intrasynaptosomal distribution of the ras, rho and smg-25a gtp-binding proteins in bovine brain

Molecular Brain Research, 6 (1989) 167-176 167 Elsevier

BRESM 70156

Intrasynaptosomal distribution of the ras, rho and smg-25A GTP-binding proteins in bovine brain

Shigekuni Kim 1, Akira Kikuchi 1, Akira Mizoguchi 2 and Yoshimi Takai 1

Departments of t Biochembtry and 2Anatomy, Kobe University School of Medicine, Kobe (Japan)

(Accepted 9 May 1989)

Key words: Guanosine triphosphate binding protein; ras protein; rho protein; smg-25A protein; Intrasynaptosomal distribution; Synaptosome

We have purified to near homogeneity and characterized three small molecular weight (Mr) GTP-binding proteins, the c-Ki-ras protein (c-Ki-ras p21), the rho protein (rho p20) and a novel smg-25A protein (smg p25A), from bovine brain crude membranes. In the present studies, the intrasynaptosomal distribution of these 3 small M r G proteins has been investigated using bovine brain. ras p21 and rho p20 are found in the synaptosomal membrane fraction but not in the synaptosomal soluble fraction. In contrast, smg p25A is found in both the synaptosomal membrane and soluble fractions. These results indicate that the intrasynaptosomal distribution of these small M r G proteins is different and suggest that they are involved in different neuronal functions.

INTRODUCTION

In addi t ion to a group of G proteins having an aft7 subunit s tructure and serving as t ransducers for

m e m b r a n e receptors , such as G s, G~, G O , and t ransducin 4'1°'43"48, there is another group of G

prote ins whose M r values are about 20,000 in

mammal i an tissues. We have designated here the

former and la t ter groups of G prote ins as large and

small M r G proteins, respectively. Small M r G

prote in group includes the proteins encoded by the

3 ras (Ki-, Ha- and N-ras) l, 3 rho (clones, 6, 9 and 12) 6"23'54, ral 7, 4 rab (rabl, -2, -3, -4) 46'55, R-ras 21,

ypt113 and arj ~6"41 genes. The rabl gene is identical

with the ypt l gene 13"46. The prote ins encoded by

these genes have the consensus amino acid se-

quences for GTP/GDP-b ind ing and GTPase do-

mains 1,44. Several small M r G prote ins including the

arf prote in have been purified from mammal ian tissues14.27,28, 49.

We have recent ly separa ted at least 15 small M r G

prote ins from bovine brain crude membranes by several column chromatographies 15"17"5~'53. A m o n g

them, we have purif ied to near homogene i ty and

character ized two novel small M r G proteins , designated as the smg p25A 17"24 and the smg p2115, the

rho (clone 6) p2051 and the c-Ki-ras p2153. We have

cloned the c D N A of smg p25A from a bovine brain

c D N A l ibrary and de te rmined its comple te nucleo-

t ide and deduced amino acid sequences 24. This

protein is composed of 220 amino acids with a

calculated M r of 24,954. Moreover , we have isolated

two o ther c D N A s highly homologous to the smg- 25A c D N A 24. The prote ins encoded by these two

c D N A s are des ignated as the stag p25B and stag

p25C, respectively. The amino acid sequence ho-

mology among the 3 smg p25s is about 80% 24.

Bovine brain smg p25A is identical with rat brain

rab3 prote in 24'55. We have also cloned the c D N A of

smg p21 from the same c D N A l ibrary and deter-

mined its pr imary structure is. This prote in is com-

posed of 184 amino acids with a calculated M r of

20,987. The smg p21 m R N A level is expressed in

many tissues and smg p21 is most abundant in human platelets 32'33. These novel small M r G prote ins have

also the consensus amino acid sequences responsible

Correspondence: Y. Takai, Department of Biochemistry, Kobe University School of Medicine, Kobe 650, Japan.

