Vol. 10, No. 6

MOLECULAR AND CELLULAR BIOLOGY, June 1990, p. 2645-2652 0270-7306/90/062645-08$02.00/0 Copyright C 1990, American Society for Microbiology

Tissue and Subcellular Distributions of the smg-211rapll Krev-1 Proteins Which Are Partly Distinct from Those of c-ras p2ls SHIGEKUNI KIM,' AKIRA MIZOGUCHI,2 AKIRA KIKUCHI,' AND YOSHIMI TAKAI1* Departments of Biochemistry' and Anatomy,2 Kobe University School of Medicine, Kobe 650, Japan Received 4 December 1989/Accepted 25 February 1990

We have made a specific antiserum recognizing both smg p21A (the raplAlKrev-l protein) and -B (the raplB protein), ras p21-like GTP-binding proteins having the same putative effector domain as ras p2ls and have used this antiserum to study the tissue and subcellular distributions of smg p2ls by immunoblot and inmunocytochemical analyses. By immunoblot analysis, smg p2ls were detected in various rat tissues and at the highest level in brain. By light microscopic immunocytochemical analysis, smg p2ls were also detected in various rat tissues. Particularly, smg p2ls in brain were found abundantly in the cytoplasmic region of most types of neuronal cell bodies and moderately in neuropil, whereas c-ras p2ls were found more abundantly in neuropil than in the cytoplasmic region of most types of neuronal cell bodies. smg p2ls in testis were found in spermatogenic cells, in which c-ras p2ls were not significantly detected. By subcellular fractionation analysis of cerebrum, smg p2ls were detected in all of the particulate fractions but not in the cytosol fraction. Among the particulate fractions, approximately 70% of smg p2ls was recovered with the highest specific content in the fraction containing mainly synaptosomes, mitochondria, and myelin. In further fractionation of this fraction, approximately 40% of smg p2ls was recovered in each of the synaptosome fraction and the mitochondrial fraction. This subceilular distribution of smg p2ls in cerebrum was partly distinct from that of c-ras p21s, which were mainly recovered in the synaptosome and microsome fractions but present at very low levels in the mitochondrial fraction. In the synaptosome fraction, smg p2ls were recovered mainly in the synaptic plasma membrane fraction and partly in the synaptic vesicle and mitochondrial fractions with similar specific contents but not in the synaptic cytosol fraction. This intrasynaptosomal distribution of smg p2ls was also partly distinct from that of c-ras p2ls, which were recovered in the synaptic plasma membrane fraction but not in the synaptic vesicle or mitochondrial fraction. These tissue and subcellular distributions of smg p2ls together with the fact that smg p2ls have the same putative effector domain as ras p2ls suggest that smg p2ls exert their own specific actions in addition to the actions similar or antagonistic to those of c-ras p2ls.

calculated Mr values are 20,987 and 20,825, respectively (11, 17). Both smg p21A and -B have the consensus amino acid sequences for GTP- and GDP-binding and GTPase activities as described for other G proteins (11, 17), and the purified proteins exhibit these activities (9, 11, 22). smg p21A and -B share 95% amino acid sequence homology and differ from each other by only 9 amino acids, 6 of which are clustered in the last 13 amino acid residues of the C terminus (11, 17). smg p21A is identical with the raplA and Krev-1 proteins, and smg p21B is identical with the raplB protein (11, 14, 17, 23, 24). The most striking observation of smg p21A and -B is that both smg p2ls have the same putative effector domain as ras p2ls (11, 17). This structural property suggests that smg p2ls exert actions similar or antagonistic to those of ras p2ls. Consistent with this assumption, the Krev-1 gene has been shown to suppress the transforming activity of v-Ki-ras p21 in NIH 3T3 cells (14). The Krev-1 and raplA genes have been shown to be expressed in various mammalian tissues (14, 23). We have also found that the smg p21B gene is expressed in various mammalian tissues (K. Sano, Y. Matsui, and Y. Takai, unpublished observation). However, the tissue distributions of the protein molecules of smg p2ls have not been studied. We have previously found that smg p21B is present abundantly in human platelet membranes and that the amount of this G protein in platelet membranes

