A Rho GTPase Controls the Rate of Protein Synthesis

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A Rho GTPase controls the rate of protein synthesis

in the sea urchin egg

Salvador Manzo, Guadalupe Martnez-Cadena, Juana Loopez-Godnez,Mario Pedraza-Reyes, and Jesuus Garca-Soto*

Instituto de Investigacioon en Biologa Experimental, Facultad de Qumica, Universidad de Guanajuato,

P.O. Box 187, Guanajuato, Gto. CP 36000, Mexico

Received 24 August 2003

Abstract

Fertilization of the sea urchin egg triggers a Ca2-dependent cortical granule exocytosis and cytoskeletal reorganization, both of

which are accompanied by an accelerated protein synthesis. The signaling mechanisms leading to these events are not completely

understood. The possible role of Rho GTPases in sea urchin egg activation was studied using the Clostridium botulinumC3 exotoxin,

which specifically ADP-ribosylates Rho proteins and inactivates them. We observed that incubation of eggs with C3 resulted in

in situ ADP-ribosylation of Rho. Following fertilization, C3-treated eggs were capable of performing cortical granule exocytosis but

not the first cytokinesis. C3 caused in both unfertilized eggs and early embryos alterations in the state of actin polymerization and

inhibition of the spindle formation. Moreover, C3 diminished markedly the rate of protein synthesis. These findings suggested that

Rho is involved in regulating the acceleration of protein synthesis that accompanies the egg activation by sperm.

2003 Elsevier Inc. All rights reserved.

Keywords: Fertilization; Microfilaments; Microtubules; Protein synthesis; G-protein; Rho; Egg; Sea urchin

Upon fertilization, the sea urchin egg is released from

the G1-phase and enters the S-phase. Egg activation by

sperm is accompanied by a raise in the cytosolic Ca2

and in the intracellular pH (pHi) [1,2]. Cytosolic Ca2

increase promotes, at least, exocytosis of the cortical

granules [3] whereas the cytoplasmic alkalinization is

partly responsible for accelerating the rate of protein

synthesis [47]. It has been widely documented that the

cytoskeleton is required for spermegg interaction [8],

egg activation, and progression of early development [9

12]. For instance, perturbation of the cytoskeleton with

cytochalasin B prevents the characteristic acid release

that follows fertilization, the elevation of the fertiliza-

tion membrane, and the first cytokinesis [11]. Although

it has been postulated that the egg cytoskeleton plays an

essential role in the transmission of the signal elicited by

sperm, the mechanisms underlying the initiation of the

cytoskeletal reorganization and its role in egg activation

are not completely understood.

In most cellular types, the state of actin polymeriza-

tion is controlled by members of the Rho family of small

GTP-binding proteins (for reviews see [1316]). The Rho

subfamily of GTPases are selectively and specifically

ADP-ribosylated by the exotoxin C3 from Clostridium

botulinum [17]. ADP-ribosylation leads to functional

inactivation of Rho proteins [18]. Hence, the functions

of Rho proteins have been in part elucidated by the use

of this toxin [19,20]. In sea urchin embryos, RhoA lo-

calizes in the contractile ring and in the cleavage furrow

that precedes the first cell division [21]. Direct evidence

that Rho is part of the molecular machinery which

controls the cytokinesis arises from experiments of C3

microinjection in sand dollar, sea urchin, and Xenopus

embryos [22,23]. In unfertilized sea urchin eggs, RhoA is

present preferentially in the egg cortex [21,24]. However,

the role played by Rho in the events which characterize

egg activation has not been completely elucidated. In the

present work unfertilized sea urchin eggs were treated

* Corresponding author. Fax: +52-47-37-35-19-03.

E-mail address: [email protected](J. Garca-Soto).

0006-291X/$ - see front matter 2003 Elsevier Inc. All rights reserved.

doi:10.1016/j.bbrc.2003.08.153

Biochemical and Biophysical Research Communications 310 (2003) 685690

BBRCwww.elsevier.com/locate/ybbrc
http://mail%20to:%[email protected]/http://mail%20to:%[email protected]/


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with C. botulinum C3 exotoxin to study its effect on the

cytoskeletal organization, microtubule formation, and

protein synthesis following fertilization.