0169-328X/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

168

for GTP/GDP-binding and GTPase domains, smg

p21 has the same putative effector domain as ras

p21s. The smg-21 gene is identical with the rapl and Krev-1 genes recently described by Pizon et al. 35 and Kitayama et al.~S, respectively. The rapl cDNA is isolated from a human lymphocyte cDNA library with the probe designed from a conservative region of the D-ras gene of Drosophila ~5, while the Krev-1

cDNA is isolated as a cDNA supressing the transforming action of v-Ki-ras p21 in NIH/3T3 cells ~.

Most of these small M r G proteins have been found in brain 5 "~" J4. ~ ~, 17,34.38.4%51 - 5 3 . Particularly, ras

p21s and smg p25A are present most abundantly in brain 934"3s. We have shown that c-ras p21s are

present mostly in both synaptosomes and microsomes in brain 2~. In synaptosomes, c-ras p21s are exclusively present in the synaptic plasma membranes and not in the synaptosomal cytosol, mitochondria or synaptic vesicles 25. As to other small M r G proteins, we have previously shown that small M r G proteins are mostly present in crude membranes and about one-sixth of total GTPvS-binding activity is found in the cytoso152. In the cytosol, there are multiple small

M r G proteins and one of them is identified as smg

p25A 52. Among many small M r G proteins in the cytosol, stag p25A is most abundant. However, the intrasynaptosomal distribution of small M r G proteins other than c-ras p21s has not been clarified.

In the present studies, we have compared the intrasynaptosomal distribution of smg p25A and rho

p20 with that of c-ras p21s in bovine brain. Since the antibodies recognizing stag p25A and rho p20 in the crude homogenate of bovine brain are not available at present, these proteins are identified by use of several column chromatographies. It is difficult at present to identify smg p2t by this method since there are too many steps to purify it and the total amount of this protein is limited in bovine brain Ls.

MATERIALS AND METHODS

Materials" and chemicals

Bovine brains obtained from the heads of freshly slaughtered cattle were employed. Botulinum toxin type C1 was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). [~-32p]NAD (spec. act., 850 Ci/mmol) and [35S]GTPvS (spec. act., 1,100

Ci/mmol) were obtained from Du Pont-New En-

gland Nuclear. ~25I-Labeled Bol ton-Hunter reagent (200 Ci/mmol) was from Amersham. Other materials and chemicals were obtained from the same sources as described previously t6'17"25.

Monoclonal antibodies

The anti-ras p21 monoclonal antibody RASK-4 was a generous gift from Dr. H. Shiku (Nagasaki University, Nagasaki, Japan). This antibody recognized not only Ki-ras p21 but also Ha- and N-ras

p21s. The anti-smg p25A monocional antibody was made by a routine method. Briefly, female BALB/c mice were immunized intraperitoneally with purified smg p25A in complete Freund's adjuvant. At 2-week intervals, 3 booster injections were given. Three days before sacrifice, mice were further boostered. Spleen cells from mice were fused with P3U1 myeloma cells. Standard fusion, screening and cloning procedures were followed. The antibody thus made specifically recognized smg p25A among the 15 small M r G proteins highly purified from bovine cerebrum ~5'w'~'53. However, when the homogenate of bovine cerebrum was used, this antibody recognized several proteins in addition to smg p25A.

Subcellular fractionation o[ bovine cerebrum

Subcellular fractionation of bovine cerebrum was carried out by the method described by Ueda et al. 47, which was a slight modification of the method of Cotman and Taylor s and that of Whittaker et al. 5°. The detailed procedures were described previously zS. Electronmicroscopic analysis of the Pz C and the CSM fractions indicated that the ultrastruc- tural characteristics of these fractions obtained by our procedures were similar to those previously reported by Ueda et al. 47.

Electron microscopy

Small amounts of the materials from the P2C and CSM fractions were transferred to test tubes and fixed in suspension by the addition of 20 vol of the fixative (0.1 M sodium cacodylate buffer at pH 7.4, 2% glutaraldehyde, I% tannic acid and 0.32 M sucrose) at 4 °C overnight. The fixed material was spun at 3000 g for 30 min and the pellets were washed by 0.1 M sodium cacodylate buffer at pH 7.4 and then postfixed in 1% OsO4 in the same buffer at 4 °C for 1 h. Thereafter. they were dehydrated in

169

graded ethanols and propylene oxide and embedded

in Spurr resin. Sections were made on an LKB ultramicrotome and then stained sequentially with

uranyl acetate and lead citrate. Sections were exam- ined on the J E O L 100 SX electron microscope.

