Kahlke D. Willer T. Kastelein R. Bazan F. Thiele H.G. J. Biol. Chem. 1996; 271: 7686-7693Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 5Tsuchiya M. Hara N. Yamada K. Osago H. Shimoyama M. J. Biol. Chem. 1994; 269: 27451-27457Abstract Full Text PDF PubMed Google Scholar), is less well understood. Recently, we have reported that brefeldin A (BFA), 1The abbreviations used are:BFAbrefeldin AGAPDHglyceraldehyde-3-phosphate dehydrogenaseERendoplasmic reticulumARFADP-ribosylation factorFITCfluorescein 5-isothiocyanatePBSphosphate-buffered salinePAGEpolyacrylamide gel electrophoresisGTPγSguanosine 5′-O-(3-thiotriphosphate)ATPγSadenosine 5′-O-(3-thiotriphosphate)GDPβSguanosine 5′-O-(2-thiodiphosphate)ARIADP-ribosylation inhibitor a fungal toxin metabolite of palmitic acid (6Harri E. Loeffer W. Sigg H.P. Stahelin H. Tamm H. Helv. Chim. Acta. 1963; 46: 1235-1243Crossref Scopus (141) Google Scholar) with potent inhibitory effects on intracellular membrane traffic (7Klausner R.D. Donaldson J.G. Lippincott-Schwartz J. J. Cell Biol. 1992; 116: 1071-1080Crossref PubMed Scopus (1548) Google Scholar), stimulates the selective mono-ADP-ribosylation of two cytosolic proteins of 38 and 50 kDa in mammalian cells (8De Matteis M.A. Di Girolamo M. Colanzi A. Pallas M. Di Tullio G. McDonald L.J. Moss J. Santini G. Bannykh S. Corda D. Luini A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1114-1118Crossref PubMed Scopus (69) Google Scholar). The 50-kDa substrate (BARS-50; see Ref. 9Di Girolamo M. Silletta M.G. De Matteis M.A. Braca A. Colanzi A. Pawlak D. Rasenik M.M. Luini A. Corda D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7065-7069Crossref PubMed Scopus (41) Google Scholar) binds GTP and is regulated by βγ-subunits of trimeric G proteins; it has therefore been proposed to be a novel G protein involved in membrane transport (9Di Girolamo M. Silletta M.G. De Matteis M.A. Braca A. Colanzi A. Pawlak D. Rasenik M.M. Luini A. Corda D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7065-7069Crossref PubMed Scopus (41) Google Scholar). The p38 substrate is glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a multifunctional protein involved in several cellular processes (10Singh R. Green M.R. Science. 1993; 259: 365-368Crossref PubMed Scopus (391) Google Scholar, 11Huitorel P. Pantaloni D. 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These observations have raised the question as to whether some of the cellular effects of BFA might be mediated by mono-ADP-ribosylation. brefeldin A glyceraldehyde-3-phosphate dehydrogenase endoplasmic reticulum ADP-ribosylation factor fluorescein 5-isothiocyanate phosphate-buffered saline polyacrylamide gel electrophoresis guanosine 5′-O-(3-thiotriphosphate) adenosine 5′-O-(3-thiotriphosphate) guanosine 5′-O-(2-thiodiphosphate) ADP-ribosylation inhibitor The cellular actions of BFA are multiple and complex. BFA selectively blocks constitutive protein secretion and causes a rapid and extensive disruption of the Golgi apparatus consisting of the transformation of the Golgi stacks into a tubular reticular network followed by the redistribution of most of the Golgi resident proteins into the endoplasmic reticulum (ER) (17Lippincott-Schwartz J. Yuan L.C. Bonifacino J.S. Klausner R.D. 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Cell. 1991; 67: 601-616Abstract Full Text PDF PubMed Scopus (683) Google Scholar). Some of these effects are most probably due to an already well documented effect of the toxin, namely the release of a set of proteins (25Robinson M.S. Kreis T.E. Cell. 1992; 69: 129-138Abstract Full Text PDF PubMed Scopus (286) Google Scholar, 26Narula N. McMorrow I. Plopper G. Doherthy J. Matlin K.S. Burke B. Stow J.L. J. Cell Biol. 1992; 117: 27-38Crossref PubMed Scopus (89) Google Scholar, 27Podos S.D. Reddy P. Ashkenas J. Krieger M. J. Cell Biol. 1994; 127: 679-691Crossref PubMed Scopus (56) Google Scholar, 28Beck K.A. Buchanan J.A. Malhotra V. Nelson W.J. J. Cell Biol. 1994; 127: 707-723Crossref PubMed Scopus (168) Google Scholar), in particular the coat proteins ADP-ribosylation factor (ARF) and coatomer (a major protein complex involved in COPI-coated vesicle formation), from Golgi membranes (7Klausner R.D. Donaldson J.G. Lippincott-Schwartz J. J. 