Proc. Nati. Acad. Sci. USA Vol. 87, pp. 5692-56%, August 1990 Cell Biology Association of the GTP-binding Rab3A with bovine adrenal chromaffin granules (catecholamine/Ras-like protein/secretion/vesicle) FRANCOIS DARCHEN*t, AHMED ZAHRAOUI*, FREDERIC HAMMEL*, MARIE-PIERRE MONTEILS*, ARMAND TAVITIAN*, AND DANIEL SCHERMAN*t *UA 1112 Centre National de la Recherche Scientifique, Institut de Biologie Physico-Chimique, 75005 Paris, France; and tU248 Institut National de la Sante et de la Recherche MWdicale, Facultd de MWdecine Lariboisiere Saint-Louis, 75010 Paris, France Communicated by Pierre Joliot, May 7, 1990 (received for review February 5, 1990)

ABSTRACT The Rab3A protein belongs to a large family The protein Rab3A, which belongs to the family of small GTP-binding that are present in eukaryotic cloned from a brain cDNA library and which is identical to cells and that share amino acid identities with the Ras proteins Smg25A isolated from brain extracts (6-8, 31, 32), is likely to (products of the ras protooncogenes). Rab3A, which is specif- be involved in regulated secretion, since it is specifically ically located in nervous and endocrine tissues, is suspected to detected in neural, endocrine, and exocrine tissues (33-36) play a key role in secretion. Its localization was investigated in and since its expression increases with neuronal differentia- bovine adrenal gland by using a polyclonal antibody. Rab3A tion (34, 37). As a first step in elucidating the function of was detected in adrenal medulla but not in adrenal cortex. In Rab3A, we have studied its cellular and subcellular localiza- cultured adrenal medulla cells, Rab3A was specifically ex- tion in adrenal medulla, a gland that secretes epinephrine and pressed in the catecholamine-secreting chromaffin cells. Sub- norepinephrine. cellular fractionation suggested that Rab3A is about 30% cytosolic and that particulate Rab3A is associated with the membrane of chromaffin granules (the catecholamine storage MATERIALS AND METHODS organelles) and with a second compartment likely to be the Materials. [3H]Dihydrotetrabenazine ([3H]TBZOH, 15 Ci/ plasma membrane. The Rab3A localization on chromaffin mmol; 1 Ci = 37 GBq) was from the Centre d'Etudes granule membranes was confirmed by immunoadsorption with Nucleaires (Gif-sur-Yvette, France). Uridine diphospho- an antibody against dopamine f-hydroxylase. Rab3A was not D-[U-14C]galactose (319 mCi/mmol), 1251 (100 mCi/ml), and extracted from this membrane by NaCl or KBr but was affinity-purified 1251-labeled protein A (30 mCi/mg) were partially extracted by urea and totally solubilized by Triton from Amersham. [a-32P]GTP (3000 Ci/mmol) was from X-100, suggesting either an interaction with an intrinsic protein NEN. Fluorescein-conjugated affinity-purified F(ab')2 frag- or a membrane association through fatty acid acylation. This ment of goat anti-rabbit IgG was from Immunotech S.A. study suggests that Rab3A, which may also be located on other (Marseille, France). Anti-rabbit IgG and anti-goat IgG, alka- secretory vesicles containing noncharacterized small GTP- line phosphatase conjugates, were from Promega and Sigma, binding proteins, is involved in their biogenesis or in the respectively. Rabbit antiperoxidase and peroxidase were regulated secretion process. from UCB-Bioproducts. Protein standards for electrophore- sis were from Bio-Rad. Protein A-Sepharose CL4B was from In addition to the GTP-binding proteins involved in signal Pharmacia. transduction, protein synthesis, or formation, a large Culture of Chromafmn Cells. Bovine chromaffin cells were family of small GTP-binding proteins has been discovered in isolated from fresh adrenal