[35S]GTPvS-binding and ADP-ribosylation assays [35S]GTPyS-binding to G proteins was determined

by use of the nitrocellulose filter method as described previously ~7. ADP-ribosylation of proteins

by an ADP-ribosyltransferase contained in botuli-

num toxin type C1 was performed by the method described previously 16.

SDS-PA GE and immunoblotting S D S - P A G E was performed by the method of

Laemmli TM using a 12% polyacrylamide gel. Proteins

on the gel were transferred electrophoretically to a nitrocellulose sheet and immunoblot analysis was carried out as described previously x7'52.

Determinations The radioactivity of 35S-labeled samples was de-

termined using a Beckman liquid scintillation sys-

tems, model LS 3801. Protein was determined with

bovine serum albumin as a standard protein by the method of Lowry et al. 22.

RESULTS

Subcellular distribution of [~SS]GTPvS-binding activity

The homogenate of bovine cerebrum was frac-

tionated into the Pl fraction containing nuclei and cell debris, the P2 fraction containing myelin, mito-

chondria and synaptosomes, the P3 fraction contain-

ing microsomes and the S fraction containing soluble cytosol. When [3sS]GTP~,S-binding activity of these

fractions was assayed, this activity was found in all 4

fractions. However, the activity was most abundant

in the P2 fraction and the specific activity was the highest in the P3 fraction (Table I). In the P2 fraction, [35S]GTP~S-binding activity was found in

all 4 fractions, the P2 A fraction containing myelin and some contamination of membrane components

in the P2 B fraction, the P2 B fraction containing

endoplasmic reticulum, Golgi complex and plasma

membranes, the P2 C fraction containing mainly

TABLE I

Subcellular distribution of [~sS]G TPvS-binding activity

Bovine cerebrum (60 g wet wt.) was fractionated and [35S]GTP~'S-binding activity of each fraction was assayed as described in Materials and Methods. The results shown are representative of 3 independent experiments.

Fraction Protein Total [35S]G TPvS-binding Specific activity Distribution (mg/g tissue) activity (nmol/g tissue) (nmol/rng) (%)

Starting material (homogenate) 114.0 35.28 0.31 Pl 16.8 4.41 0.26 P2 36.7 14.62 0.38 P3 16.0 8.33 0.52 S 22.1 5.87 0.26 Total (P~ + P2 + P3 + S) 91.6 33.23

Starting material (Pz) 36.7 14.62 0.38 P2 A 8.1 1.58 0.19 P2 B 3.3 1.17 0.36 P2 C 9.2 4.50 0.52 P2 D 10.0 3.67 0.36 Total (P2 A + P2B + P2 C + P2D) 30.6 10.92

Starting material (P2C) 9.2 4.50 0.52 CSM 4.3 1.82 0.42 CSV 0.1 0.08 0.80 SS 1.7 0.83 0.49 Total (CSM + CSV + SS) 6.1 2.73

13.3 44.0 25.1 17.6

100

14.5 10.7 41.2 33.6

100

67.2 3.0

29.8 100

170

A / , i.6 '

~ 0 . 8 ~ , 3 E-

° ! i ° E ~

: 0 "O 4> ¢ B u /"\ ¢ o 0.6 2

~-~- (n ® . :'[ \ / !,,.,,.. /*%. ~'"'"~ ~ ,~ ,, o

0.3 i I /

0 ..... T-"- ;" I I L I 10 20 30 40 50 60 70

Fraction Humber Fig. 1. Ultrogel AcA-44 column chromatography. A 20 ul aliquot of each fraction was assayed for [35S]GTPTS-binding activity. (4~---O), GTPTS-binding; ( - - - ) , absorbance at 280 nm. A: the CSM fraction. B: the SS fraction.