There is a superfamily of ras p21 and ras p21-like GTPbinding proteins (G proteins) with molecular weight (Mr) values of about 20,000 (see for reviews, references 1 and 28). smg p21 is a member of this superfamily, and we have purified it first from bovine brain membranes (11) and subsequently from human platelet membranes (22) and bovine aortic smooth muscle membranes (9). We have also cloned the cDNA of smg p21 from a bovine brain cDNA library and determined its primary structure (11). Recently, we have cloned another cDNA encoding a protein highly homologous to smg p21 from the same bovine brain cDNA library, using the smg p21 cDNA as a probe (17). We have designated the proteins encoded by the previously and newly isolated cDNAs as smg p21A and -B, respectively (17). Moreover, we have further sequenced the amino acids of smg p21 previously purified from bovine brain and human platelet membranes and identified both of them as smg p21B (17). Although we have not yet purified smg p21A from bovine brain or human platelet membranes, we have recently purified smg p21 from bovine aortic smooth muscle membranes (9) and identified it as a mixture of smg p21A and -B (M. Kawata, Y. Kawahara, S. Araki, M. Sunako, T. Tsuda, H. Fukuzaki, and Y. Takai, unpublished data). Both smg p21A and -B are composed of 184 amino acids, and their *

Corresponding author. 2645

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is at least 10 times higher than that of c-ras p21s (22). Moreover, we have found that smg p21B purified from bovine brain and human platelet membranes is phosphorylated by cyclic AMP-dependent protein kinase (protein kinase A) in a cell-free system and that it is also phosphorylated by the same protein kinase in intact human platelets in response to cyclic AMP-elevating prostaglandin E1 (6, 10). We have also found that bovine aortic smooth muscle smg p21A as well as p21B is phosphorylated by protein kinase A (Kawata, et al., unpublished data). smg p21A has recently been found in human platelet cytosol and has also been shown to be phosphorylated by protein kinase A (20, 21). In general, platelet aggregation and secretion are regulated in a bidirectional manner (27). Protein kinase C activation and Ca2'-mobilization linked to the phospholipase C-mediated hydrolysis of phosphoinositides induce platelet activation (for a review, see reference 27), whereas protein kinase A antagonizes this activation (4). Accumulating evidence suggests that in addition to the actions similar or antagonistic to those of ras p21s, smg p2ls have their own specific functions in cooperation with well-known intracellular messenger systems. To analyze the possible functions of smg p2is, we attempted as a first step to make an anti-smg p21 antibody and use it to investigate the tissue and subcellular distributions of smg p2ls. In this study, we first established an antiserum specifically recognizing both smg p21A and -B among many ras p21 and ras p21-like G proteins. By use of this antiserum, we show that smg p2is are present in various rat tissues, particularly brain, and that the tissue and subcellular distributions of smg p2is are partly distinct from those of c-ras p2ls.

MATERIALS AND METHODS Materials and chemicals. Female New Zealand White rabbits and adult male rats (200 to 250 g) of a SpragueDawley strain were used. smg p21B was purified from human platelet and bovine brain membranes as described previously (11, 22). smg p21A was purified from bovine aortic smooth muscle membranes (Kawata, et al., unpublished data). c-Ki-ras p21, rhoA p21, rhoB p20, and smg p25A were purified from bovine brain membranes as described previously (7, 12, 34, 35). v-Ki-ras p21 and c-Ha-ras p21 were generous gifts from H. Nakano (Kyowa Hakko Kogyo Co., Tokyo, Japan) and S. Hattori (University of Tokyo, Tokyo, Japan), respectively. Bovine brain ADP ribosylation factor was kindly supplied by R. A. Kahn (National Institutes of Health, Bethesda, Md.). The anti-ras p21 monoclonal antibody RASK-4 was a generous gift from H. Shiku (Nagasaki University, Nagasaki, Japan). This antibody recognized equally all Ha-, Ki-, and N-ras p2is but not smg p21A or -B even when they were used at 10-fold the concentrations of ras p2ls. The antisynaptophysin (p38) antibody was from Boehringer Mannheim Biochemicals. Gi and G. were purified from the crude membrane fraction of bovine brain (2, 26). '25I-labeled protein A (100 ,XCi/ml) and fluorescein-labeled and horseradish peroxidase-labeled goat anti-rabbit immunoglobulins were from Amersham Corp. Nitrocellulose sheets (BA-85; pore size, 0.45 ,um) were from Schleicher and Schuell, Inc. Prestained molecular weight marker proteins were from Bio-Rad Laboratories. Generation of the anti-smg p21 antiserum. The anti-smg p21 antiserum was made by a routine method. Briefly, female New Zealand White rabbits were immunized subcutaneously with 0.1 mg of smg p21B purified from human