glands by retrograde perfusion eukaryotic cells (1-12). These 20- to 27-kDa proteins, which with collagenase and purified on a self-generating Percoll present amino acid identities with the ras protooncogene gradient (25, 38). They were suspended in Dulbecco's mod- products, might be involved in central cellular processes. In ified Eagle's medium with 10% fetal bovine serum, 10 AM yeast, Ras proteins interact with adenylate cyclase and with 1-,B-D-arabinofuranosylcytosine, 10 ,uM 5-fluoro-2'-deoxy- inositolphospholipid turnover, suggesting a function in signal uridine, 50 ,ug of streptomycin per ml, and 50 units of transduction (1, 12). Moreover, an involvement in intracel- penicillin per ml (25) and cultured in plastic flasks (Nunc) at lular vesicle traffic and secretion has been shown in yeast for 360C in a water-saturated 5% CO2 atmosphere. the Ras-like YPT1 (13, 14) and SEC4 (15, 16) proteins. Polyclonal Antibody Against Rab3A. Rabbits were immu- Mutation of the SEC4 protein, which is associated with nized with purified human Rab3A expressed in Escherichia secretory vesicles and the plasma membrane, blocks a post- coli (7). The antiserum IgG fraction, obtained by DEAE Golgi step of constitutive secretion. chromatography (pH 7.2), was used at a 1:100 dilution. When The function of the Ras-like proteins in mammals is un- indicated, an immunopurified antibody was used. known. A role in analogous to that of the Iodination of Plasma Membrane External Proteins. Chro- trimeric GTP-binding proteins has been suggested (17, 18). maffin cells (50 x 106) were incubated with Na1251 and Small GTP-binding proteins may also be involved in vesicle lactoperoxidase (39) at 200C for 5 min. traffic (19), since GTP analogs affect intra-Golgi vesicle Protein Separation and Immunostaling. Samples were transport (20) and regulated secretion in neutrophils, plate- boiled for 3 min in 60 mM Tris HCI, pH 6.8/10%o glycerol/2% lets, and chromaffin cells (21-25). Moreover, association of SDS/5% 2-mercaptoethanol, and proteins were separated by noncharacterized small GTP-binding proteins with secretory vesicles has been reported (26-30). Abbreviations: BSA, bovine serum albumin; DBH, dopamine /3- hydroxylase; PBS, phosphate-buffered saline; TBZOH, dihydrotet- rabenazine. The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed at: Institut de payment. This article must therefore be hereby marked "advertisement" Biologie Physico-Chimique, 13, rue P. et M. Curie, 75005 Paris, in accordance with 18 U.S.C. §1734 solely to indicate this fact. France. 5692 Cell Biology: Darchen et al. Proc. Natl. Acad. Sci. USA 87 (1990) 5693 SDS/PAGE (40) (generally in 13% polyacrylamide or in 8% adrenal cortex, and the medullary localization of Rab3A was polyacrylamide for immunodetection) and electro- confirmed by immunohistology (Fig. 1B). Cultured dissoci- phoretically transferred to nitrocellulose sheets (0.45 um; ated cells from adrenal medulla contain endothelial cells in Schleicher & Schuell) in 20 mM Tris/150 mM glycine/20o addition to the catecholamine-secreting chromaffin cells. methanol/0.02% SDS, pH 8.3 (41). The blots were saturated Rab3A was detected exclusively in chromaffin cells identified in phosphate-buffered saline (PBS: 140 mM NaCl/5.7 mM by antibody to dopamine ,8-hydroxylase (DBH), an enzyme phosphate, pH 7.4)/0.2% Tween 20/5% powdered skim milk, located in the secretory chromaffin granules (Fig. 2). incubated for 1 hr at 20'C with antibodies in PBS/0.2% Subcellular Localization of Rab3A. Cultured chromaffin Tween 20/1% bovine serum albumin (BSA), and washed in cells were homogenized and fractionated by differential cen- the saturation medium. Proteins