Identification of a Potential Effector Pathway for the Trimeric Go Protein Associated with Secretory Granules
1998; Elsevier BV; Volume: 273; Issue: 27 Linguagem: Inglês
10.1074/jbc.273.27.16913
ISSN1083-351X
AutoresStéphane Gasman, Sylvette Chasserot‐Golaz, P. Hubert, Dominique Aunis, Michael Bäder,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoBesides having a role in signal transduction, heterotrimeric G proteins may be involved in membrane trafficking events. In chromaffin cells, Go is associated with secretory organelles, and its activation inhibits the ATP-dependent priming of exocytosis. By using permeabilized cells, we previously described that the control exerted by the granule-bound Go on exocytosis may be related to effects on the cortical actin network through a sequence possibly involving Rho. To provide further insight into the function of Rho in exocytosis, we focus here on its intracellular localization in chromaffin cells. By immunoreplica analysis, immunoprecipitation, and confocal immunofluorescence, we found that RhoA is specifically associated with the membrane of secretory chromaffin granules. Parallel subcellular fractionation experiments revealed the occurrence of a mastoparan-stimulated phosphatidylinositol 4-kinase activity in purified chromaffin granule membranes. This stimulatory effect of mastoparan was mimicked by GAP-43, an activator of the granule-associated Go, and specifically inhibited by antibodies against Gαo. In addition, Clostridium botulinum C3 exoenzyme completely blocked the activation of phosphatidylinositol 4-kinase by mastoparan. We propose that the control exerted by Go on peripheral actin and exocytosis is related to the activation of a downstream RhoA-dependent phosphatidylinositol 4-kinase associated with the membrane of secretory granules. Besides having a role in signal transduction, heterotrimeric G proteins may be involved in membrane trafficking events. In chromaffin cells, Go is associated with secretory organelles, and its activation inhibits the ATP-dependent priming of exocytosis. By using permeabilized cells, we previously described that the control exerted by the granule-bound Go on exocytosis may be related to effects on the cortical actin network through a sequence possibly involving Rho. To provide further insight into the function of Rho in exocytosis, we focus here on its intracellular localization in chromaffin cells. By immunoreplica analysis, immunoprecipitation, and confocal immunofluorescence, we found that RhoA is specifically associated with the membrane of secretory chromaffin granules. Parallel subcellular fractionation experiments revealed the occurrence of a mastoparan-stimulated phosphatidylinositol 4-kinase activity in purified chromaffin granule membranes. This stimulatory effect of mastoparan was mimicked by GAP-43, an activator of the granule-associated Go, and specifically inhibited by antibodies against Gαo. In addition, Clostridium botulinum C3 exoenzyme completely blocked the activation of phosphatidylinositol 4-kinase by mastoparan. We propose that the control exerted by Go on peripheral actin and exocytosis is related to the activation of a downstream RhoA-dependent phosphatidylinositol 4-kinase associated with the membrane of secretory granules. Studies on diverse secretory cell types have established a crucial role for GTP-binding proteins in the regulation of calcium-dependent exocytosis. Trimeric G proteins have been found associated with the membrane of various secretory granules (1Ahnert-Hilger G. Schäfer T. Spicher K. Grund C. Schultz G. Wiedenmann R. Eur. J. Cell Biol. 1994; 65: 26-38PubMed Google Scholar, 2Konrad R.J. Young R.A. Record R.D. Smith R.M. Butkerait P. Manning D. Jarett L. Wolf B.A. J. Biol. Chem. 1995; 270: 12869-12876Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 3Vitale N. Gensse M. Chasserot-Golaz S. Aunis D. Bader M.F. Eur. J. Neurosci. 