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platelet membranes in complete Freund adjuvant. At 4-week intervals, two booster injections of 0.1 mg of human platelet smg p21B were given. Five days after the last booster injection, blood was collected and the serum was screened for the presence of the anti-smg p21 antiserum by the enzyme-linked immunosorbent assay. In this way, an antiserum highly specific for smg p21A and -B was obtained. Enzyme-linked immunosorbent assay. The 96-well microdilution plates were coated with smg p21B purified from human platelets by incubation with 0.25 ,g of smg p21B in 50 R1 per well overnight at 4°C. After the antigen solution was removed, the plates were washed three times with phosphate-buffered saline (PBS) containing 0.05% Tween 20, followed by the addition of 100 RI of PBS containing 5% bovine serum albumin (BSA) to block the unsaturated plastic surface. After 1 h at room temperature, the blocking solution was removed and the plates were washed three times with PBS containing 0.05% Tween 20. A 50-pI sample of a 1/1,000 dilution of the anti-smg p21 antiserum was added to each well. After incubation for 1 h at room temperature, the plates were washed four times with PBS containing 0.05% Tween 20, and the antiserum bound to smg p21B immobilized on the plates was detected by horseradish peroxidase-labeled antirabbit immunoglobulin. Preparation of the homogenates of various rat tissues. Various tissues of adult male rats were used. Tissues (about 0.1 g [wet weight] each) were homogenized at 4°C by six strokes with a Potter-Elvehjem Teflon-glass homogenizer in 10 ml of 1 mM NaHCO3 containing 0.32 M sucrose, 1 mM MgCl2, 0.5 mM CaCl2 and 1 puM (p-amidinophenyl)methanesulfonyl fluoride. The homogenates were solubilized with Laemmli sample buffer, boiled, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) and immunoblotting as described below. Subcellular fractionation of rat cerebrum. Subceilular fractionation of rat cerebrum was carried out by the method of Ueda et al. (31), which was a slight modification of the method of Cotman and Taylor (3) and that of Whittaker et al. (32). The detailed procedures have been described previously (19). Electron microscopic analysis of all subcellular fractions indicated that the ultrastructural characteristics of these fractions obtained by our procedures were similar to those reported previously by Ueda et al. (31). The validity of data for the subcellular fractionation of synaptic vesicles was checked by measuring the distribution of synaptophysin, a synaptic vesicle marker protein (8, 32). The distribution of this protein in the fractions obtained was similar to that described previously (8, 33). SDS-PAGE and immunoblotting. SDS-PAGE was performed by the method of Laemmli (15), using 12% polyacrylamide gels. Proteins on the gels were transferred electrophoretically to nitrocellulose sheets as described by Towbin et al. (30). The sheets were incubated with Tris-buffered saline (TBS) containing 5% BSA for 2 h at room temperature. For determination of the tissue and subceilular distributions of smg p21, the sheets were first incubated for 1 h at room temperature with a 1/400 dilution of the anti-smg p21 antiserum in TBS containing 5% BSA and washed three times for 10 min each with TBS containing 0.05% Tween 20. For detection of the attached antiserum on the sheets, the sheets were incubated for 1 h at room temperature with 251I-labeled protein A (2.5 ,Ci/ml in TBS containing 5% BSA) and washed three times for 20 min each with TBS containing 0.05% Tween 20. The sheets were autoradiographed by using Kodak X-Omat AR film with intensifying screens at -80°C.