that bound antibodies were trifugation. Rab3A was abundant in the nuclear (ref. 54; revealed as indicated in the figure legends. according to the nomenclature of de Duve) fraction N, the Biochemical Assays. Binding of 4 nM [a-32P]GTP to nitro- mitochondrial fraction M, and the cytosolic fraction S but cellulose transfer blots was performed (17) in 50 mM was very low in the microsomal fraction P (Table 1). Fraction Tris-HCl, pH 7.5/0.25% Tween 20/5 mM MgCl2/0.5 mM M was enriched in plasma membrane Na+/K+ pump, in dithiothreitol/0.2% BSA and detected by autoradiography. mitochondrial cytochrome-c oxidase, in lysosomal 8-glucu- Glucose-6-phosphatase and galactosyltransferase (42), (3- ronidase, and in the monoamine transporter of chromaffin glucuronidase (43), cytochrome-c oxidase (44), and ouabain- granules assayed by [3HJTBZOH binding (46). On the other sensitive Na+/K+ pump ATPase (45) were assayed as de- hand, the specific activity of endoplasmic reticulum glucose- scribed. Binding of 3 nM [3HJTBZOH to chromaffin granule 6-phosphatase and ofGolgi galactosyltransferase was highest membranes (46) was assayed after 2 hr of incubation at 20°C in fraction P. Clathrin (a marker of coated vesicles) was high in 0.3 M sucrose/40 mM KOHHepes, pH 8.0, followed by in fractions S, N, and P but almost totally absent from filtration over GF/C fiberglass filters (Whatman). Filters fraction M (data not shown). were washed with three 2-ml portions of the same buffer The heterogeneous fraction N, known to contain nuclei, supplemented with 100 ,uM tetrabenazine and 3H was mea- unbroken cells, and debris, was not studied further. Fraction sured by liquid scintillation. Protein was determined by the M, enriched in Rab3A, was fractionated on a density gradient Lowry procedure. (Fig. 3) on which chromaffin granules are well separated from other cellular constituents (48, 49). Proteins were distributed throughout the gradient, with three peaks corresponding to RESULTS sucrose concentrations of 50% (peak 1), 35% (peak 2), and Tissue and Cell Specificity of Rab3A Expression. The rabbit antibody against human Rab3A expressed in E. coli did not recognize the other analogs of the Rab family (data not shown). On a Western blot ofa homogenate ofbovine adrenal medulla, the major band corresponded to the molecular mass of Rab3A (27 kDa; Fig. 1A). This band was not observed in + 4~~~, ..,, 1 A B "~ a C M 97_ 660- _ Cf

42 w- Cr ~,1:6 31 m $.lx:.*

21 M

14 m FIG. 2. Immunofluorescent staining of cultured bovine adrenal medulla cells. Phase-contrast micrographs (Upper) show the pres- ence of round, bright chromaffin cells (arrowheads) and of flat, spindle-shaped endothelial cells. Only chromaffin cells were deco- FIG. 1. Rab3A localization in the adrenals. (A) Homogenates (20 rated with an antibody against DBH (A Lower) or Rab3A (B Lower). ,g of protein) from bovine adrenal medulla (lane M) or cortex (lane Cells were cultured for 3 days on glass in Lab-Tek chamber slides C) were immunoblotted and stained with anti-Rab3A antibody and (Nunc). For DBH detection, cells were permeabilized and fixed with alkaline phosphatase-conjugated anti-rabbit IgG. Molecular size 5% acetic acid/95% methanol for 10 min at -20°C, washed with PBS, (kDa) standards are indicated at left; arrowhead at right points to incubated for 1 hr at 20°C with anti-DBH antibody (1:500) in PBS Rab3A band. (B) Staining of rat adrenal gland with immunopurified containing 0.1% BSA, washed with PBS, incubated with the fluo- anti-Rab3A antibody. The medulla (M) is highly labeled. Some rescein-conjugated F(ab')2 fragment of anti-rabbit IgG, and washed. labeling is detected in the adrenal cortex zona reticularis (Cr), For Rab3A detection, cells were fixed with 4% formaldehyde in PBS whereas none is observed in the cortical zona fasciculata (Cf). Rats (10 min), washed with PBS, incubated with 10 mM NH4CI, washed, were perfused through the ascending aorta with 0.1% glutaral- permeabilized during 1 hr with 0.05% saponin/0.2% BSA/PBS, dehyde/4% paraformaldehyde in PBS (pH 7.4). Adrenals were washed, incubated with immunopurified anti-Rab3A antibody (1:10), incubated for 3 hr in the same medium, then overnight in PBS. washed, and incubated with fluorescein-conjugated anti-rabbit Sections (75 gm) were incubated with the antibody (1:10) in PBS plus F(ab')2 fragment, all steps being done in the permeabilization me- 0.1% BSA, followed by peroxidase-antiperoxidase detection (47). dium. The coverslips were mounted in a glycerol/PBS mixture. (Bar (Bar = 500 gm.) = 50,um.) 5694 Cell Biology: Darchen et al. Proc. Natl. Acad. Sci. USA 87 (1990) Table 1. Distribution of Rab3A and of subcellular markers after differential centrifugation Activity, % total (and per mg of protein) Fraction N Fraction M Fraction P Fraction S Rab3A 32.9 ± 10.4 (40.1) 42.6 ± 15.8 (99.4) 4.4 ± 3.5 (31.2) 20.0 ± 7.5 (39.8) Protein 35.6 22.1 8.4 33.9 [3H]TBZOH binding (pmol) 46.2 (2.8) 42.9 (4.2) 9.4 (2.4) 1.5 (0.1) Na+/K' ATPase (nmol/min) 41.1 (39.1) 37.4 (68.8) 14.1 (24.2) 7.4 (50.2) Cytochrome-c oxidase (j.mol/min) 40.4 (85.9) 59.0 (241.5) 0.6 (2.2) <0.2 (<0.5) f3-Glucuronidase (pmol/min) 41.1 (0.79) 36.9 (1.15) 12.9 (1.06) 9.1 (0.18) Glucose-6-phosphatase (nmol/min) 35.3 (7.0) 28.3 (12.2) 23.4 (19.6) 3.0 (0.6) Galactosyltransferase (fmol/min) 28.8 (17.3) 36.4 (35.2) 26.4 (66.9) 8.4 (5.2) Chromaffin cells (3 x 108) were mechanically collected after 3 days ofculture and homogenized with a glass homogenizer in 0.3 M sucrose/10 mM NaOH-Hepes, pH 7.0/1 mM EDTA with 5 ,ug of aprotinin per ml and 6 /Ag of leupeptin per ml (sucrose buffer). The homogenate was centrifuged at 1500 x g for 5 min. The pellet was homogenized again and centrifuged identically. This step was repeated twice to give the nuclear fraction N in the final pellet. The three supernatants were pooled and centrifuged at 20,000 x g for 20 min. The pellet was resuspended in sucrose buffer and centrifuged at the same speed to yield fraction M. The two supernatants were collected and centrifuged at 140,000 x g for 60 min to give the supernatant S and the microsomal pellet P. Rab3A was quantitated by immunoblotting using 1251-labeled protein A and scanning of the autoradiograms (arbitrary units); Rab3A results are means ± SE from three independent experiments. 25-30o (peak 3). Chromaffin granules were mostly in the gradients, an immunoadsorption procedure was used. Prein- densest peak, 1. Some [3H]TBZOH binding was observed cubation of chromaffin granule membranes (purified as in higher in the gradient, presumably due to the presence of Fig. 4 on a density gradient) with an anti-DBH antibody nonmature or lysed chromaffin granules. Golgi, endoplasmic followed by protein A-Sepharose addition resulted in the reticulum, and plasma membranes (revealed by the Na+/K+ coadsorption of Rab3A, [3H]TBZOH binding sites, DBH, pump and by cell surface protein iodination) were in peaks 3 and chromaffin granule membrane cytochrome b-561. Con- and 2. Such a bimodal localization has been reported for versely, no Rab3A was adsorbed when membranes were plasma membranes (50). Mitochondria were detected in a preincubated with an antibody against mitochondrial cy- single peak (peak 2), and lysosomes were broadly distributed tochrome-c oxidase (Fig. 5). Moreover, a similar result was around a slightly higher density (39% sucrose). The binding