Materials and methods

Gamete collection and reagents. Gametes from the sea urchin

Strongylocentrotus purpuratus were collected as indicated in [24].

[35S]Methionine (1000 Ci/mmol) and [32P]NAD (1000 Ci/mmol) were

from Amersham. Recombinant C3 exoenzyme fromC. botulinumwas

from Upstate Biotechnology. Antibodies against RhoA and tubulin

were from SantaCruz and Zymed, respectively. Materials for poly-

acrylamide gel electrophoresis were from Bio-Rad. All other reagents

were obtained from Sigma.

C3 treatment of eggs. Unfertilized eggs (29,000 cells in 600ll sea-

water) were incubated for 3060min at 16C in the absence (control)

or presence of 1.5 ng/ll C3, followed by fertilization. At the times in-

dicated, samples were withdrawn and the cells were washed in seawater

by centrifugation at 1000gfor 10 min. Thereafter, unfertilized eggs and

embryos were either fixed with 4% paraformaldehyde in seawater or

lysed for in vitro [32

P]ADP-ribosylation by C3. In the latter case, thedisappearance or drastic reduction in radiolabeling of the 25 kDa

protein compared with control cells was indicative that C3 effectively

ADP-ribosylated Rho in intact cells.

Cell fractionation and in vitro ADP-ribosylation by C3. Eggs or

embryos were mixed with 100ll of a lysis buffer (50 mM TrisHCl,

150mM NaCl, 1 mM EDTA, 2.5mM MgCl2, 5% glycerol, 1% (v/v)

NP-40, 1mM Na3VO4, 1mM NaF, 5 lg/ml leupeptin, 5lg/ml anti-

pain, 5 lg/ml pepstatin, and 1lg/ml soybean trypsin inhibitor, pH 7.4)

and homogenized in a glass homogenizer with a Teflon pestle; the

extract was cleared by centrifugation at 2000g for 10 min. Cellular

fractions were kept in aliquots at )70 C. ADP-ribosylation by

C. botulinum C3 exoenzyme was assayed in cellular extracts as previ-

ously described [24]. Briefly, 2030lg of cellular protein was incubated

in 30ll of a reaction mixture (50 mM TrisHCl, pH 7.0, 1 mM ATP,

2 lM NAD, 2 mM MgCl2, 1 mM DTT, 1 mM EDTA, 0.6% Triton X-100, 15 ng exoenzyme C3, and 0.25lCi [32P]NAD). Following incu-

bation for 1h at 16 C, proteins were separated by SDSPAGE and

subjected to autoradiography.

Fluorescence microscopy. Eggs or embryos fixed with 4% parafor-

maldehyde in seawater were sedimented (2000g, 10 min), resuspended

in 1% Triton X-100 in seawater, and incubated overnight for effective

permeabilization; thereafter, they were washed three times with

phosphate buffered solution (PBS). Samples were incubated overnight

with anti-RhoA (1:25) or anti-tubulin (1:500) antibodies, respectively.

After washing to remove unbound antibody, the cells were incubated

with Alexa 459-conjugated anti-rabbit IgG (1:250 dilution) for 1 h at

room temperature. After incubation with the secondary antibody, the

cells were washed 3 times with PBS and mounted onto glass slides.

Fluorescence images were acquired with a Bio-Rad MRC600 Kr-Ar

confocal microscope attached to a Zeiss Axioskop using Comos 7.0software. Images were analyzed using the Confocal Assistant software

(Bio-Rad). Where specified, F-actin was labeled by incubating fixed

and permeabilized cells with fluorescein-conjugated phalloidin for 1 h

at room temperature and viewed under the confocal microscope.

Measurements of protein synthesis. Protein synthesis was deter-

mined by measuring [35S]methionine incorporation into TCA-precipi-

table material. In brief, eggs (48 cells/ll) were loaded with 10lCi

[35S]methionine for 1 h at 16C in 600ll seawater and centrifuged

(1000g, 2 min) to eliminate extracellular radioactive tracer, followed by

an additional 60 min-incubation without or with C3 exotoxin (1.5ng/

ll). Samples (250ll) were taken before or after addition of sperm and

extracted with 1 ml of a 20% trichloroacetic acid (TCA) solution.

