Regulation of Reversible Binding of smg p25A, a rus ... · Regulation of Reversible Binding of smg p25A, a rus p214ike GTP- binding Protein, to Synaptic Plasma Membranes and Vesicles

THE JOURNAL OF BIOLOGICAL CHEMWIXY 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 265, No. 22, Issue of August 5, pp. 13007-13015,199O Printed in U.S.A.

Regulation of Reversible Binding of smg p25A, a rus p214ike GTP- binding Protein, to Synaptic Plasma Membranes and Vesicles by Its Specific Regulatory Protein, GDP Dissociation Inhibitor*

(Received for publication, February 27, 1990)

Shin Araki, Akira Kikuchi, Yutaka Hata, Mitsuo Isomura, and Yoshimi TakaiS From the Department of Biochemistry, Kobe University School of Medicine, Kobe 650, Japan

We have previously purified from bovine brain cytosol a novel regulatory protein for smg p25A, a ras p21-like GTP-binding protein. This protein, named smg p25A GDP dissociation inhibitor (GDI), regulates the GDP/GTP exchange reaction of smg p25A by inhibiting the dissociation of GDP from and thereby the subsequent binding of GTP to it. We have also previously found that smg p25A is mainly localized in presynaptic plasma membranes and vesicles and moderately in presynaptic cytosol in rat brain synapses. In this paper, we have studied the possible involvement of smg p25A GDI in the localization of smg p25A in the cytosol, plasma membranes, and vesicles in rat brain synapses. Both the GDP- and GTP-bound forms of smg p25A bound to the synaptic membranes and vesicles. smg p25A GDI inhibited the binding of the GDP-bound form of smgp25A, but not that of the GTP- bound form, to the synaptic membranes and vesicles. Moreover, smg p25A GDI induced the dissociation of the GDP-bound form, but not that of the GTP-bound form, of both endogenous and exogenous smg p25As from the synaptic membranes and vesicles. smg p25A CD1 made a complex with the GDP-bound form of smg p25A with a molar ratio of l:l, but not with the GTP- bound or guanine nucleotide-free form. These results suggest that smg p25A reversibly binds to synaptic plasma membranes and vesicles and that this reversible binding is regulated by its specific GDI.

smg p25A is a member of a smg p25 family composed of three members which belongs to a ras p2llra.s p21-like G protein’ superfamily (for reviews, see Refs. 1 and 2). smg p25A has first been purified from bovine brain membranes (3). The primary structure of this protein has been also determined (4). smg p25A is composed of 220 amino acids with a calculated

* This investigation was supported by Grants-in-Aid for Scientific Research and Cancer Research from the Ministry of Education, Science, and Culture, Japan (1989). Grants-in-Aid for Abnormalities in Hormone Receptor Mechanisms, Cardiovascular Diseases, and for Cancer Research from the Ministrv of Health and Welfare, JaDan (1989), and by grants from the Yamanouchi Foundation for Research on Metabolic Disease (1989), the Research Program on Cell Calcium Signal in the Cardiovascular System (1989), and the Princess Taka- matsu Cancer Research Fund (1988). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertkement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be addressed. 1 The abbreviations used are: G protein, GTP-binding protein;

GDI, GDP dissociation inhibitor; DTT, dithiothreitol; GTPrS, guan- osine 5’-(3-0-thio)triphosphate; SDS-PAGE, sodium dodecyl sulfate- polyacrylamide gel electrophoresis; Chaps, 3-[(3-cholamidopropyl) dimethylammoniol-1-propanesulfonate.

M, value of 24,954 (4). smg p25A is identical with the protein of the rab3A gene (5). smg p25A is present abundantly in brain but is also found in neurocrine, exocrine, and endocrine secretory cells, but not in nonsecretory cells (6-9). In brain, smg p25A is mainly localized in synapses and partly in the cytosol (9-11). In synapses, smg p25A is mainly localized in presynaptic plasma membranes and vesicles and moderately in synaptic cytosol but not in mitochondria or postsynapses (9, 11). The functions of smg p25A have not been clarified, but available evidence suggests that smg p25A plays important roles in secretory processes.

smg p25A has a unique C-terminal sequence that is Cys- Ala-Cys (4). This structure is different from those of ras ~21s which have Cys-A-A-X where A is aliphatic amino acids and X is any amino acid (1, 2). It has recently been shown that polyisoprenoid moiety is attached to this cysteine residue of ras ~21s and essential for ras ~21s to attach to membranes and to acquire transforming activity (12). Evidence has been also presented that palmitic acid is attached to a cysteine residue upstream of this polyisoprenylated cysteine residue and that this fatty acid modification enhances the transforming activity of the polyisoprenylated rus ~21s (12). By analogy with ras ~21s and from the fact that smg p25A does not have a strong hydrophobic portion but binds to membranes (3, 4), it could be postulated that the C-terminal cysteine residue(s) of smg p25A is modified with a hydrophobic moiety(ies). Nevertheless, our previous results indicate that smg p25A is partly recovered in bovine brain cytosol and that this cytosolic smg p25A is indistinguishable from the membrane-bound one in their enzymatic and physical properties (3, 10, 11). We have previously proposed from these results that some factors may be involved in the localization of smg p25A in the cytosol and membranes (10).

We have purified from bovine brain cytosol a novel regulatory protein for smg p25A, named smg p25A GDI, that regulates the GDP/GTP exchange reaction of smg p25A by inhibiting the dissociation of GDP from and thereby the subsequent binding of GTP to it (13). This smg p25A GDI is specific for smg p25A among many ras p2l/ras p21-like G proteins. We have proposed that if the inhibitory action of smg p25A GDI is reversed by protein modification or another factor which is regulated by intracellular messenger systems such as protein kinase C-Ca” systems, the GDP-bound inactive form of smg p25A may be converted to the GTP-bound active form which then exerts its specific action(s). In this paper, we have studied the possible involvement of this smg p25A GDI in the localization of smg p25A in the cytosol, plasma membranes, and vesicles in rat brain synapses. This paper describes that smg p25A reversibly binds to synaptic plasma membranes and vesicles and that this reversible binding is regulated by smg p25A GDI.

13007

13008 GDI-regulated Reversible Binding of smg p25A to Membranes

EXPERIMENTAL PROCEDURES Materials and Chemicals-smg p25A and smg p25A GDI were

purified from bovine brain membranes and cytosol, respectively (3, 13). smg p25A (33.8 rg of protein/ml) dissolved in 20 mM Tris/HCl at uH 7.5 containing 5 mM MnCb 1 mM EDTA, 1 mM DTT, and 0.6% Chaps was used. smg p25AGDI (475 pg of protein/ml) dissolved in 25 mM Tris/HCl at pH 7.5 containing 0.5 mM EDTA and 1 mM DTT was used. These samples were stored at -80 “C until use. [35S] GTP$S (44.4 TBq/mmol) and [3H]GDP (0.99 TBq/mmol) were uurchased from Du Pont-New England Nuclear. Nitrocellulose filters IBA-85,0.45-pm pore size) were obtained from Schleicher & Schuell. Other materials and chemicals were from commercial sources.