obtained on a nonfractionated postnuclear supernatant of of [a-32P]GTP to the fractions after SDS/PAGE displayed an cultured chromaffin cells: an antiserum against purified chro- increased intensity at the three peaks (Fig. 3E), for which the maffin granule membranes allowed the preferential adsorp- major labeling was observed at the electrophoretic Rab3A tion of DBH and of Rab3A (assayed by [a-32P]GTP binding), position. Rab3A was also detected in the three peaks (Fig. whereas the anti-cytochrome-c oxidase antibody selectively 3E), and the high Rab3A concentration in peak 1 contrasted immunoadsorbed mitochondrial membranes (data not shown). with the low concentration of membrane-bound proteins (the Together, these immunoadsorption experiments show that slight discrepancy between the distributions in peak 1 of Rab3A is located either on the chromaffin granule itself or on [3H]TBZOH binding and ofRab3A was not reproducible). On membranes that bind to the granules either physiologically or the other hand, Rab6, a protein ofthe Rab family localized in during the preparation steps. the Golgi apparatus (51), was specifically detected in the In purified chromaffin granules membranes adsorbed with upper fractions of the gradient (Fig. 3F), in agreement with the anti-DBH antibody, the Rab3A band corresponded to the the galactosyltransferase distribution. highest [a-32P]GTP binding activity, suggesting that Rab3A is The density-gradient experiment was performed seven the major small GTP-binding protein of the chromaffin gran- times, leading to similar results. In peaks 2 and 3, the amount ule membrane. of Rab3A was too high to be attributed entirely to the contamination by chromaffin granule membranes, and Rab3A was thus, at least partly, associated to a different DISCUSSION membrane compartment. Among the mammalian Ras-like proteins discovered so far, Association of Rab3A with the Chromaffin Granule Mem- Rab3A distinguishes itself by its selective localization in brane. While peaks 2 and 3 contained organelles of various tissues in which secretion is regulated (i.e., is dependent origin, peak 1 was quite homogeneously constituted of chro- upon an external stimulus, as opposed to constitutive secre- maffin granules. Evidence for the association of Rab3A from tion) and occurs through exocytosis of storage vesicles. This this peak with secretory granules was obtained by stimulating is exemplified in the adrenal gland, since Rab3A is expressed cultured chromaffin cells for secretion: a sustained depolar- in the medulla but is hardly detectable in the cortex, which ization by 50 mM K+ induced (i) nearly complete depletion secretes steroids by diffusion through the plasma membrane of the catecholamine stores; (ii) the loss of [3H]TBZOH (the low Rab3A concentration in the adrenal cortex may be binding in peak 1, which reflected the disappearance of attributed to contaminating chromaffin cells or to afferent mature chromaffin granules; and (iii) the concomitant disap- nerve endings). That Rab3A may be involved in regulated pearance of Rab3A immunodetection in peak 1 (data not secretion is further supported by the lack ofRab3A in adrenal shown). Moreover, Rab3A was still detected on purified medulla endothelial cells (Fig. 2) and in rat astrocytes (37) and granules after osmotic lysis, and was thus membrane-bound by the high Rab3A concentration in regions rich in synapses (Fig. 4). Rab3A was not extracted by 1 M NaCl or by 0.6 M of the cerebellum (35). KBr, a cytoskeleton-depolymerizing agent, indicating that it The main result of this study is the preferential association was neither extrinsically bound to the chromaffin membrane of Rab3A with