synaptosomes and the P2D fraction containing

mainly mi tochondr ia , but was most abundant in the

P2C fraction (Table 1). Moreover , the specific

activity was the highest in this fraction. When the

P2C fraction was further f ract ionated into the CSM

A I ,~

I E E

" ~ °

' 0 Q

g e ~ m° 1.0 ~ ) , , , , ~ , q \,,, ~

0 {/I

0.5

/ '

0 - 0 2 4 6 8 10 12 14

Fraction Number Fig. 2. PhenyI-Sepharosc CL-4B column chromatography. A 20 ul aliquot of each fraction was assayed for [35S1GTPyS- binding activity. The symbols are the same as those used in Fig. 1. A: the CSM fiaction. B: the SS fraction.

fraction containing crude synaptic membranes , the

CSV fraction containing crude synaptic vesicles and

the SS fraction containing synaptosomal soluble

substances, about two thirds of [35S]GTPvS-binding

activity was found in the CSM fraction and about

one third was in the SS fraction (Table 1). Only a

small activity was found in the CSV fraction, but the

specific activity was the highest in this fraction.

Intrasynaptosomal distribution of smg p25A, rho p20 and c-ras p21

Since [35S]GTPvS-binding activity did not always

reflect the activity of small M r G proteins, we

purified small M r G proteins from the CSM and SS

fractions, but could not purify them from the CSV

fraction because of the l imitation of the prepara t ion

of this fraction and small amount of G proteins.

Purification of small M r G proteins from the CSM and SSfractions. During the purification procedures ,

the following 4 buffers were employed: Buffer A,

(l.32 M sucrose containing 1 mM NaHCO3; Buffer

B, 20 mM Tris-HCl at pH 8.0 containing 1 mM

E D T A and 1 mM DTT; Buffer C, 20 mM Tris-HCl

at pH 8.0 containing 1 mM E D T A , 1 mM DTT and

5 mM MgCl2; Buffer D, 20 mM Tris-HCl at pH 8.0

containing 0.1 mM E D T A , 1 mM DTT and 3 mM

MgCI 2. All the purification procedures were carried

out at 0 -4 °C.

The CSM fraction obta ined from bovine cerebrum

(60 g wet wt.) was suspended with 30 ml of Buffer

A containing 1 /~M (p-amidinophenyl )methanesul -

fonyl fluoride. The membranes were collected by

centrifugation at 32,800 g for 20 min, resuspended

with 20 ml of Buffer B containing 1 ,uM (p-

amidinophenyl)methanesul fonyl fluoride and 4.5%

sodium cholate, and incubated for 1 h with stirring.

The extracted membranes were removed by centrif-

ugation at 100,000 g for 1 h. The supernatant was

pooled and MgCI 2 was added to a final concentrat ion of 5 raM. This fraction was used as the crude extract

of the CSM fraction and was subjected to several

column chromatographies . On the o ther hand, about

600 ml of the SS fraction obta ined from bovine

cerebrum (60 g wet wt.) was concentra ted to

approximate ly 20 m] by an Amicon ultrafi l trat ion

cell equipped with a YM-5 filter membrane .

The crude extract of the CSM fraction (20 ml, 140

mg of protein) or the concentra ted SS fraction (20

AI",

,., E ! -

0 q i I I t 0 4 ) 0

~ 8

t,- 0,5 '<

0 -~ ~-- ' - - -~- - '~- - ' - ""i 0 0 !0 20 30 4o 50

Fraction Humber Fig. 3. Hydroxyapatite column chromatography. A 20 /d aliquot of each fraction was assayed for [35S]GTPTS-binding activity. The symbols are the same as those used in Fig. 1. A: the CSM fraction. B: the SS fraction.

ml, 70 mg of protein) was separately applied to an

Ultrogel AcA-44 column (3 × 70 cm) equilibrated with Buffer C containing 1% sodium cholate and 100

mM NaC1. The elution was performed with the same buffer at a flow rate of 30 ml/h. Fractions of 5 ml

each were collected. When each fraction was assayed for [35S]GTPyS-binding activity, two peaks appeared

for both the CSM and SS fractions (Fig. 1). The first and second peaks of the CSM fraction appeared in

the same fractions as those of the SS fraction. The first peak contained large M~ G proteins while the second peak containing small M r G proteins Iv. The

active fractions of the second peak (fractions 48-59) of the CSM and SS fractions were pooled and purified further.