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I 2 3 4 5 6 7 8 9 10 11 FIG. 2. Immunoblot analysis of smg p21 with the anti-smg p21 antiserum in various rat tissues. The homogenates of various rat tissues (50 ,ug each of protein) were immunoblotted with 1/400diluted antiserum. Lanes: 1, 50 ng of human platelet smg p21B; 2, cerebrum; 3, cerebellum; 4, adrenal gland; 5, thymus; 6, lung; 7, heart; 8, liver; 9, small intestine; 10, kidney; 11, testis.

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 FIG. 1. Specificity of the anti-smg p21 antiserum. (A) Human platelet smg p21B, bovine brain smg p21B, bovine aortic smooth muscle smg p21A, and rat cerebrum homogenate were separately subjected to SDS-PAGE. After SDS-PAGE, the proteins on the gels were either stained with Coomassie brilliant blue or transferred to a nitrocellulose sheet. The nitrocellulose sheet was reacted sequentially with 1/400-diluted anti-smg p21 antiserum and 251I-labeled protein A. Lanes: 1 to 4: protein staining of 100 ng of human platelet smg p21B (lane 1), 100 ng of bovine cerebrum smg p21B (lane 2), 100 ng of bovine aortic smooth muscle smg p21A (lane 3), and 100 p.g of rat cerebrum homogenate (lane 4); 5 to 9, immunoblot analysis of 100 ng of human platelet smg p21B (lane 5), 100 ng of bovine brain smg p21B (lane 6), 100 ng of bovine aortic smooth muscle smg p21A (lane 7), 100 ,ug of rat cerebrum homogenate (lane 8), and the same immunoblotted sheet as in lane 8 except that antiserum preincubated with an excess amount of smg p21B was used (lane 9). (B) Bovine brain membrane c-Ki-ras p21, rhoA p21, rhoB p20, and smg p25A and c-Ha-ras p21 and v-Ki-ras p21 produced in E. coli were separately subjected to SDS-PAGE. After SDS-PAGE, the proteins on the gels were either stained with Coomassie brilliant blue or immunoblotted as described above. Lanes: 1, 3, 5, 7, 9, 11, and 13, protein staining of 100 ng of human platelet smg p21B (lane 1), 100 ng of c-Ki-ras p21 (lane 3), 100 ng of rhoA p21 (lane 5), 100 ng of rhoB p20 (lane 7), 100 ng of smg p25A (lane 9), 100 ng of c-Ha-ras p21 (lane 9), and 100 ng of v-Ki-ras p21 (lane 13); 2, 4, 6, 8, 10, 12 and 14, immunoblot analysis of 100 ng of human platelet smg p21B (lane 2), 1 ,ug of c-Ki-ras p21 (lane 4), 1 p.g of rhoA p21 (lane 6), 1 ,ug of rhoB p20 (lane 8), 1 ,ug of smg p25A (lane 10), 1 ,ug of c-Ha-ras p21 (lane 12), and 1 ,ug of v-Ki-ras p21 (lane 14).

Light microscopic immunocytochemistry. Rats were perfused transcardially with 2% paraformaldehyde in PBS containing 8% sucrose. Brains and testes were removed, cut into small blocks, and immersed in the same fixative for an additional 2 h at 4°C. The fixed tissues were cryoprotected through a range of increasing sucrose concentrations (10, 15, and 20%), mounted in OCT embedding medium, quickly frozen, and sectioned on a cryostat. The 10-p.m-thick frozen sections collected on gelatin-coated slides were incubated with PBS containing 5% BSA for 1 h at room temperature and then overnight with a 1/400 dilution of the anti-smg p21 antiserum or with nonimmune rabbit immunoglobulin at a concentration of 10 ,ug/ml in PBS containing 5% BSA. After being washed four times with PBS, the sections were incubated for 4 h at room temperature with fluorescein-labeled goat anti-rabbit immunoglobulin at a final dilution of 1/20. After being washed four times with PBS, the sections were

mounted in PBS containing 20% glycerol under a cover glass and examined by a fluorescence microscope. Nonimmune rabbit immunoglobulin G was used as a control instead of the antiserum. Determination. Protein concentrations were determined with BSA as a standard protein by the method of Lowry et al. (16).