1996; 8: 1275-1285Crossref PubMed Scopus (44) Google Scholar), suggesting their participation in the exocytotic reaction. Accordingly, the involvement of a plasma membrane-bound Gi3 protein in the late stages of exocytosis in mast cells has been demonstrated (4Aridor M. Rajmilevich G. Beaven M.A. Sagi-Eisenberg R. Science. 1993; 262: 1569-1572Crossref PubMed Scopus (224) Google Scholar). Direct control of exocytosis by Gi and Goproteins has also been described in insulin-secreting cells (5Lang J. Nishimoto I. Okamoto T. Regazzi R. Kiraly C. Weller U. Wollheim C.B. EMBO J. 1995; 14: 3635-3644Crossref PubMed Scopus (125) Google Scholar) and in chromaffin cells (3Vitale N. Gensse M. Chasserot-Golaz S. Aunis D. Bader M.F. Eur. J. Neurosci. 1996; 8: 1275-1285Crossref PubMed Scopus (44) Google Scholar, 6Vitale N. Mukai H. Rouot B. Thierse D. Aunis D. Bader M.F. J. Biol. Chem. 1993; 268: 14715-14723Abstract Full Text PDF PubMed Google Scholar, 7Vitale N. Thierse D. Aunis D. Bader M.F. Biochem. J. 1994; 300: 217-227Crossref PubMed Scopus (35) Google Scholar, 8Vitale N. Deloulme J.C. Thierse D. Aunis D. 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EMBO J. 1994; 13: 2029-2037Crossref PubMed Scopus (186) Google Scholar, 13Caumont A.S. Galas M.-C. Vitale N. Aunis D. Bader M.-F. J. Biol. Chem. 1998; 273: 1373-1379Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar), anterior pituitary cells (14Lledo P.M. Vernier P. Vincent J.D. Mason W.T. Zorec R. Nature. 1993; 364: 540-544Crossref PubMed Scopus (179) Google Scholar), and melanotrophs (15Rupnik M. Law G.J. Northrop A.J. Mason W.T. Zorec R. Neuroreport. 1995; 6: 853-856Crossref PubMed Scopus (13) Google Scholar). In addition, recent investigations led to the idea that Rho may be a component of the molecular machinery underlying regulated secretion (16Mariot P. O'Sullivan A.J. Brown A.M. Tatham P.E. EMBO J. 1996; 15: 6476-6482Crossref PubMed Scopus (36) Google Scholar, 17Norman J.C. Price L.S. Ridley A.J. Koffer A. Mol. Biol. Cell. 1996; 7: 1429-1442Crossref PubMed Scopus (108) Google Scholar, 18O'sullivan A.J. Brown A.M. Freeman H.N.M. Gomperts B.D. Mol. Biol. Cell. 1996; 7: 397-408Crossref PubMed Scopus (52) Google Scholar, 19Komuro R. Sasaki T. Takaishi K. Orita S. Takai Y. Genes Cells. 1996; 1: 943-951Crossref PubMed Scopus (48) Google Scholar, 20Gasman S. Chasserot-Golaz S. Popoff M.R. Aunis D. Bader M.-F. J. Biol. Chem. 1997; 272: 20564-20571Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Rho belongs to the GTPase family consisting of Rho, Rac, and Cdc 42 proteins. This family has been implicated in a number of cellular functions requiring the reorganization of actin-based structures (21Takai Y. Sasaki T. Tanaka K. Nakanishi H. Trends Biochem. Sci. 1995; 20: 227-231Abstract Full Text PDF PubMed Scopus (364) Google Scholar, 22Machesky L.M. Hall A. Trends Cell Biol. 1996; 6: 304-310Abstract Full Text PDF PubMed Scopus (253) Google Scholar, 23Symons M. Trends Biochem. Sci. 1996; 21: 178-181Abstract Full Text PDF PubMed Scopus (259) Google Scholar). In secretory cells, cytoskeletal rearrangements are a prerequisite for exocytosis since actin forms a cortical barrier that must be reorganized to enable docking and/or fusion of the secretory granules with the plasma membrane (24Sontag J.M. Aunis D. Bader M.F. Eur. J. Cell Biol. 1988; 46: 316-326PubMed Google Scholar, 25Burgoyne R.D. Morgan A. O'Sullivan A.J. Cell Signalling. 