The active fractions of the second peak of the Ultrogei AcA-44 column chromatography (each 60 ml, 15 mg of protein for the CSM fraction or 8 mg

of protein for the SS fraction) were diluted with 220 ml of Buffer C containing 287.5 mM NaCI and applied to a phenyl-Sepharose CL-4B column (1 x

29 cm). The column was first equilibrated with 400 ml of Buffer B and then with 200 ml of Buffer C containing 0.2% sodium cholate and 250 mM NaC1. After the column was washed with 200 ml of Buffer C containing 0.2% sodium cholate and 250 mM NaCI, the elution was performed in a stepwise manner with 100 ml of Buffer C containing 1.3%

171

sodium cholate and 25 mM NaCI at a flow rate of 48

ml/h. Fractions of 5 ml each were collected. When each fraction was assayed for [35S]GTP~'S-binding

activity, a single peak appeared in the same fractions

for both the CSM and SS fractions (Fig. 2). The active fractions (fractions 5-10) of both fractions

were pooled and purified further. The active fractions of the phenyI-Sepharose

CL-4B column chromatography (each 30 ml, 5 mg of protein for the CSM fraction or 2.5 mg of protein for

the SS fraction) were pooled and diluted with 20 ml

of 20 mM Tris-HC1 at PH 8.0 containing 1 mM Dqq" and applied to a hydroxyapatite column (1.8 x 5.3

cm) equilibrated with 200 ml of Buffer D containing

10 mM KH2PO 4. The elution was performed first with 200 ml of Buffer D containing 0.6% CHAPS

and 10 mM KHzPO 4 and then with 50 ml of Buffer

D containing 0.6% CHAPS and 100 mM KH2PO 4 at a flow rate of 5 ml/min. Fractions of 5 ml each were collected. When each fraction was assayed for [35S]GTPTS-binding activity, one major and one

minor peak appeared for both the CSM and SS fractions (Fig. 3). The active fractions of the first

peak (fractions 4-14) were collected and concen- trated to approximately 4 ml by the same Amicon ultrafiltration cell as described above. The second

peak of this column chromatography of the CSM fraction contained c - r a s p21 (see below).

2

1

"O e-

o i .0

~, 0.s

,' ' , "' 0.4 i \ ,., :~lf~l I:

I I I I I I I

:!., i ; i , J

; , , " . I , 0 .1

- - T - - - ~ - - o

i'o 2o s'o ,'o s'o oo ¢o Fraction Humber

Fig. 4. Mono Q HR5/5 column chromatography. A 20 /A aliquot of each fraction was assayed for [35S]GTPyS-binding activity. The symbols are the same as those used in Fig. 1. A: the CSM fraction. B: the SS fraction.

172

The active fractions of the first peak of the

hydroxyapat i te column chromatography (each 4 ml,

1 mg of prote in for the CSM fraction or 0.3 mg of

protein for the SS fraction) was appl ied to a Mono

Q HR5/5 column (0.5 x 5 cm) equil ibrated with

Buffer C containing 0.6% C H A P S and 10 mM NaC1.

Af te r the column was washed with 5 ml of Buffer C

containing 0.6% C H A P S and 10 mM NaC1, the

elution was per formed with a 20-ml l inear gradient

of NaCI (0.01-0.3 M) in Buffer C containing 0.6%

CHAPS. The elution was per formed at a flow rate of

30 mi/h. Fractions of 0.5 ml each were collected.

When each fraction was assayed for [35S]GTP?S-

binding activity, 4 peaks appeared for the CSM

fraction while 3 peaks appeared for the SS fraction

(Fig. 4). The first, second and fourth peaks of the

CSM fraction appeared in the same fractions as

those of the first, second and third peaks of the SS

fraction, respectively. The first and second peaks of

both the CSM and SS fractions were not identified.

The third peak of the CSM fraction contained rho

p20 (see below). The fourth peak of the CSM

fraction and the third peak of the SS fraction

contained smg p25A.