RESULTS Specificity of the anti-smg p21 antiserum. The anti-smg p21 antiserum was made by using smg p21B purified from human platelet membranes (22). This antiserum reacted with both human platelet and bovine brain membrane smg p2lBs to the same extent (Fig. 1A). This antiserum also equally reacted with bovine aortic smooth muscle membrane smg p21A. When the total homogenate of rat cerebrum was immunoblotted with this antiserum, a single band was detected. This band migrated at the same position as that of smg p2ls. The Mr value of this band was about 22,000. This band was not observed when the electroblotted sheet was stained with the anti-smg p21 antiserum preincubated with an excess amount of smg p21B. This band was also not detected by nonimmune serum (data not shown). These results indicate that this band is smg p21A or -B. We have separated at least 15 ras p21 and ras p21-like G proteins from bovine brain membranes by several column chromatographies and purified five of them to near homogeneity, including c-Ki-ras p21, rhoA p21, rhoB p20, smg p21B, and smg p25A (7, 11, 12, 34, 35). The anti-smg p21 antiserum was not reactive with c-Ki-ras p21, rhoA p21, rhoB p20, or smg p25A even when they were used at 10-fold the concentration of smg p2ls (Fig. 1B). This antiserum was also inactive for v-Ki-ras p21 or c-Ha-ras p21 produced from Escherichia coli. Furthermore, the anti-smg p21 antiserum was inactive for any of other partially purified ras p21-like G proteins from bovine brain membranes even when they were used at 10-fold the concentration of smg p2ls (data not shown). The amounts of these partially purified G proteins were calculated on the basis of the [35S]GTP-yS-binding activity of the respective G proteins. The anti-smg p21 antiserum did not react with either Gi, G., or ADP ribosylation factor purified to near homogeneity from bovine brain membranes even when they were used at 10-fold the concentration of smg p2is (data not shown). Tissue distribution of smg p2ls. The band corresponding to smg p2is was also observed in various rat tissues, including cerebrum, cerebellum, adrenal gland, thymus, lung, heart, liver, small intestine, kidney, and testis (Fig. 2). Among these tissues, smg p2is were abundant in cerebrum, lung, testis, and particularly cerebellum. Light microscopic immunocytochemistry of smg p2ls. The

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FIG. 3. Indirect immunofluorescence staining of rat cerebellum with the anti-smg p21 antiserum (A) and RASK-4 (B). The immunoreactive materials appear white. M, Molecular layer; P, Purkinje cell layer; G, granule cell layer. Bars, 50 ,.m.

10-,um-thick frozen sections of rat organs were examined for the presence of smg p2ls by the indirect immunofluorescence method. In cerebellum, the cytoplasmic region of Purkinje cells was stained strongly, but the nuclei were unstained (Fig. 3A). Moderate fluorescence was seen homogeneously in the molecular layer, and patchy fluorescence was found in the granule cell layer. On the other hand, when the cerebellum was stained with the anti-ras p21 antibody, RASK-4, which recognized equally all Ha-, Ki-, and N-ras p21s but not smg p21A or -B, Purkinje cells were stained very weakly, but the molecular layer and granule cell layer

A

were stained strongly (Fig. 3B). In cerebrum, the cytoplasmic region of most types of neuronal cell bodies was also stained strongly, and neuropil was stained moderately by the anti-smg p21 antiserum (Fig. 4A). In contrast, when cerebrum was stained by RASK-4, the cytoplasmic region of most types of neuronal cell bodies was stained very weakly, but neuropil was stained strongly (Fig. 4B). In testis, strong immunofluorescence of smg p2ls was seen homogeneously in the cytoplasmic region of all types of spermatogenic cells by the anti-smg p21 antiserum, but the spermatogenic cells of testis were not stained significantly by RASK-4 (Fig. 5).

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FIG. 4. Indirect immunofluorescence staining of rat cerebrum with the anti-smg p21 antiserum (A) and RASK-4 (B). Bars, 50 ,um.

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FIG. 5. Indirect immunofluorescence staining of rat testis with the anti-smg p21 antiserum (A) and RASK-4 (B). The connective tissue lining the seminiferous tubules appeared to be stained nonspecifically because the incubation of RASK-4 with an excess amount of c-Ha-ras p21 did not diminish the intensity of the fluorescence. Bars, 50 j±m.