1989; 1: 323-334Crossref PubMed Scopus (84) Google Scholar, 26Vitale M.L. Seward E.P. Trifaro J.M. Neuron. 1995; 14: 353-363Abstract Full Text PDF PubMed Scopus (308) Google Scholar). Rho together with a trimeric G protein regulates the changes in the actin cytoskeleton observed in activated mast cells (17Norman J.C. Price L.S. Ridley A.J. Koffer A. Mol. Biol. Cell. 1996; 7: 1429-1442Crossref PubMed Scopus (108) Google Scholar, 27Norman J.C. Proce L.S. Ridley A.J. Hall A. Koffer A. J. Cell Biol. 1994; 126: 1005-1015Crossref PubMed Scopus (127) Google Scholar). In chromaffin cells, we recently described that the secretory granule-associated Gocontrols the priming of exocytosis by modifying the cortical actin network through a sequence of events that eventually involves Rho (20Gasman S. Chasserot-Golaz S. Popoff M.R. Aunis D. Bader M.-F. J. Biol. Chem. 1997; 272: 20564-20571Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Thus, Rho seems to be an integral component of the signaling pathway leading to the cytoskeletal redistribution necessary for secretion, although the mechanism by which Rho relates to the actin organization remains to be elucidated. To identify the partners of Rho signaling in calcium-evoked secretion, we examine here the intracellular distribution of Rho in chromaffin cells. By immunoreplica analysis and confocal immunofluorescence, we demonstrate that RhoA is a specific component of the membrane of secretory chromaffin granules. Furthermore, our data reveal that the phosphatidylinositol (PtdIns) 1The abbreviations used are: PtdIns, phosphatidylinositol; gPtdIns-4-P, glycerophosphatidylinositol 4-phosphate; HPLC, high pressure liquid chromatography; PBS, phosphate-buffered saline; DTT, dithiothreitol; CHO, Chinese hamster ovary; DβH, dopamine-β-hydroxylase; PtdInsP, phosphatidylinositol monophosphate; PtdInsP2, phosphatidylinositol 4,5-bisphosphate; DTAF, dichlorotriazinyl aminofluorescein; TRITC, tetramethylrhodamine isothiocyanate; CGA, chromogranin A. 1The abbreviations used are: PtdIns, phosphatidylinositol; gPtdIns-4-P, glycerophosphatidylinositol 4-phosphate; HPLC, high pressure liquid chromatography; PBS, phosphate-buffered saline; DTT, dithiothreitol; CHO, Chinese hamster ovary; DβH, dopamine-β-hydroxylase; PtdInsP, phosphatidylinositol monophosphate; PtdInsP2, phosphatidylinositol 4,5-bisphosphate; DTAF, dichlorotriazinyl aminofluorescein; TRITC, tetramethylrhodamine isothiocyanate; CGA, chromogranin A. 4-kinase present on chromaffin granules (28Husebye E.S. Flatmark T. Biochim. Biophys. Acta. 1988; 968: 261-265Crossref PubMed Scopus (30) Google Scholar, 29Wiedemann C. Schafer T. Burger M.M. EMBO J. 1996; 15: 2094-2101Crossref PubMed Scopus (167) Google Scholar) can be stimulated by specific activators of Gαo through a mechanism sensitive to Clostridium botulinum C3 ADP-ribosyltransferase. We propose that the granule-bound Go controls the peripheral actin cytoskeleton and exocytosis through an effector pathway involving the sequential participation of RhoA and PtdIns 4-kinase both located on the membrane of secretory chromaffin granules. Chromaffin cells were isolated from fresh bovine adrenal glands by retrograde perfusion with collagenase and purified on self-generating Percoll gradients (30Bader M.F. Trifaro J.M. Langley O.K. Thierse D. Aunis D. J. Cell Biol. 1986; 102: 636-646Crossref PubMed Scopus (55) Google Scholar). Cells were suspended in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and containing cytosine arabinoside (10 μm), fluorodeoxyuridine (10 μm), streptomycin (50 μg/ml), and penicillin (50 units/ml). Cells were cultured as monolayers on either 