The active fractions of the third peak (fractions

43-48) of the Mono Q HR5/5 column chromatog-

raphy of the CSM fraction were pooled. The pooled

fractions (2.5 ml, 20/~g of protein) were diluted with

22.5 ml of Buffer C containing 0.6% C H A P S to

decrease the NaCI concentrat ion and then subjected

to rechromatography on the same Mono Q HR5/5

column equi l ibrated with the same buffer. Af te r the

column was washed with 5 ml of the same buffer, the

m

¢~ 0.2 1,002 o

O I0 20 30 tO °O 60 Fraction Number

Fig. 5. Re-Mono Q HR5/5 column chromatography of the CSM fraction. A 20 M aliquot of each fraction was assayed for [3-sS]GTPvS-binding activity. The symbols are the same as those used in Fig. 1.

elution was per formed with a 30-ml l inear gradient

of NaC1 (0.05-0.25 M) in the same buffer at a flow

rate of 30 ml/h. Fract ions of 0.5 ml each were

collected. When each fraction was assayed for

[35S]GTPvS-binding activity, two peaks appeared

(Fig. 5). The second peak was identif ied as rho p20

(see below).

Identification as ras p21, smg p25A and rho p20.

We have previously shown that ras p21 is eluted in

the second peak of the hydroxyapat i te column

chromatography when it is purified from bovine

brain crude membranes 53. In the CSM fraction, the

second peak of the hydroxyapat i te column chroma-

tography was recognized by the anti-ras p21 anti-

body but the first peak was not (data not shown). In

the SS fraction, nei ther the first nor the second peak

was recognized by this ant ibody (data not shown).

This result was consistent with our ear l ier observa-

tions that ras p21s were highly concentra ted in the

fractions rich in synaptic plasma membranes , were

A P,

3 6 K

~ . . . .

20.1 K - -

1 7 K - -

1 2 3 1 2 3

Fig. 6. Protein staining and immunoblotting of the fourth peak of the CSM fraction and the third peak of the SS fraction of the Mono Q HR5/5 column chromatography. A: protein staining, stag p25A, the fourth peak of the CSM fraction and the third peak of the SS fraction (about 1 pg of each protein) were subjected to SDS-PAGE (12% polyacrylamide). The proteins were visualized with Coomassie brilliant blue. Lane 1, stag p25A; Lane 2. the fourth peak of the CSM fraction; Lane 3. the third peak of the SS fraction. B: immunoblot analysis. stag p25A, the fourth peak of the CSM fraction and the third peak of the SS fraction of the Mono Q HR5/5 column chromatography (about 300 ng of each protein) were subjected to SDS-PAGE (12% polyacrylamide). After the proteins on the gel were transferred electrophoretically to a nitrocellulose sheet, the sheet was immunoblotted with the anti-smg p25A monoclonal antibody under the conditions specified previously 52. Lane 1, smg p25A; Lane 2, the fourth peak of the CSM fraction; Lane 3, the third peak of the SS fraction. The protein markers used were glyceraldehyde-3-phosphate dehy- drogenase (M r - 36,000), trypsin inhibitor (M, = 20.100) and myoglobin (M,. = 17,000).

poorly present in other fractions rich in synaptic vesicles, intrasynaptosomal mitochondria or.postsyn- aptic densities and were undetectable in the SS fraction 25.

We have previously reported that the small M r G protein eluted at the NaCI concentration of about 240 mM on the first Mono Q HR5/5 column chromatography is smg p25A 17. The fourth peak of

the CSM fraction and the third peak of the SS fraction shown in Fig. 4 appeared at the same NaCI concentration, suggesting that the small M r G proteins eluted in these peaks are smg p25A. Consistent with this result, both G proteins eluted in these peaks were recognized by the antibody against smg p25A (Fig. 6).