Precipitates were filtered through glass fiber filters (Whatman), which

were washed three times with 10% TCA. Radioactivity was determined

by liquid scintillation counting. Protein synthesis was also measured by

processing the samples (40ll) for polyacrylamide gel electrophoresis

and autoradiography.

Results and discussion

Inactivation of Rho in unfertilized eggs leads to inhibitionof cytokinesis following fertilization

Lysates that were prepared from unfertilized eggs

(control) and from embryos collected at intervals after

fertilization were subjected to [32P]ADP-ribosylation

with C3. Fig. 1A shows radiolabeling of a 25 kDa pro-

tein whose intensity did not change significantly during

the period studied. No radiolabeled proteins were ob-

served in the absence of C3 (not shown). Western blot

analysis utilizing a specific antibody against human

RhoA revealed that RhoA was present after activation

and during early development (Fig. 1B); when the pri-mary antibody was omitted (control), any signal was

detected (not shown). As observed in Fig. 1B, the anti-

body also crossreacted with a lower molecular weight

protein. Taking together, these results suggest that the

overall levels of Rho do not appreciably change after

fertilization.

To study the effect of C3 on egg activation, we first

tested whether the toxin was able to in situ ADP-ri-

bosylate Rho. To this end, unfertilized eggs were

preincubated in C3-containing seawater for 60 min and

Fig. 1. RhoA expression after egg activation and in situ ADP-ribo-

sylation of Rho by C3 exotoxin in intact eggs and embryos. Unfer-

tilized eggs or embryos collected at different times after fertilization

were lysed and assayed for [32P]ADP-ribosylation by C3 (A) and

Western blot with an antibody against RhoA (B) as described in

Materials and methods. (C) lysates prepared from C3-treated eggs and

embryos (150 min postfertilization) derived from these eggs were pro-

cessed for in vitro [32P]ADP-ribosylation by C3. Each lane was loaded

with 30lg protein.

686 S. Manzo et al. / Biochemical and Biophysical Research Communications 310 (2003) 685690


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then lysed. To verify Rho ADP-ribosylation, the lysates

were processed for in vitro [32P]ADP-ribosylation with

C3 plus [32P]NAD. Results revealed that lysates from

eggs which were not preincubated with C3 showed the

typical radiolabeling of Rho (Fig. 1C, lane 1) whereas

lysates from C3-treated eggs displayed a marked de-

crease in the intensity of radiolabeling (lane 3), indi-cating a drastic reduction of potential sites for in vitro

[32P]ADP-ribosylation. Similar results were obtained in

embryos derived from C3-treated eggs (Fig. 1C, com-

pare lanes 2 and 4). Although the mechanism of C3

entry is unknown, it could occur through endocytosis

which at least in oocytes has been reported to be active

[25]. These results revealed that under the conditions

used C3 ADP-ribosylated Rho in intact eggs.

Eggs or embryos incubated with C3 were next ex-

amined by microscopy and compared with nontreated

cells. Fig. 2A shows the bright field and fluorescence,

respectively, of a control egg (a, a0) and 2 cell-stage

embryo (c, c0). Interestingly, fertilized C3-pretreated

eggs failed to reach the first cytokinesis (compare d and

c) although formation of the fertilization envelope was

not blocked. Moreover, only mononucleated cells were

observed (d0). Quantitative analysis of cytokinesis inhi-

bition by C3 is shown in Fig. 2B. Using batches of eggs

showing 100% activation by sperm, above 80% of em-

bryos divided into two cells at 150 min after fertilization

while the rest of the embryos were still in one-cell stage

(Fig. 2B). When these eggs were incubated with C3 be-

fore fertilization, the respective embryos did not divide

and cytokinesis inhibition was above 95%. These results

indicated that Rho inactivation does not affect exocy-

tosis of cortical granules but it leads to inhibition of the

first cytokinesis, arresting the embryos in the one-cellstage. The effect of C3 resulted to be reversible since

embryos incubated in fresh seawater, without the toxin,

recovered the capacity to divide around 5 h later.