Preparation of Synaptic Plasma Membranes and Vesicles-Synap- tic plasma membranes and vesicles were prepared from rat brains as described (14). The ultrastructural characteristics of the synaptic membranes and vesicles were monitored by an electron microscopy as described (14). The synaptic membranes (432 pg of protein/ml) dissolved in 0.8 M sucrose containing 1 mM NaHC03 were stored at -80 “C until use, and all experiments were done within 3 weeks after the preparation. The synaptic vesicles (168 pg of protein/ml) dissolved in 0.2 M sucrose containing 1 mM NaHC03 were stored on ice, and all experiments were done within 4 days after the preparation. When synaptic membranes and vesicles were used, they were diluted with 0.2 M sucrose containing 1 mM NaHC03.

Preparation of Intrasynaptosomal Mitochondria and Human Eryth- rocyte Ghosts-Intrasynaptosomal mitochondria were prepared from rat brains as described (15). Human erythrocyte ghosts were prepared as described (16). The intrasynaptosdmal mitochondria (3.45 mg of nrotein/ml) dissolved in 0.32 M sucrose containing 1 mM NaHCOs

I

and human erythrocyte ghosts (1.97 mg of protein/ml) dissolved in 0.2 M sucrose containing 1 mM NaHC03 were stored at -80 “C until use. When used, they were diluted in 0.2 M sucrose containing 1 mM NaHC03.

Preparation of the f’H]GDP- and f’S]GTPyS-bound Forms of smg n25A-To make the 13H]GDP- and F’S]GTPrS-bound forms of smg p25A, smg p25A (675 ng of protein) was incubated with 3 pM [3H] GDP and 3 UM l?SlGTPrS (1.2 x lo6 or 3.0 X lo6 cnm). resnectivelv. for 20 min at 3d “C’in 50 hl of Reaction Mixture A &'&M !l’ris/HCl at pH 7.5 containing 5 mM MgC12, 10 mM EDTA, and 1 mM DTT). After the incubation, 1 ~1 of MgCl, was added to give a final concentration of 20 mM and the mixture was cooled on ice to prevent the dissociation of the radioactive guanine nucleotides from smg p25A. The [3H]GDP- and [35S]GTPyS-bound forms of smg p25A (675 ng of protein, 2.2 X lo5 or 5.4 X lo6 cpm) were obtained in this way.

Binding of the PHJGDP- and p’S]GTPrS-bound Forms of smg 25A to the Svnnotic Plasma Membranes and Vesicles-The 13H1GDP- and [35S]GTPy’S-bound forms of smg p25A (675 ng of protein; 2.2 X 10 or 5.4 x lo6 cpm) were incubated for 5 min at 30 “C with the synaptic plasma membranes (27 pg of protein) or vesicles (42 pg of protein) in the presence or absence of smg p25A GDI (48 pg of protein) in 500 ~1 of Buffer A (25 mM Tris/HCl at pH 7.5 containing I pM GTP, 5 mM MgCl*, and 1 mM DTT) containing 100 pM GDP and 100 uM GTP. resnectivelv. Under these conditions, the final

-

concentrations of the [3H]GDP: and [?S]GTPyS-bound forms of smg p25A were 5.4 X 10-s M. After the incubation, 400 ~1 of each mixture was centrifuged on a discontinuous sucrose density gradient at 64,700 x g for 2 h at 4 “C. The discontinuous gradient consisted of 1.4 ml of Buffer A containing 2 M sucrose, 2.8 ml of Buffer A containing 0.5 M sucrose, 400 ~1 of the sample containing 0.1 M sucrose, and 400 ~1 of Buffer A in a 5-ml tube from the bottom in this order. After the centrifugation, fractions of 200 ~1 each were collected. The radioac- tivitv of a loo-u1 aliquot of each fraction was counted after filtration through the nitrocellulose filters. During the ultracentrifugation, the dissociation of 13HlGDP and 135SlGTPrS bound to smz u25A from * > .~ -_ it was prevented as maximally as possible by adding to the buffer a high concentration of MgCl* to give a final concentration of 5 mM and by cooling the buffer at 4 “C. Even under these conditions, however, about 50 and 10% of [3H]GDP bound to smg p25A were dissociated from it in the absence and presence of smg p25A GDI, respectively. [?S]GTP$S bound to smg p25A was not practically dissociated from it.

Detection of smgp25A GDZ-smg p25A GDI of each fraction of the discontinuous sucrose density gradient ultracentrifugation was detected by SDS-PAGE followed by protein staining. A 40-~1 aliquot of each fraction was subiected to SDS-PAGE (12% polyacrylamide gel) as described (17). The proteins on the gel were stained-with silver. The relative intensity of the smg p25A GDI protein band was quan-

tified by densitometric tracing at 600 nm using a Shimadzu dual wave length chromatogram scanner, model CS-930.

Detection of smg p25A-smg p25A of the membrane and soluble fractions obtained by the discontinuous sucrose density gradient ultracentrifugation was detected by immunoblotting with the specific anti-smg p25A monoclonal antibody, mAb SG-11-7 (8, 9). The assay samnles were subiected to SDS-PAGE. Proteins on the gel were transferred electrophoretically to the nitrocellulose sheet for 12 h at 8 V/cm as described (18). The sheet was incubated with Tris-buffered saline (50 mM Tris/HCl at pH 7.5, containing 0.2 M NaCl) containing 5% bovine serum albumin for 2 h at room temperature. After the incubation, the sheet was incubated for 2 h at room temperature with the ‘251-labeled anti-smg p25A monoclonal antibody (3.1 X lo6 cpm/ pg of protein, 2 fig of protein/ml in Tris-buffered saline containing 5% bovine serum albumin) and washed three times for 30 min each with Tris-buffered saline containing 0.05% Tween 20. The sheet was dried and autoradiographed with a Kodak X-Omat AR film using an intensifying screen at -80 “C. The radioactive bands were excised from the sheet and the incorporated radioactivity was counted. To quantify the content of smg p25A, the purified sample of smg p25A was used as a standard.

Determinations-The radioactivity of ?S- and 32P-labeled samples and that of Y-labeled samples were determined using a Beckman liquid scintillation system, model LS3801, and Aloka Auto-well Gamma system, ARC-300, respectively. Protein concentrations were determined with bovine serum albumin as a reference protein as described (19). The membrane phospholipid was extracted from various membrane preparations with chloroform/methanol by the method of Bligh and Dyer (20). The amount of phospholipid phos- phorus was determined with KH,POI as a standard by-the method of Bartlett (21).