We have also reported that the small M r G proteins eluted at the NaCI concentration of about 180 mM on the first Mono Q HR5/5 column chromatography contains rho p20 and an unidenti- fied small M r G protein 5~. These two G proteins are separated by re-Mono Q HR5/5 column chromatography, and the second peak has been identified as rho p205~. Moreover, we have shown that rho p20 is

A B

36K - -

2 0 . I K ~

1 7 K ~

1 2 1 2

Fig. 7. Protein staining and the ADP-ribosylation by an ADP-ribosyltransferase contained in botulinum toxin of the second peak of the re-Mono Q HR5/5 column chromatography of the CSM fraction. A: protein staining, rho p20 and the second peak of the CSM fraction (about 300 ng of each protein) were subjected to SDS-PAGE (12% polyacrylamide). The proteins were visualized with silver. Lane 1, rho p20; Lane 2, the second peak of the CSM fraction. B: autoradiogram. About 160 ng of rho p20 and the second peak of the CSM fraction were ADP-ribosylated by an ADP-ribosyltransferase contained in botulinum toxin type C1 under the conditions described previously 16. The ADP-ribosylated proteins were subjected to SDS-PAGE (12% polyacrylamide) followed by autoradiography. Lane 1, rho p20; Lane 2, the second peak of the CSM fraction. The protein markers used were the same as described in Fig. 6.

173

ADP-ribosylated by an ADP-ribosyltransferase contained in botulinum toxin type C116. As shown in Fig. 5, the third peak of the first Mono Q HR5/5 column chromatography was eluted at the same NaC1 concentration as rho p20. This peak was eluted in two peaks on the re-Mono Q HR5/5 column chromatography and the second peak was eluted at the same NaC1 concentration as rho p20. Moreover, the second peak was ADP-ribosylated (Fig. 7). These results indicate that the third peak of the Mono Q HR5/5 column chromatography of the CSM fraction contains rho p20 and that the second peak of the re-Mono Q HR5/5 column chromatography is rho p20. In the SS fraction, the third peak of the CSM fraction containing rho p20 was not observed, indicating that rho p20 is not present in this fraction.

DISCUSSION

We have previously shown that c-ras p21(s) is found in the synaptic membranes and not in the synaptosomal cytosol or the synaptic vesicles 25. We

have also shown previously that smg p25A is present not only in the crude membranes but also in the cytosol of bovine brain 52. In the present paper, we have extended these earlier observations and shown that smg p25A is found in both the synaptic membranes and synaptosomal cytosol. Moreover,

we have shown here that rho p20 is found in the synaptic membranes but not in the synaptosomal cytosol. It remains to be clarified whether these small M r G proteins are present in the synaptic vesicles, but our present results indicate that among the three well-characterized small M r G proteins the c-ras p21 and -rho p20 show a similar intrasynaptosomal distribution while smg p25A shows a different distribution.

The mechanism of different intrasynaptosomal distribution of these small M r G proteins is unknown at present. However, many small M r G proteins have unique C - t e r m i n a l sequences 1"6'7"13"15"21'23'24'37"44'46'

54. All of Ha-, Ki- and N-ras p21s have a common C-terminal sequence, that is Cys-X-X-X, where X is any amino acid TM. This cysteine residue is palmitoylated 1. This palmitoylation is essential for ras

p21s to attach to plasma membranes and to acquire transforming activity 1. The R-ras, 3 rho, ral and smg-21 proteins also have a similar C-terminal

174

sequence ~.~,.7.~5.2~.23.44.54. In contrast, the ypt l , rab2

and SEC4 proteins have a different common C- terminal sequence, that is Cys-Cys 133744"4~'. The

YPT1 protein, the yeast counterpart of the mam-

malian yp t l protein, is also palmitoylated at one or both of these cysteine residues 26. The Y P T I protein

attaches to membranes through this fatty acid. The

YPT1 protein has been shown to be localized in Golgi complex 4~ while the SEC4 protein in secretory

vesicles and plasma membranes ~. Three smg p25s have also a common C-terminal sequence different

from those of other small M r G proteins described above, that is Cys-X-Cys 24"44. It is possible that these

small M r G proteins are also modified with palmitic

acid at either of these cysteine residues and attach to membranes through this fatty acid. Thus, there are

at least 3 groups of small M r G proteins which have different unique C-terminal sequences. It is not known at present why there are different types of