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First, BFA activates ADP-ribosylation both in intact and Triton-solubilized Golgi membranes through a site exhibiting a ligand selectivity identical to that involved in the BFA effects on the Golgi structure (9Di Girolamo M. Silletta M.G. De Matteis M.A. Braca A. Colanzi A. Pawlak D. Rasenik M.M. Luini A. Corda D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7065-7069Crossref PubMed Scopus (41) Google Scholar). This suggests that the same (or very similar) BFA-binding components may be involved in the two BFA-induced events. Second, nonspecific inhibitors of mono-ADP-ribosylation such as nicotinamide (33Rankin P.W. Jacobson E.L. Benjamin R.C. Moss J. Jacobson M.K. J. Biol. Chem. 1989; 264: 4312-4317Abstract Full Text PDF PubMed Google Scholar, 34Banasik M. Komura H. Shimoyama M. Ueda K. J. Biol. Chem. 1992; 267: 1569-1575Abstract Full Text PDF PubMed Google Scholar) can reduce the effects of the toxin on the morphology of the Golgi complex (35Mironov A. Sciulli M.G. Flati S. Colanzi A. Santini G. Fusella A. Mironov Jr., A. Corda D. De Matteis M.A. Luini A. Eur. J. Cell Biol. Suppl. 1995; 5: 155Google Scholar). It is therefore of interest to develop a set of specific inhibitors of BFA-induced mono-ADP-ribosylation as they would greatly help to define the cellular role of this reaction. Here, we report the characterization and development of a series of chemical inhibitors of the BFA-dependent mono-ADP-ribosylation reaction. They belong to two chemical classes, containing either a coumarin (dicumarol, coumermycin A1, and novobiocin) or a quinone (ilimaquinones and analogs) group. These drugs, when tested in vivo at concentrations similar to those effective in vitro mono-ADP-ribosylation assays, antagonized the ability of BFA to induce the disassembly of the Golgi complex. BFA, NAD+, nucleotides,dl-dithiothreitol, and fluorescein 5-isothiocyanate (FITC)-conjugated Helix pomatia lectin were from Sigma. Coenzymes Q2, Q4, and Q6 were from Fluka. All the other inhibitors, unless otherwise specified, were from Sigma. BFA analogs were prepared by Dr. A. Greene (J. Fourier University, Grenoble, France) as described previously (36Corey E.J. Wollenberg R.H. Tetrahedron Lett. 1976; 51: 4701-4704Crossref Scopus (32) Google Scholar, 37Le Drian C. Green A.E. J. Am. Chem. Soc. 1982; 104: 5473-5483Crossref Scopus (121) Google Scholar); ilimaquinone was a gift from Dr. V. Malhotra (University of California, San Diego, CA); and AA861 was from Dr. T. Shimizu (University of Tokyo, Tokyo, Japan). [32P]NAD+ was from DuPont NEN or Amersham Corp. Rabbit skeletal muscle GAPDH was from Calbiochem or Sigma. The monoclonal antibody against ARF (1D9) was a gift of Dr. R. Kahn (Emory School of Medicine, Atlanta, GA). The35S-labeled rabbit antibody against mouse IgG was from Amersham Corp. Polyclonal antibodies against the calcium-binding protein (CaBP1) and calreticulin were gifts of Dr. J. Füllekrug (University of Göttingen, Göttingen, Germany). The polyclonal antibody against mannosidase II was a gift of Dr. K. Moremen (University of Georgia, Athens, GA). Monoclonal antibodies against Rab5 and Rab7 were gifts from Dr. M. Zerial (EMBL, Heidelberg, Germany); the monoclonal antibody against TGN38 was donated by Dr. G. Banting (School of Medical Science, Bristol, United Kingdom). FITC- and tetramethylrhodamine 5-isothiocyanate-conjugated secondary antibodies were from Sigma or Cappel. The materials for cell culture, including plasticware, chamber slides (Nunc, Roskilde, Denmark), and medium (Life Technologies, Inc.), were purchased from Mascia Brunelli S. p. A. (Milano, Italy). Rat basophilic leukemia 2H3 cells were grown in Eagle's minimal essential medium supplemented with 16% fetal calf serum and 1 mm l-glutamine as described (38De Matteis M.A. Di Tullio G. Buccione R. Luini A. J. Biol. Chem. 1991; 266: 10452-10460Abstract Full Text PDF PubMed Google Scholar). Harlan Sprague Dawley male rats (150–250 g) were killed by decapitation. Tissues were immediately removed and placed in ice-cold homogenization buffer (0.32 m sucrose, 4 mmHEPES, and 1 mm EDTA, pH 7.3), washed four to five times with the same buffer, minced with scissors, and homogenized in a Teflon-glass Potter-Elvehjem homogenizer (15–20 strokes). All procedures were carried out at 4 °C. The homogenate was centrifuged at 700 × g for 10 min, and the post-nuclear supernatant was collected and ultracentrifuged at 150,000 ×g for 90 min. The supernatant was discarded, and the pellet (suspended in 3 m KCl and incubated for 30 min with stirring) was centrifuged at 12,700 × g in a microcentrifuge. The pellet obtained, termed "total membranes," was washed and suspended in phosphate-buffered saline (PBS), pH 7.4; aliquoted; frozen in liquid nitrogen; and then stored at −80 °C until needed. The reproducibility of these preparations was satisfactory. Each experiment was performed with at least two preparations, with similar results. Rat brain cytosol was prepared as described (39Malhotra V. Serafini T. Orci L. Sheperd J.C. Rothman J.E. Cell. 1989; 58: 329-336Abstract Full Text PDF PubMed Scopus (321) Google Scholar). Subcellular fractions were prepared as described (39Malhotra V. Serafini T. Orci L. Sheperd J.C. Rothman J.E. Cell. 1989; 58: 329-336Abstract Full Text PDF PubMed Scopus (321) Google Scholar, 40Borgese N. Meldolesi J. J. Cell Biol. 1980; 85: 501-515Crossref PubMed Scopus (34) Google Scholar). In some experiments, mitochondria were further purified as described by Rusinol et al. (41Rusinol A.E. Cui Z. Chen M.H. Vance J.E. J. Biol. Chem. 1994; 269: 27494-27502Abstract Full Text PDF PubMed Google Scholar). To check the composition of each fraction (post-nuclear supernatant, rough ER, smooth ER, Golgi membranes, mitochondria, and cytosol), equal amounts of protein (80 μg) were separated by SDS-PAGE, blotted onto nitrocellulose, and probed with polyclonal antibodies against specific markers of intracellular organelles by Western blotting. The purity of the mitochondrial fraction was also tested by electron microscopy. Mitochondria were pelleted and processed as described previously (42Buccione R. Bannykh S. Santone I. Baldassarre M. Facchiano F. Bozzi Y. Di Tullio G. Mironov A. Luini A. De Matteis M.A. J. Biol. Chem. 1996; 271: 3523-3533Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) and then photographed with a Zeiss 109 transmission electron microscope. They were >90% pure. Unless otherwise specified, 10 μg of total membranes and 50 μg of cytosol, both from rat brain (or alternatively, 50 μg of post-nuclear supernatant), were incubated at 37 °C for 1 h with 30 μg/ml BFA in 50 mmphosphate buffer, pH 7.4, 30 μm NAD+, 0.01 μCi/μl [32P]NAD+, 2.5 mmMgCl2, 5 mm dl-dithiothreitol, and 10 mm thymidine in a final volume of 50 μl. The reaction was stopped by the addition of Laemmli sample buffer (51Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar). Samples were boiled for 5 min and analyzed by 10% SDS-PAGE. Proteins were then transferred to a nitrocellulose sheet by electroblotting, and the radiolabeled proteins were quantified by autoradiography or by an Instant Imager (Canberra Packard). A rapid filtration assay was set up to measure the labeling of purified commercial GAPDH (8De Matteis M.A. Di Girolamo M. Colanzi A. Pallas M. Di Tullio G. McDonald L.J. Moss J. Santini G. Bannykh S. Corda D. Luini A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1114-1118Crossref