24 multiple 16-mm Costar plates (Cambridge, MA) at a density of 2.5 × 105 cells/well or fibronectin-coated glass coverslips at a density of 2 × 105 cells or 100-mm Costar plates at a density of 5 × 106 cells/plate. Experiments were performed 3–7 days after plating. Cultured chromaffin cells (50 × 106cells) were washed with Locke's solution, rapidly frozen and thawed, and collected in 3.7 ml of buffer A containing 20 mm HEPES, pH 7.5, 1 mm EDTA, 1 mm DTT, 20 μg/ml pepstatin, 20 μg/ml aprotinin, 20 μg/ml trypsin inhibitor, 20 μg/ml RNase, 0.1 mm phenylmethylsulfonyl fluoride (total homogenate). Following 5 min incubation on ice, 2.5 ml of the homogenate was centrifuged for 45 min at 100,000 × g. The supernatant was saved (cytosol) and the pellet homogenized in 500 μl of buffer A (membrane-bound fraction). Plasma and chromaffin granule membranes were purified from bovine adrenal medulla as described previously (3Vitale N. Gensse M. Chasserot-Golaz S. Aunis D. Bader M.F. Eur. J. Neurosci. 1996; 8: 1275-1285Crossref PubMed Scopus (44) Google Scholar). Briefly, adrenal medullary glands were homogenized in 0.32 m sucrose (10 mmTris-HCl, pH 7.4) and then centrifuged at 800 × g for 15 min. After centrifugation at 20,000 × g for 20 min, the pellet was resuspended in 0.32 m sucrose (10 mm Tris-HCl, pH 7.4), layered on a continuous sucrose density gradient (1–2.2 m sucrose, 10 mmTris-HCl, pH 7.4), and centrifuged for 1 h at 100,000 ×g. Twelve 1-ml fractions were collected from top to bottom and analyzed for protein by the Bradford procedure. The distribution of dopamine-β-hydroxylase (DβH) and Na+/K+ATPase was estimated as described previously (31Aunis D. Miras-Portugal M.T. Mandel P. Biochem. Pharmacol. 1973; 22: 2581-2589Crossref PubMed Scopus (21) Google Scholar, 32Ishii T. Takeyasu K. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8881-8885Crossref PubMed Scopus (33) Google Scholar). Plasma membranes were purified from fractions 2 and 3, which contained the highest Na+/K+ ATPase activity. Chromaffin granule membranes were recovered from fractions 11 and 12, which contained the highest DβH activity. Fractions were diluted 10 times in TED buffer (20 mm Tris-HCl, pH 8.0, 1 mm EDTA, 1 mm DTT), and membranes were collected by centrifugation for 30 min at 100,000 × g. Membranes were suspended in TED buffer and stored at −20 °C. ADP-ribosylation with C. botulinum exoenzyme C3 ADP-ribosyltransferase (5 μg/ml) was performed with 15 μg of proteins in 20 mm HEPES, pH 7.5, 10 mmthymidine, 1 mm DTT, 1 mm EDTA, 5 mm MgCl2, 1 mm ATP, 100 μm GTP, 0.5 μCi of [32P]NAD (0.15 μm) (30 Ci/mmol, 2 mCi/ml, NEN Life Science Products). The reaction was carried out for 30 min at 37 °C in a final volume of 120 μl. Proteins were then precipitated with 10% trichloroacetic acid, centrifuged (15 min at 12,000 rpm), and dissolved in sample buffer for SDS-polyacrylamide gel separation. Labeled proteins were analyzed by autoradiography with a Bio-Imaging Analyzer FUJIX BAS1000 (Fuji, Tokyo, Japan). PtdIns kinase activity was detected as described by Husebye and Flatmark (28Husebye E.S. Flatmark T. Biochim. Biophys. Acta. 1988; 968: 261-265Crossref PubMed Scopus (30) Google Scholar). Chromaffin granule membrane proteins (50 μg) were preincubated for 10 min in the presence or absence of the indicated concentrations of mastoparan in assay buffer containing 30 mm HEPES, pH 7.0, 0.1 mm EGTA, 5 mm MgCl2 in a final volume of 300 μl. The reaction was started by addition of 30 μl of the ATP solution containing 2.5 μCi of [γ-32P]ATP (3000 Ci/mmol, Amersham Pharmacia Biotech) and 5 mm MgATP. After 2 min, the reaction was stopped by addition of 800 μl of