the membrane of chromaffin granules. This nor attached to this membrane by cytoskeleton fibers. It was localization differs from that of other Ras-like proteins re- slightly extracted by 6 M urea and completely solubilized by ported so far, except for SEC4 association with yeast secre- 1% (vol/vol) neutral detergent Triton X-100. tory vesicles. Since Rab3A appears to be the major small To exclude the association of Rab3A with a contaminant GTP-binding protein of the chromafin granule membrane, it that comigrated with chromaffin granules in the density may be responsible for the GTP-binding activity reported on Cell Biology: Darchen et al. Proc. Natl. Acad. Sci. USA 87 (1990) 5695 Pellet Supernatant I 0 0 31_..__W_-

21._ 1 2 3 4 5 1 2 3 4 5 FIG. 4. Extraction of Rab3A from chromaffin granule mem- toal branes. Chromaffin granules were purified from adrenal medulla mmbrabowd homogenate by differential centrifugation followed by a 45% sucrose (density = 1.2 g/ml) discontinuous density gradient (49). Membranes I obtained by osmotic lysis and pelleting were incubated (10 mg of protein per ml) for 15 min at 40C in 20 mM Hepes (pH 7.4) (lanes 1) supplemented with 1 M NaCl (lanes 2), 6 M urea (lanes 3), 0.6 M KBr (lanes 4), or 1% Triton X-100 (lanes 5) and centrifuged at 140,000 x g for 1 hr. Identical volumes were analyzed for Rab3A by immu- ._ 0 notransfer, using anti-rabbit IgG alkaline phosphatase conjugate for [3-HIn 7BZOH binding detection. In the experiment shown, less Rab3A was immunode- a kTPase tected in the Triton X-100 supernatant than would be expected from -0-o- MfeabriAnebound[125-I1 the loss of signal in the pellet. This artifact was not observed in two :E subsequent control experiments.

0 secretory vesicles from other systems (28-30). We believe that the Rab3A association with the chromaffin granule a membrane cannot be attributed to membrane fusion or to artifactual binding occurring during homogenization and frac- tionation, since (i) the highest Rab3A concentration per mass f*W 0 ofmembrane-bound protein was in chromaffin granules (peak - B 0 glucuronidase 1, Fig. 3), in which comparatively very little Rab6 was D- Glc 6 phosphatase detected, and (ii) Rab3A was not extracted from chromaffin _- Galactosyl zansferase 0- Cytochrome-c oxidase granule membranes by 1 M NaCl or 0.6 M KBr. Moreover, the association of Rab3A with an unidentified membrane compartment comigrating with chromaffin granules in the density gradients is unlikely, since Rab3A was immunoad- sorbed using an antibody against DBH. Rab3A was partly extracted from chromaffin granule mem- branes by chaotropic 6 M urea, suggesting that it might be partially associated noncovalently with a membranous o [32-P]GTP binding protein. On the other hand, the Ras proteins have been -.--* rab3A S reported to be attached to membranes through polyisopre- nylation (farnesylation) of a C-terminal cysteine (52). A similar mechanism might occur for Rab3A, whose complete 'I solubilization required detergent. This point was not inves- 0 tigated. Rab3A is not exclusively located on chromaffin granules. The results of Table 1 and Figs. 3 and 5 tend to exclude a 10 15 preferential association of Rab3A with mitochondria, lyso- Fractions somes, Golgi apparatus, endoplasmic reticulum, and clath- F rin-coated vesicles but reveal the existence of a soluble form 31 - (probably cytosolic) and suggest also a localization on the