RESULTS

Binding of smg p25A to the Synaptic Plasma Membranes and Vesicles-When the [3H]GDP- or [35S]GTPyS-bound form of smg p25A was incubated with the synaptic plasma membranes or vesicles and each mixture was subjected to the discontinuous sucrose density gradient ultracentrifugation to separate smg p25A in the membrane and soluble fractions, both forms of smg p25A were mostly recovered in the membrane fraction (Fig. l), whereas they were all recovered in the soluble fraction in the absence of the synaptic membranes and vesicles (data not shown). This binding of both the [3H] GDP- and [35S]GTPyS-bound forms of smg p25A was dose- dependent (data not shown). In Fig. 1, both the [3H]GDP- and [35S]GTPyS-bound forms of smg p25A were used at a final concentration of 5.4 x lo-’ M. However, even when they were used at a final concentration of 2.2 x 10m7 M in the present assay system, the saturable binding was not observed. The concentration of smg p25A in intact synapses could be calculated to be about 1.1 x 10T6 M (9). We could not obtain this concentration of smg p25A in the present assay system just because such a large amount of smg p25A could not be practically purified. Therefore, it remains to be clarified whether the binding of smg p25A to the synaptic membranes and vesicles is saturable or not.

When the synaptic plasma membranes and vesicles were boiled or digested with trypsin and the binding of the [35S] GTPyS-bound form of smg p25A to the boiled samples was examined by the discontinuous sucrose density gradient ultracentrifugation, most of smg p25A still bound to them (Fig. 2). In the experiments using the trypsin-digested samples, a part of smg p25A was recovered in the soluble fraction, but this partial recovery of smg p25A in the soluble fraction might be due to the denaturation of the synaptic membranes and vesicles because the digested synaptic membranes and vesicles were also partly recovered in the soluble fraction. The same results were obtained when the [3H]GDP-bound form of smg p25A was used instead of the [35S]GTPyS-bound form (data not shown).

Both the [3H]GDP- and [35S]GTPyS-bound forms of smg

GDI-regulated Reversible Binding of smg p25A to Membranes 13009

Fraction Number

FIG. 1. Binding of the [‘H]GDP- and [36S]GTPrS-bound forms of smg p25A to the synaptic plasma membranes and vesicles. The [3H]GDP- and [?S]GTPrS-bound forms of smg p25A were prepared and the binding of each form of smg p25A to the synaptic plasma membranes or vesicles was assayed by the discontinuous sucrose density gradient ultracentrifugation. Over 90% synaptic membrane and vesicle phospholipids were recovered at the 0.5-2 M sucrose interface and phospholipid was not detected at the 0.1-0.5 M sucrose interface as estimated by measuring the phospholipid phos- phorus. A, synaptic membranes; B, synaptic vesicles. 0, the [3H) GDP-bound form of smg p25A; 0, the [35S]GTPrS-bound form of smg p25A. Long and short arrows indicate the positions of the 0.5-2 and 0.1-0.5 M sucrose interfaces, respectively. The results shown are representative of three independent experiments.

p25A bound to not only the synaptic plasma membranes and vesicles but also intrasynaptosomal mitochondria (Table I). In our previous subcellular fractionation analysis and histo- chemical studies, smg p25A was not found in intrasynaptosomal mitochondria (9). Moreover, both forms of smg p25A bound to human erythrocyte ghosts where smg p25A was not found (Table I).

All of these results suggest that the specific protein molecule is not essential for the binding of the GDP- and GTP-bound forms of smg p25A to the synaptic plasma membranes and vesicles, although they do not necessarily neglect the possibility that smg p25A interacts with a certain specific membrane protein.

Inhibition by smgp25A GDZ of the Binding of the PH]GDP- bound Form of smg p25A to the Synaptic Plasma Membranes and Vesicles-When the [3H]GDP- or [35S]GTPrS-bound form of smg p25A was first incubated with the synaptic plasma membranes in the presence of smg p25A GDI and each mixture was then subjected to the discontinuous sucrose density gradient ultracentrifugation, the [3H]GDP-bound form was recovered in the soluble fraction, but the [35S]GTPyS-bound form of smg p25A was recovered in the membrane fraction (Fig. 3, A and B). Under these conditions, all amount of smg p25A GDI was recovered in the soluble fraction (data not shown). Similar results were obtained when the synaptic vesicles were used instead of the synaptic membranes (Fig. 3, C and D). These results indicate that both the GDP- and GTP-bound forms of smg p25A bind to the synaptic plasma membranes and vesicles, and that smg p25A GDI inhibits the binding of only the GDP-bound form to them.

Dissociation by smg p25A GDZ of Exogenous smgp25A from the Synaptic Plasma Membranes and Vesicles-The synaptic plasma membranes and vesicles which prebound the [3H] GDP- or [35S]GTPyS-bound form of exogenous smg p25A were prepared. The radiolabeled synaptic membranes were incubated with smg p25A GDI and the mixture was subjected

Fmctlon Number Fmctlon Number

FIG. 2. Binding of the [?3]GTPyS-bound form of smg p25A to the boiled or trypsin-digested synaptic plasma membranes and vesicles. The synaptic plasma membranes (27 pg of protein) and vesicles (42 pg of protein) were boiled for 3 min in 250 ~1 of 0.2 M sucrose containing 1 mM NaHC03. Other samples of the synaptic membranes (27 pg of protein) and vesicles (42 pg of protein) were digested with trypsin (1.4 and 2.1 pg of protein, respectively) for 20 min at 30 “C in 250 ~1 of the same mixture. Under these conditions, membrane proteins were markedly degraded as estimated by SDS- PAGE followed by protein staining with Coomassie Brilliant Blue. The digestion was stopped by the addition of trypsin inhibitor (4.1 and 6.3 pg of protein for the synaptic membranes and vesicles, respectively). The [35S]GTPrS-bound form of smg p25A was prepared and its binding to the boiled or trypsin-digested samples was assayed by the discontinuous sucrose density gradient ultracentrifugation. Over 90% synaptic membrane and vesicle phospholipids were recovered at the 0.5-2 M sucrose interface and phospholipid was not detected in the 0.1-0.5 M sucrose interface when intact or boiled samples were used, whereas 60-80 and 20-30% synaptic membrane and vesicle phospholipids were recovered at the 0.5-2 and 0.1-0.5 M sucrose interfaces, respectively, when the trypsin-digested samples were used. A, synaptic membranes; B, synaptic vesicles. 0, intact samples; 0, boiled samples; A, trypsin-digested samples. Long and short arrows indicate the 0.5-2 and 0.1-0.5 M sucrose interfaces, respectively. The results shown are representative of three independent experiments.

TABLE 1

Binding of the [-H/GDP- and r’S]GTPyS-bound forms of smg p25A to the synaptic plasma membranes, synaptic vesicles,

intrasynaptosomal mitochondria, and erythrocyte ghosts [3H]GDP- and [?S]GTPyS-bound forms of smg p25A were pre-

pared and the binding of each form of smg p25A to the synaptic plasma membranes, synaptic vesicles, intrasynaptosomal mitochondria, and erythrocyte ghosts (2.5 pg of phospholipid each) was examined by the discontinuous sucrose density gradient ultracentrifu- eation. Values are means + SE. from three indenendent exneriments.