PubMed Scopus (69) Google Scholar, 9Di Girolamo M. Silletta M.G. De Matteis M.A. Braca A. Colanzi A. Pawlak D. Rasenik M.M. Luini A. Corda D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7065-7069Crossref PubMed Scopus (41) Google Scholar). Unless otherwise specified, 10 μg of GAPDH were incubated with 10 μg of total membranes from rat brain as described above for the ADP-ribosylation assay. Samples were then pelleted at 12,000 ×g for 10 min, and the supernatant was mixed with 100 μl of ice-cold 1 mm NAD+, 1 mm AMP, and 0.6 mm sodium pyrophosphate (final concentrations) and then filtered on a 96-well Bio-Rad dot-blot apparatus through a nitrocellulose sheet. Each well was filled with ice-cold 20% trichloroacetic acid (200 μl/well) prior to the addition of the sample. GAPDH was allowed to adhere to the nitrocellulose by a gentle filtration and washed twice by filtering ice-cold 20% trichloroacetic acid (100 μl/well) and four times with low salt Tris-buffered saline (20 mm Tris-HCl, pH 7.5, and 500 mm NaCl). The nitrocellulose sheet was then boiled in distilled water for 10 min and stained with Ponceau S. Radiolabeled GAPDH was quantified by an Instant Imager. GAPDH was recovered quantitatively on the filters. This was established by processing in parallel a variety of identical samples by both the filter and SDS-PAGE-based assays (8De Matteis M.A. Di Girolamo M. Colanzi A. Pallas M. Di Tullio G. McDonald L.J. Moss J. Santini G. Bannykh S. Corda D. Luini A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1114-1118Crossref PubMed Scopus (69) Google Scholar, 9Di Girolamo M. Silletta M.G. De Matteis M.A. Braca A. Colanzi A. Pawlak D. Rasenik M.M. Luini A. Corda D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7065-7069Crossref PubMed Scopus (41) Google Scholar). None of the treatments described under "Results and Discussion" affected the amount of protein recovered on the filter. As previously reported (15Zhang J. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9382-9385Crossref PubMed Scopus (229) Google Scholar,16McDonald L.J. Moss J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6238-6241Crossref PubMed Scopus (176) Google Scholar), GAPDH is nonenzymatically mono-ADP-ribosylated in the absence of BFA. Thus, for each condition tested in this study, control experiments using the BFA vehicle (dimethyl sulfoxide) were carried out to evaluate the basal nonenzymatic ADP-ribosylation of GAPDH. The pmol of ADP-ribosylated GAPDH reported in this study were calculated by subtracting the values of nonenzymatically ADP-ribosylated GAPDH from those obtained in the presence of BFA. Membranes (10 μg of protein) were incubated for 10 min with 100 μg of rat brain cytosol in a final volume of 100 μl containing 25 mm HEPES-KOH, pH 7, 25 mm KCl, 2.5 mm MgCl2, 1 mm ATP, 1 mm dl-dithiothreitol, and 40 μg/ml BFA where indicated; 10 μm GTPγS was then added, and incubation was continued for 5 min. The reaction was stopped by centrifugation at 14,000 × g at 4 °C, and the pellets were boiled for 5 min in Laemmli sample buffer (51Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar). Membrane proteins were separated by 12% SDS-PAGE, blotted onto nitrocellulose, and probed with a mouse monoclonal antibody against ARF (1D9) and a 35S-labeled rabbit antibody against mouse IgG. Radioactivity was quantified by an Instant Imager. Rat basophilic leukemia cells (grown on glass chamber slides) were fixed in 4% paraformaldehyde in PBS at room temperature for 10 min; quenched with 10 mm NH4Cl for 10 min; washed with PBS; and permeabilized with 0.05% saponin and 0.2% bovine serum albumin in PBS for 30 min at room temperature. The cells were stained with FITC-conjugated H. pomatia lectin (100 μg/ml in PBS containing 0.2% bovine serum albumin) for 45 min or were incubated with primary antibody for 1 h at room temperature, washed thoroughly