chloroform, methanol, 12 m HCl (20/40/1, v/v/v). The chloroform phase was extracted, evaporated under vacuum, and then redissolved in 20 μl of chloroform/methanol (2/1, v/v). Phospholipids were separated on one-dimensional TLC oxalate-coated silica gel plates in chloroform, methanol, 4 m NH4OH (9/7/2, v/v/v). Labeled phospholipids were visualized with32P-imaging plates using the Bio-Imaging Analyzer FUJIX BAS1000. Labeled phospholipids were compared with standard lipids stained with iodine vapor. PtdIns kinase assays in the presence of LY294002 (Lilly) or quercetin (Sigma) were performed with a reduced concentration of MgATP (20 μm). Membranes were preincubated for 15 min in assay buffer containing either LY294002 (100 μm) or quercetin (100 μm) before incubation for 10 min in the presence or absence of mastoparan (20 μm). The reaction was subsequently started with [γ-32P]ATP. For definitive identification of the phosphatidylinositol monophosphates detected on the TLC plates, the radioactive spots were excised, deacylated (33Serunian L.A. Auger K.R. Cantley L.C. Methods Enzymol. 1991; 198: 78-87Crossref PubMed Scopus (130) Google Scholar), and subjected to HPLC analysis using a 10-μm Partisil SAX ion-exchange column (Interchim, Montluçon, France). After sample injection, the column was equilibrated with water for 5 min, and32P-labeled glycerophosphatidylinositol monophosphates were separated using a 60-min linear gradient of 0–0.18 mammonium phosphate, pH 3.8 (33Serunian L.A. Auger K.R. Cantley L.C. Methods Enzymol. 1991; 198: 78-87Crossref PubMed Scopus (130) Google Scholar). A mixture of AMP and ADP was applied with the deacylated samples, and their 260-nm absorbance served to standardize elution times that were similar from one run to the next. Radioactivity was detected by assaying 200-μl eluate fractions.32P-Labeled glycerophosphatidylinositol 4-phosphate (gPtdIns-4-P) standard was prepared by deacylation of [32P]PtdIns-4-P obtained by lipid extraction and TLC separation of 32P-labeled quiescent CHO cells as described (33Serunian L.A. Auger K.R. Cantley L.C. Methods Enzymol. 1991; 198: 78-87Crossref PubMed Scopus (130) Google Scholar). The 32P-labeled glycerophosphatidylinositol 3-phosphate (gPtdIns-3-P) standard was prepared by deacylation of [32P]PtdIns-3-P produced in vitro by phosphatidylinositol 3-kinase (p85) immunoprecipitated from CHO cells and incubated in the presence of PtdIns and [γ-32P]ATP (33Serunian L.A. Auger K.R. Cantley L.C. Methods Enzymol. 1991; 198: 78-87Crossref PubMed Scopus (130) Google Scholar). The GTPase activity of purified chromaffin granule membranes was estimated according to the procedure previously described (3Vitale N. Gensse M. Chasserot-Golaz S. Aunis D. Bader M.F. Eur. J. Neurosci. 1996; 8: 1275-1285Crossref PubMed Scopus (44) Google Scholar, 6Vitale N. Mukai H. Rouot B. Thierse D. Aunis D. Bader M.F. J. Biol. Chem. 1993; 268: 14715-14723Abstract Full Text PDF PubMed Google Scholar). Briefly, granule membrane proteins (10 μg) were incubated for 10 min at 30 °C in 100 μl of 50 mmHEPES, pH 8.0, 1 mm EDTA, 1 mm DTT, 100 mm NaCl, and 1 mm MgCl2 in the presence of the indicated concentrations of mastoparan. The GTPase reaction was started by the addition of GTP at a final concentration of 0.8 μm with 1 nCi [γ-32P]GTP (NEN Life Science Products, 30 Ci/mmol) and stopped by the addition of 250 μl of cold 5% (w/v) Norit in phosphate buffer. After centrifugation, the32Pi in the supernatant was determined by32P Cerenkov counting. Non-enzymatic hydrolysis of [γ-32P]GTP during the assay was subtracted from all data. Assays were performed in triplicate. Chromaffin cells grown on