Influence of C3-pretreatment on the cytoskeletal organi-

zation and spindle formation

It has been widely documented that in many cellular

types the cytoskeletal organization is largely regulated

by Rho proteins [13,26]. In sea urchin eggs, the state of

polymerization of actin is fundamental for the early

events that follow fertilization [812]. Therefore, the

effect of Rho inactivation by C3 on the distribution of

F-actin in both unfertilized eggs and in embryos was

studied. As observed in Fig. 3a, fluorescein-phalloidin

staining demonstrated that C3 affected the global

amount of filamentous actin in unfertilized eggs (com-

pare d and a) and in embryos derived from the C3-

treated eggs (compare e and b). At 120 min, C3-treated

cells did not form the cleavage furrow (f). This result

Fig. 2. Eggs preincubated with C3 exotoxin perform the cortical granule exocytosis following fertilization but do not reach the first cytokinesis. Eggs

were preincubated for 1 h without (control) or with C3 exotoxin, followed by fertilization. (A) photographs showing the morphology of the cells.

(ad) Phase-contrast optics; (a0d0) nuclear staining with Hoechst 33258. (B) quantitative analysis of embryos reaching the two-cell stage in the

absence and presence of C3. Results shown represent meansSD of three experiments.

S. Manzo et al. / Biochemical and Biophysical Research Communications 310 (2003) 685690 687


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was similar to that obtained with embryos injected with

C3 just a few minutes before the cleavage furrow for-

mation [22,23]. It has been reported that conditions

which inhibit early events at fertilization lead to inhi-

bition of the spindle formation [27]. Therefore, the effect

of C3 on the spindle formation was further evaluated by

indirect immunofluorescence. As noted, following fer-tilization C3-pretreated eggs were incapable of orga-

nizing the microtubules that form the spindle (Fig. 3b, l)

while in the control embryos the spindle formation was

normal (i).

Inhibition by C3 exotoxin of protein synthesis

Protein synthesis increases markedly within a few

minutes after fertilization [28]. Therefore, we studied

whether protein synthesis was affected by Rho inacti-

vation. To this end, eggs were loaded with [35S]methio-

nine during 1 h and then washed to remove theextracellular tracer. Following 1 h-incubation with C3

exotoxin, [35S]methionine-loaded eggs were fertilized.

Fig. 4A shows the extent of protein synthesis as a

function of time after fertilization. Unfertilized eggs

(zero time) displayed a basal level of [35S]methionine

incorporation into proteins, which was consistently re-

duced in the presence of C3. After fertilization, protein

synthesis in C3-pretreated eggs was drastically reduced

while control cells continued with the expected rate of

protein synthesis. Protein synthesis was also analyzed by

Fig. 3. Visualization of polymerized actin and microtubules in C3-

treated eggs and embryos. Eggs were pretreated or not (control) with

C3 and then fertilized. After fixation, cells were stained with FITC-

conjugated phalloidin for visualization of filamentous actin or pro-

cessed for microtubule immunolabeling. (af) Images, representing

whole cells, were generated from optical sections throughout the cell

depth that were collected by confocal microscopy. (gl) Images were

generated from the equatorial region of the cells.

Fig. 4. C3 exotoxin interferes with protein synthesis in fertilized eggs. The eggs were first loaded with [35S]methionine and then preincubated with C3

before fertilization. (A) protein synthesis was monitored by measuring the radioactivity incorporated into trichloroacetic acidprecipitable material.

Points represent means of three measurements made with eggs of a single female. (d) Control; (j) C3-treated eggs. (B) [35S]methionine-labeled

proteins were processed for SDSPAGE and autoradiography as indicated in Materials and methods section. This experiment was repeated three

times with identical results.