with PBS, and incubated with specific FITC- or tetramethylrhodamine 5-isothiocyanate-conjugated secondary antibody for 30 min at room temperature. After thorough washing, the slides were mounted in Mowiol 4-88 (Calbiochem) and examined using a Zeiss Axiophot microscope equipped with a Plan-Neofluar 100× objective. So far, work on BFA-stimulated mono-ADP-ribosylation has been carried out by labeling the cytosolic substrates with radioactive NAD+, separation by SDS-PAGE, and autoradiography (8De Matteis M.A. Di Girolamo M. Colanzi A. Pallas M. Di Tullio G. McDonald L.J. Moss J. Santini G. Bannykh S. Corda D. Luini A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1114-1118Crossref PubMed Scopus (69) Google Scholar, 9Di Girolamo M. Silletta M.G. De Matteis M.A. Braca A. Colanzi A. Pawlak D. Rasenik M.M. Luini A. Corda D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7065-7069Crossref PubMed Scopus (41) Google Scholar). In this study, to characterize the reaction in a quantitative fashion, we have developed a novel simplified assay based on the use of salt-washed membranes as the source of enzyme, pure commercial GAPDH as the substrate, a rapid filtration method to separate free from GAPDH-bound radioactivity, and counting of the radiolabeled GAPDH by an Instant Imager (see "Experimental Procedures"). This approach afforded a high sample output and reproducibility as well as the possibility to study modulatory effects of added cytosolic factors. Fig. 1 shows that BFA stimulated the reaction with an EC50 of 17 ± 3 μg/ml. The apparentKm of the reaction with respect to NAD+was 154 ± 13 μm (Fig. 1, inset). TheVmax was 510 ± 150 pmol/h/mg of membrane protein (at 30 μg/ml BFA). BFA regulated the reaction velocity by increasing the Vmax, whereas theKm was not detectably modified by varying the toxin concentration (Fig. 1, inset). Of note, the EC50of BFA was similar to that found by us in assays of ARF binding to Golgi membranes, but higher than the BFA EC50 in vitro. The difference between the in vivo and in vitro potencies of BFA has been noticed and discussed before by several authors (31Donaldson J.G. Finazzi D. Klausner R.D. Nature. 1992; 360: 350-352Crossref PubMed Scopus (596) Google Scholar, 43Orci L. Tagaya M. Amherdt M. Perrelet A. Donaldson J.G. Lippincott-Schwartz J. Klausner R.D. Rothman J.E. Cell. 1991; 64: 1183-1195Abstract Full Text PDF PubMed Scopus (357) Google Scholar). A comparison of the results of the new assay (Fig. 1) with data obtained using the old method (where the cytosol was the source of substrate; see Ref. 8De Matteis M.A. Di Girolamo M. Colanzi A. Pallas M. Di Tullio G. McDonald L.J. Moss J. Santini G. Bannykh S. Corda D. Luini A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1114-1118Crossref PubMed Scopus (69) Google Scholar) did not reveal evident differences, suggesting that cytosolic factors have no major effects on the reaction kinetics. The BFA-stimulated ADP-ribosylation of GAPDH was maximal near pH 7.6 at temperatures between 37 and 40 °C, with a second smaller peak at 30 °C, and was inhibited by higher than physiological levels of NaCl (Fig. 2). The double peak in the temperature profile is unusual. At present, we do not have a simple explanation for it. Of the major physiological divalent ions that were tested, Ca2+, Mg2+, and Mn2+ were inactive, whereas Zn2+ and Cu2+ were inhibitory (with IC50 values of ∼75 and 500 μm, respectively). Several nucleotides (GTP, GDP, GTPγS, GDPβS, ATPγS, and cAMP and different combinations of these molecules) had no effect on GAPDH mono-ADP-ribosylation at concentrations up to 1 mm, whereas ATP was inhibitory (with an IC50 of ∼1 mm). Rat brain, liver, heart, spleen, lung, skeletal muscle, and kidney were tested for their content of enzymatic mono-ADP-ribosylating activity (using commercial GAPDH as a substrate) and of endogenous cytosolic substrates (GAPDH and BARS-50) (using