fibronectin-coated glass coverslips were washed with Locke's solution and subsequently fixed for 15 min in 4% paraformaldehyde in 0.12 msodium/potassium phosphate, pH 7.0, and for a further 10 min in fixative containing 0.1% Triton X-100. Following several rinses with phosphate-buffered saline (PBS), cells were pretreated with 3% bovine serum albumin, 10% normal goat serum in PBS to reduce nonspecific staining. Cells were incubated for 2 h at 37 °C with the primary antibodies in PBS containing 3% bovine serum albumin in a moist chamber. Cells were then washed with PBS and subsequently incubated for 1 h at 37 °C with the respective secondary antibodies diluted to 1:200 in PBS containing 3% bovine serum albumin. Finally, coverslips were extensively washed with PBS, rinsed with water, and mounted in Moviol 4–88 (Hoechst). Immunofluorescence staining was monitored with a Zeiss laser scanning microscope (LSM 410 invert) equipped with a planapo oil (63 ×) immersion lens (numerical aperture =1.4). DTAF emission was excited using the argon laser 488-nm line, whereas TRITC was excited using the helium/neon laser 543-nm line. The emission signals were filtered with a Zeiss 515–565-nm filter (DTAF emission) or with a long pass 595-nm filter (TRITC signal). Nonspecific fluorescence was assessed by incubating cells with the secondary fluorescent antibodies and measuring the average intensity value for each fluorochrome. This value was subtracted from all images. Chromaffin granule membrane proteins (250 μg) were subjected to ADP-ribosylation with recombinant C3 transferase. Granule membranes were subsequently immunoprecipitated with 30 μl of anti-DβH antiserum and 100 μl of ImmunoPure Immobilized protein A (Pierce) (10Darchen F. Zahraoui A. Hammel F. Monteils M.P. Tavitian A. Scherman D. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5692-5696Crossref PubMed Scopus (163) Google Scholar). The beads were collected by centrifugation, and washed and bound proteins were analyzed by SDS-gel electrophoresis and autoradiography. One-dimensional SDS-polyacrylamide gel electrophoresis was performed on 12% acrylamide gels in Tris glycine buffer (30Bader M.F. Trifaro J.M. Langley O.K. Thierse D. Aunis D. J. Cell Biol. 1986; 102: 636-646Crossref PubMed Scopus (55) Google Scholar). Proteins were transferred to nitrocellulose sheets at a constant current of 140 mA for 45 min. Blots were developed with secondary antibodies coupled to alkaline phosphatase (Bio-Rad). Immunoreactive bands were detected with 5-bromo-4-chloro-3-indolylphosphate (0.15 mg/ml) and nitro blue tetrazolium (0.3 mg/ml) in 40 mm sodium carbonate, pH 9.8, and 5 mm MgCl2. The rabbit polyclonal anti-DβH antiserum used for chromaffin granule membrane immunoprecipitation was prepared in our laboratory and its specificity demonstrated (24Sontag J.M. Aunis D. Bader M.F. Eur. J. Cell Biol. 1988; 46: 316-326PubMed Google Scholar). Rabbit polyclonal anti-bovine chromogranin A (CGA) antibodies (34Ehrhart M. Grube D. Bader M.F. Aunis D. Gratzl M. J. Histochem. Cytochem. 1986; 34: 1673-1682Crossref PubMed Scopus (91) Google Scholar) were used at 1:5000 dilution. Mouse monoclonal antibodies against RhoA and rabbit polyclonal antibodies against RhoB (Santa Cruz Biotechnology, INC) were used at 1:10 dilution for immunocytochemical experiments and at 1:100 for immunodetection on nitrocellulose sheets. Donkey anti-rabbit IgGs conjugated to dichlorotriazinyl aminofluorescein (DTAF) were used at 1:200 dilution (Chemicon International Inc.). Goat anti-mouse IgG conjugated to tetramethylrhodamine (TRITC) were used at 1:200 dilution (Chemicon International Inc.). Affinity purified antibodies