Membrane samples

smg p25A bound

[%]GTPyS-bound [3H]GDP-bound form form

Synaptic membranes Synaptic vesicles lntrasynaptosomal

mitochrondria Erythrocyte ghosts

pmol/pg phospholipid 2.72 f 0.38 2.55 + 0.42 2.03 f 0.35 2.00 f 0.29 2.72 + 0.52 2.21 + 0.42

4.57 + 0.62 4.32 + 0.54

to the discontinuous sucrose density gradient ultracentrifugation. The [3H]GDP-bound form of smg p25A in the membrane fraction mostly disappeared and that in the soluble fraction markedly increased only in the presence of smg p25A GDI (Table II). In contrast, the [35S]GTPyS-bound form of


smg p25A in the membrane fraction did not decrease in the presence of smg p25A GDI. Under these conditions, all amount of smg p25A GDI was recovered in the soluble fraction (data not shown). The similar results were obtained when the synaptic vesicles were used instead of the synaptic membranes (Table II). These results indicate that smg p25A GDI induces the dissociation of only the GDP-bound form of exogenous smg p25A from the synaptic plasma membranes and vesicles.

Dissociation by smg p25A GDI of Endogenous smg p25A from the Synaptic Plasma Membranes and Vesicles-The synaptic plasma membranes contained endogenous smg p25A as well as other G proteins (9, 11). The endogenous G proteins were first labeled with either [3H]GDP or [35S]GTPyS and then incubated with smg p25A GDI followed by the discontinuous sucrose density gradient ultracentrifugation. The radioactivity of [3H]GDP in the membrane fraction decreased and

Fraction Number Fmciian Number

FIG. 3. Effect of smg p25A GDI on the binding of the [3H] GDP- and [36S]GTPyS-bound forms of smg p25A to the synaptic plasma membranes and vesicles. The [‘H]GDP- and [%I GTPyS-bound forms of smg p25A (675 ng of protein, 2.2 X lo5 cpm each) were prepared and the binding of each form of smg p25A to the synaptic plasma membranes and vesicles was assayed in the presence or absence of smg p25A GDI (48 pg of protein) by the discontinuous sucrose density gradient ultracentrifugation. A and B, synaptic membranes; C and D, synaptic vesicles; A and C, the [3H]GDP-bound form of smg p25A; B and D, the [%]GTPyS-bound form of smg p25A. 0, in the presence of smg p25A GDI; 0, in the absence of smg p25A GDI. Long and short arrows indicate the 0.5-2 and 0.1-0.5 M sucrose interfaces, respectively. The results shown are representative of three independent experiments.

that in the soluble fraction inversely increased only in the presence of smg p25A GDI (Table III). In contrast, the radioactivity of [35S]GTPyS in the membrane fraction did not decrease in the presence of smg p25A GDI. Under these conditions, all amount of smg p25A GDI was recovered in the soluble fraction (data not shown). Since smg p25A GDI is specific for smg p25A (13), this result suggests that smg p25A GDI induces the dissociation of only the GDP-bound form of endogenous smg p25A from the synaptic plasma membranes. In fact, it was confirmed by use of the specific anti-smg p25A antibody that the radioactivity of [3H]GDP recovered in the soluble fraction was mostly derived from smg p25A (Table IV). The partial dissociation of the radioactivity from the synaptic membranes may be explained by the fact that the synaptic membranes contain many ras p2l/ras p21-like G proteins other than smg p25A and large molecular weight G proteins such as G,, Gi, and G, (9, 11). When the similar experiments were done with the synaptic vesicles instead of the synaptic plasma membranes, the essentially same results were obtained (Tables III and IV). Thus, it is likely that smg p25A GDI selectively induces the dissociation of endogenous smg p25A from the synaptic plasma membranes and vesicles.

Properties of Endogenous and Exogenous smg p25As Bound to the Synaptic Plasma Membranes and Vesicles-smg p25A GDI induced the dissociation of the GDP-bound form of both endogenous and exogenous smg p25As bound to the synaptic plasma membranes and vesicles as described above. Both endogenous and exogenous smg p25As bound to the synaptic membranes and vesicles were not dissociated from the synaptic membranes or vesicles by the addition of a low or high concentration of NaCl (Fig. 4, A and B). Other G proteins were not dissociated from the synaptic membranes or vesicles, either. Both endogenous and exogenous smg p25As were, however, dissociated from the synaptic membranes and vesicles by the addition of Triton X-100 (Fig. 4, C and D). The concentrations of this detergent necessary for the dissociation of exogenous smg p25A from the synaptic membranes and vesicles were similar to those for the dissociation of endogenous smg p25A and other endogenous G proteins. These results suggest that both endogenous and exogenous srng p25As bind to the synaptic plasma membranes and vesicles in a similar manner.

Estimation of the M, Values of smg p25A and smg p25A GDI by Continuous Sucrose Density Gradient Ultracentrifugation

TABLE II

Dissociation by smg p25A GDI of exogenous smg p25A from the synaptic plasma membranes and vesicles The [3H]GDP- and [%]GTP+-bound forms of smgp25A (675 ng of protein, 5.4 x lo6 cpm each) were prepared.

Each form of smg p25A was incubated with the synaptic plasma membranes (27 pg of protein) or vesicles (42 pg of protein) and subjected to the discontinuous sucrose density gradient ultracentrifugation. The membrane fractions were collected, diluted with 3 volumes of Buffer A, and centrifuged at 200,000 X g for 60 min at 4 “C. The synaptic membranes and vesicles recovered in the precipitate were suspended in 400 ~1 of Buffer A containing 0.2 M sucrose. The dissociation of the [3H]GDP- or [%]GTPrS-bound form in smg p25A (2.2 X lo5 cpm each) from the synaptic membranes and vesicles was assayed in the presence or absence of smg p25A GDI (48 pg of protein) by the discontinuous sucrose density gradient ultracentrifugation. The results shown are representative of three independent experiments.

In the absence of smg p25A GDI In the presence of smg p25A GDI

Form of sm# p25A Synaptic membranes

or vesicles Membrane Soluble Membrane Soluble fraction fraction fraction fraction

CPm [3H]GDP-bound Membranes 24,910 3,420” 4,390 51,160 [%]GTP$S-bound Membranes 46,050 9,050” 42,980 11,510 [3H]GDP-bound Vesicles 23,510 6,270” 6,120 49,130 [%]GTPrS-bound Vesicles 38,530 16,620” 36,110 18,380

’ The radioactivity partly recovered in the soluble fraction in the absence of smg p25A GDI was not due to the dissociation of smg p25A but due to the partial denaturation of the synaptic membranes and vesicles because a small amount of phospholipid was detected in the soluble fraction.


TABLE III Dissociation by smg p25A GDI of pH/GDP from the radiolabeled synaptic plasma membranes and vesicles

Endogenous G proteins in the synaptic plasma membranes (27 pg of protein) and vesicles (42 pg of protein) were labeled with 3 FM [3H]GDP or 3 PM [?S]GTPrS (7.2 X lo6 cpm each) for 2 h at 30 “C in 300 ~1 of 20 mM Tris/HCl at pH 7.5 containing 6 mM MgCl*, 1 mM EDTA, and 1 mM DTT. The dissociation of [3H]GDP or [3”S] GTPyS from the radiolabeled synaptic membranes (6.0 X lo4 cpm each) and vesicles (8.0 X 10’ cpm each) was assayed in the presence or absence of smg p25A GDI (48 fig of protein) by the discontinuous sucrose density gradient ultracentrifugation. The radioactivity of a 150-~1 aliquot of each fraction was counted after filtration through the nitrocellulose filters. The results shown are reuresentative of three indenendent exneriments.