a b a b a b a b 21 FIG. 3. Isopycnic centrifugation of fraction M from bovine chro- 97_ 31_ 66_ m -w maffin cells. The fraction was layered over a 1-1.8 M sucrose linear '0 density gradient containing 10 mM NaOH-Hepes (pH 7.0), 1 mM 21_ EDTA, 5 ug of aprotinin per ml, and 6 Ag of leupeptin per ml. The gradient was centrifuged for 3 hr at 250,000 x g. Fractions were DBH Cyt b rab3A GTP collected from the bottom. Data are expressed in percent of total activity of the gradient. (A) Density. (B) Total proteins (*), and FIG. 5. Immunoadsorption of chromaffin granule membranes. membrane-bound proteins (o) obtained by hypoosmotic lysis, 5 sec of Membranes (800 ,ug prepared as in Fig. 4) were incubated at 4°C for sonication, and 1 hr ofcentrifugation at 140,000 x g. (C) [3H]TBZOH 1 hr with an antiserum against DBH (lanes a) or cytochrome-c binding (o), Na+/K+ ATPase activity (9), and membrane-bound oxidase (lanes b), washed with PBS, pelleted at 160,000 x g for 20 radioactivity after [125I]iodination of intact chromaffin cells, and min, and incubated for 2 hr at 4°C with 80 ,ul of protein A-Sepharose fractionation on a parallel sucrose gradient (o) (fractions were sub- CL-4B in PBS (pH 7.4) containing 5 Ag of aprotinin per ml and 6 ,ug mitted to hypoosmotic lysis and centrifuged for 1 hr at 140,000 x g, of leupeptin per ml. After five washes with PBS, proteins linked to and radioactivity of pellets was measured by liquid scintillation). (D) the protein A-Sepharose were eluted with SDS/PAGE buffer, elec- f3-Glucuronidase (m), glucose-6phophatase (o), galactosyltransferase trophoresed, transferred, and analyzed for [a-32P]GTP binding or (e), and cytochrome-c oxidase (o). (E) [a-32P]GTP binding (o) and stained with antibodies against Rab3A, DBH, and cytochrome b561 Rab3A immunodetection using 125I-labeled protein A (0) on Western (Cyt b) followed by anti-rabbit IgG alkaline phosphatase conjugate. blots quantitated by autoradiogram scanning; values of [a-32P]GTP Membranes attached to the protein A-Sepharose were also assayed binding refer to the major 27-kDa band identical to that of Rab3A. (F) for [3H]TBZOH binding: 290 and 26 pmol ofbinding sites were found Staining with a polyclonal antibody against Rab6 followed by alkaline with membranes preincubated with the antibodies against DBH or phosphatase-conjugated second antibody. cytochrome-c oxidase, respectively. 5696 Cell Biology: Darchen et al. Proc. Natl. Acad. Sci. USA 87 (1990) plasma membrane or on noncharacterized organelles§. Cau- 7. Zahraoui, A., Touchot, N., Chardin, P. & Tavitian, A. (1989) J. Biol. tion should be taken with glucose-6-phosphatase as a reticu- Chem. 264, 12394-12401. 8. Kikuchi, A., Yamashita, T., Kawata, M., Yamamoto, K., Ikeda, K., lum endoplasmic marker in adrenal medulla, since this enzyme Tanimoto, T. & Takai, Y. (1988) J. Biol. Chem. 263, 2897-2904. is known to display a low activity in this tissue and may be 9. Kawata, M., Matsui, Y., Kondo, J., Hishida, T., Teranishi, Y. & Takai, contaminated by nonspecific phosphatases. However, the Y. (1988) J. Biol. Chem. 263, 18965-18971. 10. Polakis, P. G., Snyderman, R. & Evans, T. (1989) Biochem. Biophys. distribution ofthis markerin Table 1 and Fig. 3 agrees with that Res. Commun. 160, 25-32. of endoplasmic reticulum in liver cells (54, 55). In our exper- 11. Sewell, J. L. & Kahn, R. A. (1988) Proc. Nadl. Acad. Sci. USA 85, iments, cultured bovine chromaffin cells were preferred as 4620-4624. starting material over adrenal medulla tissue, which is less 12. Tamanoi, F. (1988) Biochim. Biophys. Acta 94, 1-15. 