Sertoli cells in testis were not stained by the anti-smg p21 antiserum. When all of these sections were stained with nonimmune rabbit immunoglobulin G as the control, no significant fluorescence was observed (data not shown). Subcellular distribution of smg p2is in rat cerebrum. The homogenate of rat cerebrum was fractionated into subcellular fractions as described previously (31), and the contents of smg p2is in each fraction were semiquantified by immunoblot analysis using the anti-smg p21 antiserum. The homogenate was first fractionated into the P1 fraction containing nuclei and cell debris, the P2 fraction containing synaptosomes, mitochondria, and myelin, the P3 fraction containing microsomes, and the S fraction containing soluble cytosol. smg p2is were almost exclusively associated with the particulate fractions (Fig. 6A). Approximately 70% of smg p2ls in the homogenate was recovered in the P2 fraction with the highest specific content. The P2 fraction was further fractionated into four fractions: the P2A fraction containing myelin and some contamination of membrane components in the P2B fraction, the P2B fraction containing a mixture of endoplasmic reticulum, Golgi complex, and plasma membranes, the P2C fraction containing mainly synaptosomes, and the P2D fraction containing mainly mitochondria. smg p2is were recovered in all four fractions. Approximately 40% of smg p2ls in the P2 fraction was recovered in the P2C fraction with the highest

1 2 3 4 FIG. 6. Immunoblot analysis of smg p21 with the anti-smg p21 antiserum in the subcellular fractions of rat cerebrum. Samples of each fraction (60 ,g each of protein) were used. (A) Primary four sections. The amounts of protein in the P1 (lane 1), P2 (lane 2), P3 (lane 3), and S (lane 4) fractions were 336, 734, 320, and 442 mg, respectively. (B) Subfractions of the P2 fraction. The amounts of protein in the P2A (lane 1), P2B (lane 2), P2C (lane 3), and P2D (lane 4) fractions were 162, 66, 184, and 200 mg, respectively. (C) Subfractions of the P2C fraction. The amounts of protein in the CSM (lane 1), CSV (lane 2), and SS (lane 3) fractions were 106, 2.08, and 34.5 mg, respectively. (D) Subfractions of the CSM fraction. The amounts of protein in the SM1 (lane 1), SM2 (lane 2), SM3 (lane 3), and SM4 (lane 4) fractions were 12.8, 22.4, 50.4, and 12.4 mg, respectively. (E) Subfractions of the CSV fraction. The amounts of protein in the SV1 (lane 1), SV2 (lane 2), SV3 (lane 3), and SV4 (lane 4) fractions were 812, 456, 238, and 272 pg, respectively. The results shown are representative of three independent blots obtained by three independent subcellular fractionation experiments. specific content, and another 40% of smg p2is in the P2 fraction was in the P2D fraction (Fig. 6B). Since the relatively large total content and high specific content of smg p2ls were observed in the P2C fraction, this fraction was further separated into three fractions: the CSM fraction containing crude synaptic membranes, the CSV fraction containing crude synaptic vesicles, and the SS fraction containing synaptosomal soluble substances. smg p2is were recovered mainly in the CSM fraction and partly in the CSV fraction with similar specific contents (Fig. 6C). In the SS fraction, smg p2ls were undetectable. When the CSM fraction was further fractionated into fractions SM1 to -4, smg p21s were found in fractions SM1 to -3 containing mainly synaptic membranes of various sizes and postsynaptic densities and fraction SM4 containing intrasynaptosomal mitochondria. Among these fractions, SM1, -3, and -4 showed similar specific contents, and approximately 70% of smg p2is in the CSM fraction was recovered in fraction SM3 (Fig. 6D). The CSV fraction was further fractionated into four fractions. Fraction SV1 contained synaptic vesicles almost free of synaptic membranes. The contents of synaptic plasma membranes were increased in fractions SV2 to -4. smg p2ls were recovered in fractions SV1 to -4. Approximately 40% of smg p2ls in the CSV fraction was recovered with the