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SDSPAGE and autoradiography. Results shown in

Fig. 4B revealed an inhibitory effect in the synthesis of

protein by C3. Considering that Rho mainly controls

the cytoskeleton, these data suggest that Rho might

regulate the rate of protein synthesis through the mi-

crofilaments. This latter possibility was explored by

measuring protein synthesis in eggs pretreated with thecytoskeleton inhibitor cytochalasin D. [35S]Methionine-

loaded eggs were pretreated with cytochalasin D for

15 min and fertilized. Following a 60-min incubation in

seawater, protein synthesis was measured. We observed

that, with respect to control cells, cytochalasin D re-

duced in 92% the [35S]methionine incorporated into

proteins and inhibited 89% the first cytokinesis (data not

shown). Taken together, these results suggest that Rho

plays, through the cytoskeleton, a critical regulatory role

in the rate of protein synthesis following fertilization.

Taking into account that an imposed intracellular

alkalinization with ammonia stimulates the protein

synthesis [46], we studied whether C3-treated eggs were

capable of accelerating the protein synthesis upon ad-

dition of 20 mM NH4Cl. As shown in Table 1, ammo-

nium ions did stimulate protein synthesis in eggs

preincubated with C3 exotoxin, at a similar level of

control eggs. Therefore, Rho might be acting in an early

site of the signaling pathway that regulates the rate of

protein synthesis, possibly before the event involving

activation of the Na/H exchanger.

The data presented herein suggest that Rho is linked

to the initiation of protein synthesis. It has been claimed

that protein synthesis is triggered by the cytoplasmic

alkalinization that follows the fertilization [46], al-though it may also occur through a pHi-independent

pathway [7]. It is likely that the linkage between the

active state of Rho and protein synthesis is the Na/H

exchange which is activated after fertilization. In epi-

thelial cells, for instance, the state of polymerization of

actin controls the activity of the exchanger, which is the

major mechanism responsible for pHi regulation [29].

Moreover, it has been demonstrated that RhoA is nec-

essary for adequate Na/H exchange activity [30,31].

Thus, it is possible that after fertilization Rho becomes

active to direct a new organization of the cytoskeleton

which in turn promotes the activation of the Na/H

exchanger. This contention is partly supported by the

following observations: (i) cytochalasin D inhibits pro-tein synthesis (this paper); (ii) cytochalasin B blocks the

sperm-induced acid release, which in part reflects Na/

H exchange activity, and the first cytokinesis [8]; and

(iii) protein synthesis triggered by an artificial alkalin-

ization with NH3 is insensitive to C3 exotoxin (Table 1).

Nevertheless, the regulation of protein synthesis by Rho

can be more complex than this simple model and might

involve multiple factors [7]. Previous work has provided

evidence for the indispensable role of Rho proteins in

the formation of the cleavage furrow that precedes cy-

tokinesis [22,23]. This has been demonstrated by in-

jecting C3 toxin into sand dollar and sea urchin embryos

just a short time before formation of the cleavage furrow

[22]. Similar observations have been obtained in Xeno-

pus embryos microinjected with either C3, dominant

negative forms of Rho or accessory proteins as Rho-

GDI [23]. The fact that C3 inhibits cytokinesis when

added either before fertilization or injected into devel-

oping embryos suggests that Rho has a critical role at

various stages of development, as observed in other

organisms [32]. Other observation from the present

work is that C3 does not affect the Ca2-dependent

cortical granule exocytosis, which is similar to the result

obtained in mouse eggs [33], and allows us to infer that

Rho does not participate in the signaling pathway that isresponsible for the cytosolic Ca2 increase in sea urchin

eggs. Unlike Ascidian eggs, C3 did not induce sponta-

neous egg activation in the absence of sperm [34]. In

conclusion, we have shown that functional inactivation

of Rho in the sea urchin egg through ADP-ribosylation

by C3 exotoxin leads to adverse effects on activation by

sperm, particularly in the initiation of protein synthesis.

As a consequence, the early inactivation of Rho affects

late events of the first cell cycle including the correct

assembly of the spindle and the first cytokinesis.

Acknowledgments

This investigation was supported by the Consejo Nacional de

Ciencia y Tecnologa (CONACYT) of Meexico through a Grant-in-Aid

(28196N) to J.G.S. and by the University of Guanajuato. A scholar-

ship to S.M.A. was from CONACYT and the Consejo de Ciencia y

Tecnologa del Estado de Guanajuato.