Endogenous G proteins Synaptic membranes or vesicles

In the absence of snag p25A GDI

Membrane Soluble fraction fraction

In the presence of smg p25A GDI

Membrane Soluble fraction fraction

[3H]GDP-bound Membranes 22,140 240 [35S]GTPyS-bound Membranes 29,120 520 [3H]GDP-bound Vesicles 27,830 330 [?l]GTPyS-bound Vesicles 39,280 810

12,890 16,620 27,370 1,840

4,910 35,130 37,630 1,630

TABLE IV Dissociation by smg p25A GDI of endogenous smg p25A from the synaptic plasma membranes and vesicles

Endogenous G proteins in the synaptic plasma membranes and vesicles were labeled and the dissociation of endogenous smg p25A from the synaptic membranes and vesicles was assayed as described in the legend to Table III except that smg p25A was detected by immunoblotting with the anti-smg p25A antibody. Namely, the radioactivity of a 50-~1 aliquot of each fraction was counted after filtration through the nitrocellulose filters to detect the membrane and soluble fractions. The membrane and soluble fractions were then collected and concentrated to 40 ~1 using Centricon- microconcentrator. Each concentrate was subjected to SDS-PAGE followed by immunoblotting with the anti-smg p25A antibody. The results shown are means + S.E. from three indeoendent exneriments.

In the absence of smg p25A GDI In the presence of smg p25A GDI Form of smg p25A Synaptic membranes

or vesicles Membrane Soluble Membrane Soluble fraction fraction fraction fraction

[3H]GDP-bound Membranes [35S]GTPyS-bound Membranes [3H]GDP-bound Vesicles [35S]GTPyS-bound Vesicles

ng ofprotein 41 -t 6 ND” ND 36 + 5 47 + 5 ND 42 f 6 ND 92 + 7 ND ND 75 + 6 89 f 6 ND 83 + 8 ND

’ ND, not detectable.

and Gel Filtration-When the [?S]GTPyS-bound form of smg p25A was subjected to the continuous sucrose density gradient ultracentrifugation in the presence of 0.1% sodium cholate, it appeared in a single peak with a M, value of 140,000 (Fig. 5A). In the presence of 1% sodium cholate, however, smg p25A appeared in a single peak with a M, value of about 65,000. On gel filtration analysis, the [35S]GTPyS-bound form of smg p25A appeared in a single peak with a M, value of about 70,000 in the presence of 0.1 and 1% sodium cholate (Fig. 5B). When the similar experiments were performed with the [3H]GDP-bound form of smg p25A, the essentially same results were obtained (data not shown). Since the n/r, values of smg p25A estimated by SDS-PAGE and calculated from its primary structure were about 24,000 and 25,000, respectively (3,4), it is likely that smg p25A is an oligomerized form under these conditions.

When smg p25A GDI was subjected to the continuous sucrose density gradient ultracentrifugation and gel filtration in the presence of 0.1 and 1% sodium cholate under the same conditions, it appeared in a single peak with a M, value of about 59,000’ by the ultracentrifugation and also in a single peak with a M, value of about 82,000 on gel filtration (data

’ The M, value of smg p25A GDI was estimated to be about 65,000 by the continuous sucrose density gradient ultracentrifugation under the conditions described previously (13), but to be 59,000 under the conditions described in the legend to Fig. 6. In the experiment of Fig. 6, the ultracentrifugation was performed in the buffer containing 0.1 or 1% sodium cholate. The exact reason for this difference of the M, values is not known, but might be due to the presence or absence of sodium cholate.

not shown). These results together with the fact that the M, value of smg p25A GDI estimated previously by SDS-PAGE is about 54,000 (13) suggest that smg p25A GDI is a monomeric form under these conditions.

Complex Formation of smg p25A GDI with the [JHJGDP- bound Form of smg p25A-When the [3H]GDP-bound form of smg p25A was mixed with smg p25A GDI and the mixture was then subjected to the continuous sucrose density gradient ultracentrifugation in the presence of 0.1% sodium cholate, smg p25A at the position with a M, value of about 140,000 disappeared and that at the position with a M, value of about 85,000 appeared (Fig. 6A). Concomitantly, smg p25A GDI at the position with a M, value of about 59,000 partly shifted to the position with a M, value of about 85,000.’ The partial shift was due to the addition of a large excess of smg p25A GDI against smg p25A.

On the same continuous sucrose density gradient ultracentrifugation, both the [35S]GTPyS-bound and guanine nucleotide-free forms of smg p25A appeared in a single peak with a M, value of 140,000 in the presence of 0.1% sodium cholate under the same conditions (Fig. 6, B and C). When either the [?S]GTPyS-bound or guanine nucleotide-free form of smg p25A was first mixed with smg p25A GDI and each mixture was subjected to the ultracentrifugation, both forms of smg p25A and smg p25A GDI appeared at their respective original positions.3

3 The reason why the recovery of the guanine nucleotide-free form estimated by the binding of [Yl]GTP+ was lower than that of the [3H]GDP- or [35S]GTP+-bound form shown in Fig. 3, A and B, was that the guanine nucleotide-free form was inactivated during its preparation.


NaCl (M) Trlton X-100 (%)

FIG. 4. Effects of NaCl and Triton X- 100 on the dissociation of endogenous and exogenous smg p25As hound to the synaptic plasma membranes and vesicles. Endogenous G proteins in the synaptic plasma membranes and vesicles were labeled as described in the legend to Table III. The synaptic membranes and vesicles which prebound the [35S]GTPyS-bound form of exogenous smg p25A were prepared by the discontinuous sucrose density gradient ultracentrifugation as described in the legend to Table II. The radiolabeled synaptic membranes and vesicles were incubated for 2 h on ice in 500 ~1 of Buffer A containing the indicated concentration of NaCl or Triton X-100. Each mixture was subjected to the discontinuous sucrose density gradient ultracentrifugation and the radioactivity of a 100-~1 aliquot of each fraction was counted after filtration through the nitrocellulose filters. The radioactivity represents the amount of endogenous G proteins or exogenous smg p25A. The residual 100~~1 aliquot of the membrane and soluble fractions were collected and concentrated to 40-~1 and endogenous smg p25A was quantified by immunoblotting with the specific anti-smg p25A antibody as described in the legend to Table IV. A and C, synaptic membranes; B and D, synaptic vesicles; A and i?, NaC1; C and D, Triton X-100.0, endogenous smg p25A; 0, exogenous smg p25A; A, endogenous G proteins; -, the membrane fraction; ------, the soluble fraction. The results shown are representative of three independent experiments.