C-terminal sequences in small M r G proteins, but we have previously proposed that different C-terminal

sequences determine different compartmentalization

and functions of each group of small M r G proteins 44. Consistent with this earlier proposal, c-ras

p21(s) and rho p20, which have the same consensus

C-terminal sequence, show similar intrasynaptosomal distribution, and smg p25A, which has a different consensus C-terminal sequence, shows dif-

ferent intrasynaptosomal distribution. It is well established that ras p21 is found in most

mammalian tissues and is involved in the regulation of various cell functions including cell-transforming action ~. but there are several lines of evidence indicating that they play important roles in neuronal functions 3"123~3'~. ras p21s are present most abun-

dantly in nerve tissues '). Microinjection of the acti-

vated ras p21 or the GTPyS-bound c-ras p21 induces differentiation of pheochromocytoma PC-12 cells into neuron-like cells 3"~9. An anti-ras p21 antibody inhibits the nerve growth factor-induced differentiation of this cell line ~z. As to the rho proteins, the clone 12 protein mRNA has been shown to be expressed in many mammalian tissues 34 but the functions of the rho proteins have not been clarified. We have recently reported that bovine brain rho p20 is ADP-ribosylated by an ADP-ribosyltransferase(s) contained in botulinum toxin type C1~6, known to inhibit neurotransmitter release from peripheral

cholinergic neurons 42. Similarly, the bovine adrenal

gland protein ADP-ribosylated by an ADP-ribosyl-

transferase contained in botulinum toxin type C1 and D has been identified as the rho protein 2̀ ). Moreover, the porcine brain protein ADP-ribosy-

lated by a botulinum ADP-ribosyltransferase C3, known to lack the neurotoxin activity, has been

identified as the rho protein 5. Although it is not clear

whether the ADP-ribosylation of the rho proteins is involved in the neurotoxin action of botulinum

toxins type C1 and D, these toxins have been shown to inhibit the secretory process occuring downstream of the Ca 2+ system l`). Evidence is also available that

smg p25A may play important roles in neuronal functions. We have reported that the srng p25A mRNA is specifically expressed m brain and adrenal

medulla 3s. The similar results have been also shown

for the rab3 mRNA which is identical with the smg

p25A mRNA 34. We have also shown that the smg

p25A mRNA level increases after differentiation of pheochromocytoma PC-12 cells into neuron-like cells in response to nerve growth factor or dibutyryl cyclic AMP 3s. There is another line of evidence that

some small M,. G proteins are involved in the regulation of exocytosis 2.11,37.4o Particularly in

yeast, the small M r G proteins encoded by the SEC4

and YPT1 genes have been shown to be involved in this regulation 11"37"4°. In mammalian tissues, exocyt-

osis is regulated by two intracellular messenger s y s t e m s , C a 2+ and protein kinase C 3°'45. it has been

suggested that G proteins are involved in the

secretory processes occuring downstream of these two intracellular messenger systems 2. On the basis of these earlier observations together with the present

findings, it is likely that rho p20 and smg p25A as well as c-ras p21(s) are involved in the regulation of neurotransmitter release from synapses. The precise

roles of these small M,. G proteins in neuronal

functions remain to be clarified.

ACKNOWLEDGEMENTS

This investigation was supported by Grants-in- Aid for Scientific Research and Cancer Research from the Ministry of Education, Science, and Cul- ture, Japan (1988), Grants-in-Aid for Abnormalities in Hormone Receptor Mechanisms (1988), Cardio- vascular Diseases (1988), and for Cancer Research

f r o m the Minis t ry of H e a l t h and Wel fa re , J apan , and

by grants f rom the Y a m a n o u c h i F o u n d a t i o n for

R e s e a r c h on M e t a b o l i c Disease (1988) and the

R e s e a r c h P r o g r a m on Cell Ca lc ium Signal in the

ABBREVIATIONS

CHAPS

c-Ki-ras p21 DTF G protein GTPvS Mr

3-[(3-cholamidopropyl)dimethylammonio]- 1-propane-sulfonate c-Ki-ras protein dithiothreitol GTP-binding proteins guanosine 5"-(3-O-thio)triphosphate relative molecular weight

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intrasynaptosomal distribution of the ras, rho and smg-25a gtp-binding proteins in bovine brain

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