against the COOH-terminal Gαo peptide (ANNLRGCGLY) or Gαi peptide (KNNLKDCGLF) were prepared as described previously (6Vitale N. Mukai H. Rouot B. Thierse D. Aunis D. Bader M.F. J. Biol. Chem. 1993; 268: 14715-14723Abstract Full Text PDF PubMed Google Scholar), and their specificity against non-denaturated Gα protein was demonstrated. Mouse monoclonal antibodies against the p85 subunit of phosphatidylinositol 3-kinase were purchased from Upstate Biotechnology (Lake Placid, NY). Mastoparan was obtained from Sigma. Cytosolic and membrane-bound forms of GAP-43 were purified from bovine brain according to a previously published procedure (8Vitale N. Deloulme J.C. Thierse D. Aunis D. Bader M.F. J. Biol. Chem. 1994; 269: 30293-30298Abstract Full Text PDF PubMed Google Scholar).C. botulinum exoenzyme C3 ADP-ribosyltransferase (C3 transferase) prepared and purified as described (35Perelle S. Gibert M. Boquet P. Popoff M.R. Infect. Immun. 1993; 61: 5147-5156Crossref PubMed Google Scholar) was a generous gift of Dr. M. R. Popoff (Institut Pasteur, Paris). To investigate the intracellular distribution of Rho in chromaffin cells, we made use of the specific ADP-ribosylation reaction with C. botulinum C3 ADP-ribosyltransferase, in which [32P]ADP-ribose is incorporated on residue asparagine 41 of Rho (36Rubin E.J. Gill D.M. Boquet P. Popoff M.R. Mol. Cell. Biol. 1988; 8: 418-426Crossref PubMed Scopus (233) Google Scholar, 37Aktories K. Braun U. Rosener S. Just I. Hall A. Biochem. Biophys. Res. Commun. 1989; 158: 209-213Crossref PubMed Scopus (213) Google Scholar). Plasma membranes, chromaffin granule membranes, and cytosolic proteins were prepared from bovine adrenal medulla by subcellular fractionation on sucrose density gradients, and the presence of substrates for C3 transferase was analyzed by gel electrophoresis and autoradiography. We found that C3 transferase consistently labeled a 21-kDa protein present in chromaffin granule membranes and to a smaller extent in the cytosol (Fig. 1 A). In contrast, the plasma membrane contained virtually no detectable ADP-ribosylated product when incubated with C3 transferase (Fig. 1 A). Control experiments performed in the absence of C3 transferase confirmed that the granule-associated 21-kDa protein was specifically labeled in a toxin-dependent fashion (Fig. 1 A). The specific association of the C3 substrate with chromaffin granule membranes was further substantiated by analyzing the distribution pattern of [32P]ADP-ribosylated proteins in fractions collected from a sucrose density gradient layered with a crude membrane preparation. As illustrated in Fig. 1 B, the radiolabeled 21-kDa protein was not evenly distributed throughout the sucrose gradient but showed a major peak in fractions 10–12. These fractions contained the chromaffin granule membranes as revealed by immunodetection with anti-DβH antibodies, a specific marker for chromaffin granules. We also used an immunoadsorption procedure to exclude the association of the 21-kDa C3 substrate with a contaminating organelle that may comigrate with chromaffin granules in the sucrose density gradients. Fig. 1 C shows that preincubation of purified granule membranes with anti-DβH antibodies followed by protein A-Sepharose addition resulted in the co-adsorption of the 21-kDa [32P]ADP-ribosylated protein, in agreement with the idea that secretory granule membranes do contain a specific protein substrate for C3 transferase. Since Rho and Rac are potential substrates of C3 transferase (37Aktories K. Braun U. Rosener S. Just I. Hall A. Biochem. Biophys. Res. Commun. 