These results suggest that smg p25A GDI makes a complex with only the GDP-bound form of smg p25A, but not with the GTP-bound or guanine nucleotide-free form. In the presence of 1% sodium cholate, the complex formation of smg p25A GDI with the GDP-bound form of smg p25A was inhibited (data not shown).

Molar Ratio of smg p25A and smg p25A GDI in the Com- plex-To calculate the molar ratio of smg p25A and smg p25A GDI in the complex, various doses of smg p25A and smg p25A GDI were used. We have previously shown that at least about 70-fold of smg p25A GDI was needed for the full inhibition of the dissociation of [3H]GDP from smg p25A (13). This vast excess of smg p25A GDI necessary for the full action was due to a low concentration of smg p25A (1.4 X lo-’ M) used in the assay (Fig. 7). When a higher concentration of smg p25A (1.4 X 10e7 M) was used, the molar ratio of smg p25A GDI to smg p25A necessary for its full action was markedly reduced.

In the experiments for the formation of the complex of smg p25A GDI with smg p25A in Fig. 6, smg p25A was used at 1.3 X lo-? M. When smg p25A was used at 6.7 X 10e7 M, a lower concentration of smg p25A GDI was sufficient for the formation of the complex (Fig. 8). Under these conditions, the same molar concentrations of smg p25A GDI and smg p25A were used, and all smg p25A GDI and smg p25A made a complex. When smg p25A GDI was used at a lower concentration than that of smg p25A, all smg p25A GDI made a complex with smg p25A with a molar ratio of 1:l and the remaining smg p25A was observed (data not shown). These results together with those shown in Fig. 6 indicate that smg

Fraction Numb

1

20 40 60 Fnctbn Number

FIG. 5. Estimation of the M, values of smg p25A on continuous sucrose density gradient ultracentrifugation and gel filtration. The [35S]GTP+bound form of smg p25A (1 pg of protein, 8.0 x lo5 cpm) was dissolved in 75 ~1 of Reaction Mixture A and incubated for 10 min at 30 “C in 300 ~1 of 20 mM Tris/HCl at pH 7.5 containing 300 pM GTP, 10 mM MgCl,, 0.5 mM EDTA, and 1 mM DTT. The mixture was subjected to the continuous sucrose density gradient ultracentrifugation or gel filtration. The ultracentrifugation was performed at 219,000 X g for 13.8 h using 4.8 ml of a continuous sucrose density gradient (0.15-0.58 M sucrose in 20 mM Tris/HCl at pH 7.5 containing 1 pM GTP, 5 mM MgC12, 1 mM DTT, and 0.1 or 1% sodium cholate). After the centrifugation, fractions of 150 ~1 each were collected. The radioactivity of a 50-~1 aliquot of each fraction was counted after filtration through the nitrocellulose filters. The gel filtration was performed using a Sepharose CL-6B column (0.7 x 74 cm) equilibrated with 20 mM Tris/HCl at pH 7.5 containing 1 pM GTP, 5 mM MgC12, 1 mM DTT, and 0.1 or 1% sodium cholate. The elution was performed with the same solution. Fractions of 1 ml each were collected. The radioactivity of a lOO-pl aliquot of each fraction was counted after filtration through the nitrocellulose filters. A, ultracentrifugation; B, gel filtration. 0, in the presence of 0.1% sodium cholate; 0, in the presence of 1% sodium cholate. Arrows l- 4 in A indicate the positions of horse y-globulin (7.4 S, M, = 150,000), human hemoglobin (4.5 S, M, = 64,500), chicken ovalbumin (3.6 S, M, = 45,000), and horse myoglobin (1.9 S, M, = 17,000), respectively. Arrows I-5 in B indicate the positions of blue dextran (M, = 2,000,000), horse y-globulin, human hemoglobin, chicken ovalbumin, and cyanocobalamine (Mr = 1,350), respectively. The results shown are representative of three independent experiments.

p25A GDI makes a complex with the GDP-bound form of smg p25A with a molar ratio of 1:l. When the rate of the dissociation of [3H]GDP from smg p25A complexed with smg p25A GDI was compared with that from free smg p25A, the former was slower than the latter (Fig. 9).

In the experiments for the inhibition by smg p25A GDI of the binding of smg p25A to the synaptic plasma membranes and vesicles in Fig. 3, smg p25A was used at 5.4 X 10e8 M. When smg p25A was used at 2.2 X 10m7 M, a lower concentration of smg p25A GDI was effective to inhibit the binding of smg p25A to the synaptic membranes and vesicles (Table V).

DISCUSSION

We have previously shown that smg p25A GDI regulates the GDP/GTP exchange reaction of smg p25A by inhibiting the dissociation of GDP from and thereby the subsequent binding of GTP to it (13). We have shown here that smg p25A GDI makes a complex with the GDP-bound form of smg

GDI-regulated Reversible Binding of srng p25A to Membranes 13013

La ,P! 4, 0 0

0 10 20 30 FmcUanRntu

FIG. 6. Complex formation of emg p25A GDI with the [3H] GDP-bound form of smg p25A. The [3H]GDP- and [35S]GTP$!& bound forms of smg p25A (1 pg of protein, 8.0 X 10’ cpm each) were prepared. The nucleotide-free form of smg p25A was prepared as described previously (22). Each form of smg p25A was incubated with or without smg p25A GDI (29 pg of protein) for 10 min at 30 “C in 300 ~1 of 20 mM Tris/HCl at pH 7.5 containing 300 pM GTP, 10 mM MgClz, 0.5 mM EDTA, and 1 mM DTT. In the experiment for the guanine nucleotide-free form, 300 /IM GTP was removed. Under these conditions, the final concentrations of each form of smg p25A and smg p25A GDI were 1.3 X 10e7 and 1.9 X 10e6 M, respectively. Each mixture was subjected to the same continuous sucrose density gradient ultracentrifugation as used in Fig. 5 except that 1 pM GTP was removed in the experiment for the guanine nucleotide-free form. After the centrifugation, fractions of 150 ~1 each were collected. The radioactivity of a 20-~1 aliquot of each fraction was counted after filtration through the nitrocellulose filters. The guanine nucleotide- free form of smg p25A was detected by measuring the [?!J]GTPrS binding activity of a 2O-~.d aliquot of each fraction. In another experiment, smg p25A GDI (29 pg of protein) was separately subjected to the same ultracentrifugation. A 40-~1 aliquot of each fraction was used for the detection of smg p25A GDI. A, the [3H]GDP-bound form of smg p25A, B, the [35S]GTP+-bound form of smg p25A, C, the guanine nucleotide-free form of smg p25A. O-O, smg p25A in the presence of smg p25A GDI; O-O, smg p25A in the absence of smg p25A GDI; O---O, smg p25A GDI in the presence of smg p25A; O---O, smg p25A GDI in the absence of smg p25A. Arrows l-4 indicate the positions of the same M, markers as used in Fig. 5A. The results shown are representative of three independent experiments.