References

[1] L.L. Runft, L.A. Jaffe, L.M. Mehlmann, Egg activation at

fertilization: where it all begins, Dev. Biol. 245 (2002) 237254.

Table 1

C3 exotoxoin does not affect the protein synthesis stimulated by an

imposed cytoplasmic alkalinization with NH3

Condition [35S]Methionine incorporation

(cpm)

Eggs 103,398

Eggs+ 20mM NH4Cl 191,707

Eggs + C3 98,721

Eggs+C3+20mM NH4Cl 184,170

Eggs were incubated with [35S]methionine for 1 h at 16C. Two

samples of [35S]methionine-loaded eggs (1000 cells in 60ll seawater)

were incubated for 30min with 7 ng/ll C3, followed by the addition of

1.2 ll of either seawater (control) or NH4Cl (stock of 1.0M in sea-

water), and incubation proceeded for 1 h. Thereafter, radioactivity

incorporated into proteins was measured as described in Materials and

methods. As controls, two other samples were processed identically

except that C3 was omitted. Results are in triplicate obtained with a

single female. Two other experiments gave the same results.

S. Manzo et al. / Biochemical and Biophysical Research Communications 310 (2003) 685690 689


6/6

[2] M.J. Whitaker, R.A. Steinhardt, Ionic signaling in the sea urchin

egg at fertilization, in: C.B. Metz, A. Monroy (Eds.), Biology of

Fertilization, Academic Press, New York, 1985, pp. 167221.

[3] R.S. Zucker, R.A. Steinhardt, Prevention of the cortical reaction

in fertilized sea urchin eggs by injection of calcium-chelating

ligands, Biochim. Biophys. Acta 541 (1978) 459466.

[4] D. Nishioka, N.F. McGwin, Relationships between the release of

acid, the cortical reaction, and the increase of protein synthesis in

sea urchin eggs, J. Exp. Zool. 212 (1980) 215223.

[5] M.M. Winkler, R.A. Steinhardt, J.L. Grainger, L. Minning, Dual

ionic controls for the activation of protein synthesis at fertiliza-

tion, Nature 287 (1980) 558560.

[6] J.L. Grainger, M.M. Winkler, S.S. Shen, R.A. Steinhardt,

Intracellular pH controls protein synthesis rate in the sea urchin

egg and early embryo, Dev. Biol. 68 (1979) 396406.

[7] B.B. Rees, C. Patton, J.L. Grainger, D. Epel, Protein synthesis

increases after fertilization of sea urchin eggs in the absence of an

increase in intracellular pH, Dev. Biol. 169 (1995) 683698.

[8] G. Schatten, H. Schatten, Effects of motility inhibitors during sea

urchin fertilization. Microfilament inhibitors prevent sperm in-

corporation and restructuring of fertilized egg cortex, whereas

microtubule inhibitors prevent pronuclear migrations, Exp. Cell

Res. 135 (1981) 311330.

[9] D.A. Begg, L.I. Rebhun, pH regulates the polymerization of actin

in the sea urchin egg cortex, J. Cell Biol. 83 (1979) 241248.

[10] S. Yonemura, I. Mabuchi, Wave of cortical actin polymerization

in the sea urchin egg, Cell Motil. Cytoskeleton 7 (1987) 4653.

[11] D. Allemand, B. Ciapa, G. De Renzis, Effect of cytochalasin B on

the development of membrane transports in sea urchin eggs after

fertilization, Dev. Growth Differ. 29 (1987) 3333240.

[12] G.K. Wong, P.G. Allen, D.A. Begg, Dynamics of filamentous

actin organization in the sea urchin egg cortex during early

cleavage divisions: Implications for the mechanism of cytokinesis,

Cell Motil. Cytoskeleton 36 (1997) 3042.

[13] S. Narumiya, T. Ishizaki, N. Watanabe, Rho effectors and

reorganization of actin cytoskeleton, FEBS Lett. 410 (1997) 6872.

[14] D.J.G. Mackey, A. Hall, Rho GTPases, J. Biol. Chem. 273 (1998)

2068520688.[15] M. Symons, J. Settleman, Rho family GTPases: more than simple

switches, Trends Cell Biol. 10 (2000) 415419.