rat brain membranes as the enzyme source). Fig. 3 A shows that the enzymatic activity was present in all tissues examined, with the highest levels in the brain and the lowest (nearly undetectable) in the kidney. It is unclear whether this reflects a real lack of expression of the enzyme in kidney tissue rather than, for instance, the presence of inhibitory factors or of degradative enzymes. The protein substrates were ubiquitous, with the highest levels in the brain, spleen, and heart (data not shown). Among the subcellular fractions, one fraction enriched in Golgi membranes and one containing the smooth ER as well as Golgi membranes were highly active; a lower but significant activity was found in mitochondria and the lowest in the rough ER (Fig. 3 B), whereas the cytosol was completely devoid of enzyme (data not shown). Since the ability of BFA to prevent the activation and binding of ARF to intracellular membranes is a well known in vitro activity of the toxin and is considered responsible for at least some of the cellular BFA effects, it was interesting to compare the subcellular distribution of this activity with the distribution of BFA-dependent mono-ADP-ribosylation. Fig. 3 C shows that ARF binding was enriched in the Golgi membrane-containing subcellular fractions and was present (at lower levels) in the ER and mitochondria. This distribution profile was quite similar to that of BFA-dependent mono-ADP-ribosylation and is therefore compatible with the idea that the two activities of BFA (ARF binding and mono-ADP-ribosylation) might be associated (this point is discussed further below). The meaning of the presence of both activities in mitochondria is not clear at present. It is very unlikely to be due to contamination by Golgi or smooth ER membranes, as indicated by a comparison between their distribution with that of marker enzymes (CaBP1 for the endoplasmic reticulum, mannosidase II for Golgi membranes, and Rab5 for early endosomes; see Fig. 3 D). The binding of ARF to mitochondria and other subcellular fractions has been observed by others (44Cavenagh M.M. Whitney J.A. Carrol K. Zhang C.J. Boman A.L. Rosenwald A.G. Mellman I. Kahn R.A. J. Biol. Chem. 1996; 271: 21767-21774Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). Since mitochondria are capable of fission and fusion (45Bereiter-Hahn J. Int. Rev. Cytol. 1989; 122: 1-63Google Scholar), it is possible that ARF binding and mono-ADP-ribosylation might reflect the presence of a membrane transport machinery in this organelle. As noted above, BFA is nearly equipotent in in vitro mono-ADP-ribosylation and ARF binding assays. Moreover, as previously reported, two synthetic analogs of BFA known to be inactive in preventing ARF binding to isolated Golgi membranes, B5 and B36, are also devoid of mono-ADP-ribosylation-stimulating activity (8De Matteis M.A. Di Girolamo M. Colanzi A. Pallas M. Di Tullio G. McDonald L.J. Moss J. Santini G. Bannykh S. Corda D. Luini A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1114-1118Crossref PubMed Scopus (69) Google Scholar). Based on these observations, it has been suggested that the BFA-binding sites involved in the two activities are similar or identical (8De Matteis M.A. Di Girolamo M. Colanzi A. Pallas M. Di Tullio G. McDonald L.J. Moss J. Santini G. Bannykh S. Corda D. Luini A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1114-1118Crossref PubMed Scopus (69) Google Scholar). We wanted to perform a more stringent test of parallelism between the two activities by using a large variety of BFA-derived analogs, including the biologically inactive stereoisomer of BFA (7Klausner R.D. Donaldson J.G. Lippincott-Schwartz J. J. Cell Biol. 1992; 116: 1071-1080Crossref PubMed Scopus (1548) Google Scholar). Fig.4 shows that the BFA analogs B5, B17, B18, B23, B27, and B36, reported to lack BFA
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