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highest specific content in fraction SV2 (Fig. 6E). Approximately 40% of smg p2is in the CSV fraction was recovered in fraction SV1, which had a specific content similar to that of fraction SM1. DISCUSSION There is a superfamily of ras p21 and ras p21-like G proteins, the members of which are still increasing in number. In most cases the cDNAs have been isolated, but their protein molecules have not been purified or characterized from mammalian tissues as native forms. The tissue distribution of expression of their mRNAs has been examined, but that of their protein molecules, except ras p2ls (29), the yptl protein (5), and smg p25A (18; A. Mizoguchi, S. Kim, T. Ueda, A. Kikuchi, H. Yorifuji, N. Hirokawa, and Y. Takai, submitted for publication), has not been examined. ras p2is and the yptl protein have been shown to be ubiquitous (5, 29). We have recently shown that smg p25A is also highly expressed in secretory cells such as brain, endocrine glands, and exocrine glands (18; Mizoguchi et al., submitted). In this study, we first established an anti-smg p21 antiserum by use of smg p21B purified from human platelets. The antiserum obtained reacts equally with both human platelet and bovine brain membrane smg p2lBs. This antiserum also equally recognizes bovine aortic smooth muscle membrane smg p21A, which shares about 95% homology with smg p21B. This antiserum does not, however, react with other non-G proteins of rat brain homogenate that are separated by SDS-PAGE or with any of c-Ki-ras p21, rhoA p21, rhoB p20, smg p25A, ARF, Gi, or G., which have been purified to near homogeneity from bovine brain membranes. This antiserum also does not recognize other ras p21-like G proteins partially purified from bovine brain membranes, nor does it recognize v-Ki-ras p21 and c-Ha-ras p21 produced from E. coli. However, it remains to be clarified whether this antiserum is reactive with the rap2 protein (23), since the protein molecule of this G protein has not been identified or purified from mammalian tissues, including brain. By use of this specific antiserum against smg p2ls, we studied the tissue and subcellular distributions of this ras p21-like G protein by immunoblot and immunocytochemical methods. smg p2is are ubiquitously detected in various rat tissues by both methods. This result is consistent with the tissue distribution of the smg p21A mRNA (14, 23; Sano, et al., unpublished observation). The most characteristic structural feature of smg p2is is that these G proteins have the same putative effector domain as ras p2ls (11, 17). We have proposed from this property that smg p2ls can theoretically exert actions similar or antagonistic to those of ras p2ls (11). Consistently, the Krev-1 gene encoding the same G protein as smg p21A has been shown to suppress the transforming action of the activated ras p2ls in NIH 3T3 cells (14). The finding that smg p2is are ubiquitous as described for ras p2is is consistent with this proposed function of smg p2ls. Despite this ubiquitous distribution of smg p21s, they show tissue and subcellular distributions partly distinct from those of ras p2ls. smg p21B is abundant in human platelets, and its amount is at least 10 times higher than that described for ras p21s (22). Both smg p2is and ras p2is are abundant in synaptic areas of the brain, but smg p2is are detected most abundantly in the cytoplasmic region of most of neuronal cell bodies, where c-ras p2is are poorly detected. The physiological significance of the presence of smg p2ls in the neuronal cell bodies is not known, but it is conceivable that