p25A, but not with the GTP-yS-bound or the guanine nucleotide-free form. Therefore, it is likely that smg p25A GDI first makes a complex with the GDP-bound form of smg p25A and thereby inhibits the dissociation of GDP from and the subsequent binding of GTP to smg p25A. In addition to this regulatory function of smg p25A GDI, we have shown here that smg p25A GDI regulates the reversible binding of the GDP-bound form of smg p25A to the synaptic plasma membranes and vesicles. Both the GDP- and GTP-bound forms of smg p25A bind to the synaptic membranes and vesicles, but smg p25A GDI inhibits this binding of only the GDP- bound form. smg p25A GDI by itself does not bind to the synaptic membranes or vesicles. Therefore, it is likely that smg p25A GDI makes a complex with the GDP-bound form of smg p25A and thereby inhibits its binding to the synaptic membranes and vesicles. Moreover, smg p25A GDI induces the dissociation of the GDP-bound form of endogenous and exogenous smg p25As from the synaptic membranes and vesicles. It is likely that smg p25A GDI makes a complex with the GDP-bound form of the membrane-bound smg p25A and

im-

loo smp p28A GDI I m p25A (mol /mot)

FIG. 7. Effect of smg p25A GDI on low and high concentrations of smg p25A. The activity of smg p25A GDI to inhibit the dissociation of [3H]GDP from smg p25A was assayed with 1.4 X lOua or 1.4 x lo-’ M smg p25A in the presence of various concentrations of smg p25A GDI under the conditions described previously (13). smg p25A GDI activity was calculated as described (13). The value of the radioactivity of [3H]GDP bound to smg p25A after the dissociation reaction in the absence of smg p25A GDI was subtracted from that before the dissociation reaction and this value was defined as 100%. The former value was subtracted from the values of the radioactivity of [3H]GDP bound to smg p25A after the dissociation reaction in the presence of various amounts of smg p25A GDI, and smg p25A GDI activity was expressed as percent of these values to the value defined as 100%. 0, 1.4 x lo-* M smg p25A; l , 1.4 x lo-’ M smg p25A. The results shown are representative of three independent experiments.

FIG. 8. Complex formation of smg p25A GDI with a high concentration of smg p25A. The [3H]GDP-bound form of smg p25A (5 pg of protein, 4.0 x lo6 cpm) was prepared, incubated with or without smg p25A GDI (10 gg of protein), and subjected to the continuous sucrose density gradient ultracentrifugation under the same conditions as used in Fig. 6 except that a larger amount of smg p25A and a smaller amount of smg p25A GDI were used. Under these conditions, the final concentrations of smg p25A and smg p25A GDI during the complex formation were 6.7 X lo-’ M. The radioactivity of a 20-~1 aliquot of each fraction was counted after filtration through the nitrocellulose filters. In another experiment, smg p25A GDI (10 pg of protein) was separately subjected to the same ultracentrifugation. A 40-pl aliquot of each fraction was used for the detection of smg p25A GDI. O-O, smg p25A in the presence of smg p25A GDI; O-O, smg p25A in the absence of smg p25A GDI; O-----O, smg p25A GDI in the presence of smg p25A; O-----O, smg p25A GDI in the absence of smg p25A. Arrows l-4 indicate the positions of the same M, markers as used in Fig. 5A. The results shown are representative of three independent experiments.

that the complex is then dissociated from the synaptic membranes and vesicles.

In most experiments shown here, we have used a vast excess of smg p25A GDI versus smg p25A to make a bimolecular complex of smg p25A GDI and smg p25A because it is practically difficult to prepare a large amount of pure smg p25A and easy to prepare a large amount of pure smg p25A GDI. For instance, 230 rg of smg p25A and 1.8 mg of smg p25A GDI are purified from 100 g wet weight of bovine brain as described previously (3, 13). However, in key experiments shown in Figs. 7-9 and Table V, various concentrations of smg p25A and smg p25A GDI are used. When we use a higher concentration of smg p25A, a lower concentration of smg p25A GDI is enough to inhibit the dissociation of [3H]GDP

13014 GDI-regulated Reuersible Binding of smg p25A to Membranes

from smg p25A and makes a complex with the GDP-bound form of smg p25A. Therefore, the effect of a vast excess of smg p25A GDI on smg p25A in most experiments is due to smg p25A GDI itself and not simply due to a nonspecific protein contaminating the smg p25A GDI sample used.

Evidence has previously been presented that smg p25A is present in both the membrane and cytosol fractions of bovine brain, and that smg p25As recovered in both fractions are

:

0

Tilnoe (min) 20

FIG. 9. Dissociation of t3H]GDP from smg p25A complexed with smg p25A GDI. The [3H]GDP-bound form of smg p25A was prepared, incubated with or without smg p25A GDI, and subjected to the continuous sucrose density gradient ultracentrifugation under the same conditions as used in Fig. 8. The radioactivity of a 20-~1 aliquot of each fraction was counted after filtration through the nitrocellulose filters. The peaks of the radioactivity of the [3H]GDP-bound form of smg p25A complexed with smg p25A GDI (fractions 14-19) and free smg p25A (fractions 10-16) were collected. To each sample (50 ~1 each), 50 ~1 of a mixture of 20 mM Tris/HCl at pH 7.5 containing 500 FM GTP, 5 mM MgC12, 20.4 mM EDTA, and 1 mM DTT was added, and the mixtures were incubated at 30 “C to dissociate [3H] GDP from smg p25A. The reaction was stopped at the indicated times as described (13). The values of the radioactivity of [3H]GDP bound to smg p25A at the indicated times were expressed as percent of that at time 0. The values of the radioactivity of [3H]GDP bound to smg p25A complexed with smg p25A GDI and free smg p25A at time 0 were 11,520 and 6,630 cpm, respectively. The reason why the latter value was smaller than the former value was that the rate of the dissociation of [“H]GDP from free smg p25A was faster than that from smg p25A complexed with smg p25A GDI during the ultracentrifugation. 0, smg p25A complexed with smg p25A GDI; 0, free smg p25A. The results shown are representative of three independent experiments.

indistinguishable from each other in their enzymatic and physical properties (3,10,11). We have shown here that both the GTPrS- and GDP-bound forms of smg p25A purified from bovine brain membranes bind to the synaptic plasma membranes and vesicles. Once exogenous smg p25A binds to the synaptic membranes and vesicles, it is not extracted from them by a low or high concentration of NaCl and is extracted only by a detergent such as Triton X-100. The concentrations of this detergent necessary for this extraction is similar to that for the extraction of endogenous smg p25A from the synaptic membranes and vesicles. Moreover, both endogenous and exogenous smg p25As bound to the synaptic membranes are dissociated by smg p25A GDI. These results suggest that both endogenous and exogenous smg p25As bind to the synaptic plasma membranes and vesicles in a similar manner, and support the idea that smg p25A GDI regulates the reversible binding of smg p25A to the synaptic membranes and vesicles.