13. Segev, N., Mulholland, J. & Botstein, D. (1988) Cell 52, 915-924. reliable due to postmortem collection delay. Indeed, in several 14. Schmitt, H. D., Wagner, P., Pfaff, E. & Gallwitz, D. (1986) Cell 47, preliminary experiments using tissue homogenates, the pro- 401-412. portion of soluble Rab3A was higher than that observed with 15. Goud, B., Salminen, A., Walworth, N. C. & Novick, P. J. (1988) Cell 53, cultured cells, and Rab3A was 753-768. detected in Western blots as a 16. Walworth, N. C., Goud, B., Kabceneli, A. K. & Novick, P. J. (1989) doublet (data not shown and Fig. 1). A similar phenomenon EMBO J. 8, 1685-1693. has been described with yeast SEC4, for which the appearance 17. Lapetina, E. G., Lacal, J. C., Reep, B. R. & Molina y Vedia, L. (1989) of the lower molecular weight form was attributed to proteo- Proc. Nadl. Acad. Sci. USA 86, 3131-3134. 18. Maly, K., Uberall, F., Loferer, H., Doppler, W., Oberhuber, H., Groner, lytic cleavage (15). If one does not take into account the B. & Grunicke, H. H. (1989) J. Biol. Chem. 264, 11839-11842. noncharacterized N fraction (Table 1), the proportion of 19. Bourne, H. R. (1988) Cell 53, 669-671. soluble Rab3A is about 30%o. This high value agrees with 20. Melancon, P., Glick, B. S., Maihotra, V., Weidman, P. J., Serafini, T., results obtained in rat brain (36) and with the Gleason, M. L., Orci, L. & Rothman, J. E. (1987) Cell 51, 1053-1062. isolation of 21. Gomperts, B. D. (1986) Trends Biol. Sci. 11, 290-292. Rab3A from bovine brain cytosol (31). Although one cannot 22. Cockcroft, S., Howell, T. H. &Gomperts, B. D. (1987) J. Cell. Biol. 105, exclude a solubilization of membrane-bound Rab3A during 2745-2750. differential centrifugation, the hydrophilicity of the Rab3A 23. Penner, R. & Neher, E. (1989) Trends Neurosci. 12, 159-163. sequence (31) is 24. Knight, D. E., von Grafenstein, H. & Athayde, C. M. (1989) Trends compatible with a partial cytosolic localiza- Neurosci. 12, 451-458. tion, suggesting the existence of a recycling process between 25. Bader, M. F., Sontag, J. M., Thierse, D. & Aunis, D. (1989) J. Biol. different membrane compartments (16, 19). Chem. 264, 16426-16434. In conclusion, the present data identify Rab3A as a GTP- 26. Burgoyne, R. D. & Morgan, A. (1989) FEBS Lett. 245, 122-126. binding 27. Doucet, J. P., Fournier, S., Parulekar, M. & Trifaro, J. M. (1989) FEBS protein associated with a secretory vesicle, and point Lett. 247, 127-131. to an involvement ofthis protein in vesicle biogenesis, traffic, 28. Wildey, G. M., Im, M. J., Homcy, C. J. & Graham, R. M. (1989) J. Cell or exocytosis. Two working hypotheses for the role of Biol. 109, 293 (abstr.). GTP-binding proteins on vesicles have been proposed. Wal- 29. Rubins, J., Shannon, T. M. & Dickey, B. F. (1989) J. Cell Biol. 109, 298 worth et al. (16), based on genetic and (abstr.). subcellular localization 30. Padfield, P. J. & Jamieson, J. D. (1989) J. Cell Biol. 109, 299 (abstr.). studies, have suggested that SEC4 promotes the binding of 31. Matsui, Y., Kikuchi, A., Kondo, J., Hishida, T., Teranishi, Y. & Takai, secretory vesicles to the plasma membrane and is recycled Y. (1988) J. Biol. Chem. 263, 11071-11074. under its soluble form. By analogy with the prokaryotic 32. Yamamoto, K., Kim, S., Kikuchi, K. & Takai, Y. (1988) Biochem. Biophys. Res. Commun. 155, 1284-1292. EF-Tu, Bourne (19) has proposed that the 33. Olofsson, B., Chardin, P., Touchot, N., Zahraoui, A. & Tavitian, A. GTP-binding protein interacts with a budding donor mem- (1988) Oncogene 3, 231-234. brane and then remains bound to the vesicle to target its 34. Sano, K., Kikuchi, A., Matsui, Y., Teranishi, Y. & Takai, Y. (1989) selective attachment to an acceptor membrane. For a chro- Biochem. Biophys. Res. Commun. 158, 377-385. 35. Mizoguchi, A., Kim, S., Ueda, T. & Takai, Y. (1989) Biochem. Biophys. maffin granule, the donor membrane is either the trans-Golgi Res. Commun. 162, 1438-1445. network or shuttle vesicles originating from it. Alternatively, 36. 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