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smg p21s exert their own specific actions in these cell bodies. Moreover, in the subcellular fractions of cerebrum, smg p2ls are recovered in all of the particulate fractions. Among these, smg p2ls are recovered mainly in the synaptosome, mitochondrial, and microsome fractions. In the synaptosome fraction, smg p2ls are present mainly in the synaptic plasma membrane fraction and partly in the synaptic vesicle and intrasynaptosomal mitochondria fractions but not in the synaptic cytosol fraction. It is noteworthy that smg p2ls are detected in a significant amount in the synaptic vesicle fraction and that their specific content in this fraction is similar to that in the synaptic plasma membrane fraction. These results suggest that smg p2ls are present in synaptic plasma membranes, synaptic vesicles, and intrasynaptosomal mitochondria and in the components of the neuronal cell bodies such as endoplasmic reticulum and mitochondria. We have previously shown that ras p2is are found in the synaptic plasma membrane fraction but not in the vesicle, mitochondrial, or cytosol fraction (19). Moreover, we have found that smg p25A is abundant in the synaptosome fraction (13; Mizoguchi et al., submitted). Among the components of synapses, smg p25A is most highly concentrated in synaptic vesicles; over 75% of smg p25A in synapses is located in synaptic plasma membranes, with lesser amounts in the synaptic cytosol. The results presented here together with these earlier observations suggest that at least two ras p21-like G proteins, smg p25A and smg p2ls, are present in synaptic vesicles as well as synaptic plasma membranes and that many ras p21-like G proteins, including smg p2ls, ras p2ls, smg p25A, rhoA p21, and rhoB p20 are present in synapses but that smg p2ls play roles different from those of other ras p21 and ras p21-like G proteins in synaptic functions such as exo-endocytotic recycling of synaptic vesicles. The physiological significance of the presence of smg p2is in microsomes and mitochondria remains to be clarified, but it has recently been demonstrated that smg p21A or -B (or both) is associated with cytochrome b purified from human neutrophil mitochondria (25). It may be noted that smg p2is are detected in spermatogenic cells of testis, where c-ras p2ls are undetectable. The seminiferous tubules are lined by epithelia composed of two major categories of cells: supporting cells and spermatogenic cells. The supporting cells are Sertoli cells, while spermatogenic cells include various stages in the continuous process of the differentiation of male germ cells. smg p2is are detected in various stages of spermatogenic cells but are undetectable in Sertoli cells. The functions of smg p2ls in spermatogenic cells are not known, and we cannot rule out the possibility that c-ras p2ls are expressed at a very low level in these cells. The results presented here, however, raise the possibility that smg p2is rather than c-ras p2ls are related to spermatogenesis. In conclusion, smg p2ls are ubiquitous as described for ras p2ls, but the tissue and subcellular distributions of smg p2is are partly distinct from those of c-ras p2ls. It is likely that smg p2ls exert their own specific actions in addition to the actions similar or antagonistic to those of ras p2ls, depending on cell type. However, it remains to be clarified whether the tissue and subcellular distributions of smg p21A and -B are similar or different, since the anti-smg p2is antiserum used in this study reacts equally with smg p21A and -B.

VOL. 10, 1990

TISSUE AND SUBCELLULAR DISTRIBUTIONS OF smg p2ls

ACKNOWLEDGMENTS We are grateful to H. Nakano, S. Hattori, and R. A. Kahn for supplying us with v-Ki-ras p21, c-Ha-ras p21, and bovine brain ARF, respectively. We are indebted to J. Yamaguchi for skillful secretarial assistance. This investigation was supported by a grant-in-aid for scientific research and cancer research from the Ministry of Education, Science, and Culture, Japan, grants-in-aid for abnormalities in hormone receptor mechanisms, for cardiovascular diseases, and for cancer research from the Ministry of Health and Welfare, Japan, and by grants from the Yamanouchi Foundation for Research on Metabolic Disease, the Research Program on Cell Calcium Signal in the Cardiovascular System, and the Princess Takamatsu Cancer Research Fund. LITERATURE CITED 1. Barbacid, M. 1987. ras genes. Annu. Rev. Biochem. 56:779827. 2. Bokoch, G. M., T. Katada, J. K. Northup, M. Ui, and A. G. Gilman. 1984. Purification and properties of the inhibitory guanine nucleotide-binding regulatory component of adenylate cyclase. J. Biol. Chem. 259:3560-3567. 3. Cotman, C. W., and D. Taylor. 1972. Isolation and structural studies on synaptic complexes from rat brain. J. Cell Biol.

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localization of synaptophysin, an integral membrane glycoprotein of Mr 38,000 characteristic of presynaptic vesicles. Cell 41:1017-1028. 34. Yamamoto, K., J. Kondo, T. Hishida, Y. Teranishi, and Y. Takai. 1988. Purification and characterization of a GTP-binding protein with a molecular weight of 20,000 in bovine brain

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membranes-identification as the rho gene product. J. Biol. Chem. 263:9926-9932. 35. Yamashita, T., K. Yamamoto, A. Kikuchi, M. Kawata, J. Kondo, T. Hishida, Y. Teranishi, H. Shiku, and Y. Takai. 1988. Purification and characterization of c-Ki-ras p21 from bovine brain crude membranes. J. Biol. Chem. 263:17181-17188.