1989; 158: 209-213Crossref PubMed Scopus (213) Google Scholar), we characterized further the granule-associated 21-kDa protein by immunodetection on nitrocellulose sheets. Fig. 2 illustrates a Western blot analysis of adrenal medullary plasma membranes, chromaffin granule membranes, and cytosolic proteins using anti-RhoA and anti-RhoB antibodies. RhoA was detected in the granule membrane fraction, displaying an apparent molecular mass of 21 kDa (Fig. 2 A). We also observed the presence of a substantial amount of RhoA in the cytosolic fraction but not in the plasma membrane (Fig. 2 A), in agreement with the results obtained by C3-induced ADP-ribosylation. In contrast, no specific immunosignal for RhoB was detectable among the three subcellular fractions (Fig. 2 B). To evaluate the portion of RhoA present in the cytosol, cultured chromaffin cells were collected, and the content of RhoA was estimated in three fractions defined as the total homogenate, the cytosol, and the membrane-bound compartment. By C3-catalyzed ADP-ribosylation and immunoreplica analysis using the anti-RhoA antibodies, we found that the cytoplasmic pool represented approximately 15–20% of the total RhoA present in chromaffin cells (Table I).Table IQuantitative analysis of the distribution of RhoA in chromaffin cellsTotal proteinC3 substrateRhoA immunoreactivitymgunitsunitsHomogenate8.85617 (100%)157 (100%)Cytosol4.19119 (19%)23 (14%)Membrane-bound3.15389 (63%)115 (73%)Cultured chromaffin cells (50 × 106 cells) were homogenized and processed to separate the cytosol from the membrane-bound compartment. Fractions were then subjected to protein determination, C3-catalyzed [32P]ADP-ribosylation, and RhoA immunodetection on nitrocellulose sheets. Values obtained by autoradiography (PhosphorImager program) and scanning densitometry analysis are expressed as arbitrary units from one representative experiment. Values in parentheses correspond to the distribution of RhoA relative to the total amount detected in the cell homogenate. Similar results were obtained in two separate experiments. Open table in a new tab Cultured chromaffin cells (50 × 106 cells) were homogenized and processed to separate the cytosol from the membrane-bound compartment. Fractions were then subjected to protein determination, C3-catalyzed [32P]ADP-ribosylation, and RhoA immunodetection on nitrocellulose sheets. Values obtained by autoradiography (PhosphorImager program) and scanning densitometry analysis are expressed as arbitrary units from one representative experiment. Values in parentheses correspond to the distribution of RhoA relative to the total amount detected in the cell homogenate. Similar results were obtained in two separate experiments. Two-dimensional gel electrophoresis of purified chromaffin granule membranes revealed the presence of two spots labeled with the anti-RhoA antibody. These two spots displayed only a different isoelectric point value (respectively, 6.2 and 6.5) but no difference in the apparent molecular mass (data not shown). Occasionally, the RhoA-labeled band present in chromaffin granule membranes could also be resolved into two components on monodimensional gels (Fig. 2 A). This observation may reflect some post-translational modification, such as phosphorylation. Consistent with this idea, studies in human lymphocytes (38Lang P. Gesbert F. Delespine-Carmagnat M. Stancou R. Pouchelet M. Bertoglio J. EMBO J. 1996; 15: 510-519Crossref PubMed Scopus (477) Google Scholar) indicate that membrane-bound RhoA is a target for protein kinase A-mediated phosphorylation, whereas cytosolic RhoA is protected from phosphorylation by its binding to the GDP dissociation inhibitor. Next
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