The precise mechanism of the binding of smg p25A to the synaptic plasma membranes and vesicles is not known, but it is likely that a specific protein in the synaptic membranes or vesicles is not essential for this binding, since the binding of smg p25A to the synaptic membranes and vesicles is not eliminated by the prior boiling or tryptic digestion, and smg p25A binds to intrasynaptosomal mitochondria and human erythrocyte ghosts where smg p25A could not be found by our previous studies on the tissue and subcellular distributions of smg p25A (9, 11). We have previously raised the possibility that smg p25A contains a hydrophobic moiety(ies), such as lipid, at its C-terminal cysteine residue(s) and binds to the membranes through this hydrophobic moiety(ies) (3,4). The present results support this earlier assumption although they do not necessarily neglect the possibility that smg p25A interacts with a certain specific membrane protein.

It has not been established whether the putative hydrophobic moiety(ies) is indeed responsible for the binding of smg p25A to synaptic plasma membranes and vesicles. If this is the case, however, smg p25A GDI may block the binding of smg p25A to and dissociate it from the synaptic membranes and vesicles by masking this moiety(ies). It has been suggested that the hydrophobic moiety(ies) of the C-terminal cysteine residues of ras p2l/rm p21-like G proteins is involved in their localization in membranes and cytosol (12, 23, 24). The present results, however, have raised the possibility that removal of the hydrophobic moiety(ies) of smg p25A is not essential for localization of this protein in the synaptic cytosol, and that smg p25A GDI is involved in the regulation of this localization.

TABLE V Effect of a low concentration of smgp25A GDI on the binding of the pH]GDP-bound form of a high concentration

of smg p25A to the synaptic plasma membrarxs and vesicles The [3H]GDP-bound form of smg p25A was prepared, and its binding to the synaptic plasma membranes and

vesicles was assayed by the discontinuous sucrose density gradient ultracentrifugation. The concentrations of smg p25A and smg p25A GDI were used as indicated. The results shown are means + S.E. from three independent experiments.

In the absence of smg p25A GDI In the presence of smg p25A GDI Synaptic membranes

or vesicles

Synaptic membranes

Synaptic vesicles

smg p25A smg p25A GDI Membrane Soluble Membrane Soluble fraction fraction fraction fraction

M % 5.4 x 10-a 1.9 x 1o-6 90 + 4 10 f 3 10 f 4 90 + 4 5.4 x lo-+ 2.2 x lo+ 88 f 3 12 f 4 70 + 5 30 + 3 2.2 x 1o-7 2.2 x 1o-7 91 f 3 9*3 52 + 4 48 + 3

5.4 x 1o-8 1.9 x lo+ 90 x?z 4 10 + 3 9+3 91 + 4 5.4 x 1o-8 2.2 x 1o-7 88 k 4 12 f 3 72 f 4 28 + 2 2.2 x lo-’ 2.2 x lo-’ 89 f 3 11% 4 59 f 3 41 * 3


We have previously proposed that if the inhibitory action of smg p25A GDI in the GDP/GTP exchange reaction is reversed by protein modification or another factor, the GDP- bound inactive form of smg p25A may be converted to the GTP-bound active form which then exerts its specific action(s). Therefore, it is conceivable that smg p25A binds to the synaptic plasma membranes and vesicles in a cyclical manner. Namely, smg p25A is present in the GDP-bound form complexed with smg p25A GDI in the cytosol of presy- napses. When the inhibitory action of smg p25A GDI is reversed by an unidentified way, the GDP-bound form is dissociated from smg p25A GDI. The GDP-bound form free of smg p25A GDI is first converted to the GTP-bound form and then binds to the synaptic membranes and vesicles or first binds to the synaptic membranes and vesicles and is then converted to the GTP-bound form. The GTP-bound form bound to the synaptic membranes and vesicles is converted to the GDP-bound form by its intrinsic GTPase activity. The GDP-bound form is associated with the cytosolic smg p25A GDI to make a complex. The complex is then dissociated from the synaptic membranes and vesicles and translocated to the synaptic cytosol. It remains, however, to be clarified whether smg p25A indeed binds to the synaptic plasma membranes and vesicles in a cyclical manner and this binding is regulated by smg p25A GDI in intact synapses.

an oligomerized form under these conditions. The exact number of protein molecules of smg p25A in the oligomerized form with a M, value of 65,000-70,000 is not known, but two or three protein molecules appear to be included in this form as calculated from their M, values. It is presumed that, when the oligomerized form of smg p25A makes a complex with smg p25A GDI, smg p25A is present in the monomeric form in this complex as judged from the M, values of smg p25A, smg p25A GDI, and the complex. It has recently been shown that c-Ki-ras p21 is present in an oligomerized form in intact cells and that two to four protein molecules constitute the oligomer (26). Our present result is consistent with this recent finding. It remains, however, to be clarified how smg p25A is oligomerized even in the presence of 1% sodium cholate, but it could be speculated that the putative hydrophobic moiety(ies) of the C-terminal region is involved in this oligomerization of smg p25A. This speculation is consistent with the idea that smg p25A GDI may mask the hydrophobic moiety(ies) of smg p25A and thereby inhibit its binding to the synaptic plasma membranes and vesicles. It also remains to be clarified in which form, the oligomerized or monomeric form of smg p25A, binds to the synaptic plasma membranes and vesicles. Studies on the C-terminal structure of smg p25A are essential to understand the mechanism of the oligomerization of smg p25A and the mode of action of smg p25A GDI.

We have previously raised the possibility that smg p25A plays important roles in synaptic functions, particularly endo- exocytotic recycling of the synaptic vesicles (9, 11). It has been shown that protein kinase C-Ca2+ systems are involved in the extracellular signal-dependent endo-exocytotic processes (for a review, see Ref. 25). It could be, therefore, speculated that smg p25A GDI may be regulated directly or indi- rectly by these intracellular messenger systems. The mode of regulation of smg p25A GDI is being currently investigated.

Acknowledgment-We are grateful to J. Yamaguchi for her skillful secretarial assistance.

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It may be noted that the membrane specificity of the binding of smg p25A is different in in viva and in vitro systems. In the present in vitro systems using the purified smg p25A, smg p25A GDI, and various synaptosomal membranes includ- ing plasma membranes, vesicles, and mitochondria, smg p25A binds to all of them. In our previous in viva studies on intrasynaptosomal distribution of smg p25A, however, smg p25A is present mostly in plasma membranes and partly in vesicles and cytosol but not in mitochondria (9). The exact reason for these inconsistent in uitro and in uiuo results is not known, but it is conceivable that some crucial element in the cycling system of smg p25A on and off of membranes is missing or nonfunctional in our in vitro systems. It is likely that smg p25A GDI may be one of several components needed to properly regulate the localization of smg p25A in synapses.

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Another point to be discussed here is that the M, values of smg p25A estimated by the continuous sucrose density gradient ultracentrifugation and gel filtration analyses are much larger than those estimated previously by SDS-PAGE and the primary structure (3,4). The M, values of smgp25A estimated by SDS-PAGE and calculated from the primary structure are about 24,000 and 25,000, respectively (3, 4), whereas those estimated by the continuous sucrose density gradient ultracentrifugation in the presence of 0.1 and 1% sodium cholate are 140,000 and 65,000, respectively, and those estimated by gel filtration in the presence of 0.1 and 1% sodium cholate

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