[16] Y. Takai, T. Sasaki, T. Matozaki, Small GTP-binding proteins,

Physiol. Rev. 81 (2001) 153208.

[17] U. Braun, B. Habermann, I. Just, K. Aktories, J. Vandeker-

ckhove, Purification of the 22 kDa protein substrate of botulinum

ADP-ribosyl transferase C3 from porcine brain cytosol and its

characterization as a GTP-binding protein highly homologous to

the rho gene product, FEBS Lett. 243 (1989) 7076.

[18] E.J. Rubin, D.M. Gill, P. Boquet, M.R. Popoff, Functional

modification of a 21-kilodalton G protein when ADP-ribosylated

by exoenzyme C3 ofClostridium botulinum, Mol.Cell. Biol.8 (1988)

418426.

[19] I. Just, F. Hofmann, H. Genth, R. Gerhard, Bacterial protein

toxins inhibiting low-molecular-mass GTP-binding proteins, Int.J. Med. Microbiol. 291 (2001) 11871195.

[20] J.T. Barbieri, M.J. Riese, K. Aktories, Bacterial toxins that

modify the actin cytoskeleton, Annu. Rev. Cell Dev. Biol. 18

(2002) 315344.

[21] Y. Nishimura, K. Nakano, I. Mabuchi, Localization of Rho

GTPase in sea urchin eggs, FEBS Lett. 441 (1998) 121126.

[22] I. Mabuchi, Y. Hamaguchi, N. Fujimoto, M. Morii, M. Mishima,

S. Narumiya, A rho-like protein is involved in the organisation of

the contractile ring in dividing sand dollar eggs, Zygote 1 (1993)

325331.

[23] K. Kishi, T. Sasaki, S. Kuroda, T. Itoh, Y. Takai, Regulation of

cytoplasmic division of Xenopus embryo by rho p21 and its

inhibitory GDP/GTP exchange protein (rho GDI), J. Cell Biol.

120 (1993) 11871195.

[24] P. Cueellar-Mata, G. Martnez-Cadena, J. Loopez-Godnez, A.

Obregoon, J. Garca-Soto, The GTP-binding protein RhoA local-

izes to the cortical granules ofStrogylocentrotus purpuratus sea

urchin egg and is secreted during fertilization, Eur. J. Cell Biol. 79

(2000) 8191.

[25] G.M. Wessel, S.D. Conner, L. Berg, Cortical granule translo-

cation is microfilament mediated and linked to meiotic matu-

ration in the sea urchin oocyte, Development 129 (2002)

43154325.

[26] K. Kaibuchi, S. Kuroda, M. Amano, Regulation of the cytoskel-

eton and cell adhesion by the Rho family GTPases in mammalian

cells, Annu. Rev. Biochem. 68 (1999) 459486.

[27] G. Schatten, R. Bestor, R. Balczon, J. Henson, H. Chatten,

Intracellular pH shift leads to microtubule assembly and micro-

tubule-mediated motility during sea urchin fertilization: correla-

tions between elevated intracellular pH and microtubule activity

and depressed and microtubule disassembly, Eur. J. Cell Biol. 36

(1985) 116127.

[28] D. Epel, Protein synthesis in sea urchin eggs: a late response to

fertilization, Proc. Natl. Acad. Sci. USA 57 (1967) 899906.

[29] K. Kurashima, S. DSouza, K. Szaaszi, R. Ramjeesingh, J.

Orlowski, S. Grinstein, The apical Na/H exchanger isoform is

regulated by the actin cytoskeleton, J. Biol. Chem. 274 (1999)

2984329949.

[30] T. Tominaga, T. Ishizaki, S. Narumiya, D.L. Barber, p160ROCKmediates RhoA activation of NaH exchange, EMBO J. 17 (1998)

47124722.

[31] K. Szaaszi, K. Kurashima, S. DSouza, A. Kapus, A. Paulsen, K.

Kaibuchi, S. Grinstein, J. Orlowski, RhoA and Rho kinase

regulate the epithelial Na/H exchanger NH3, J. Biol. Chem. 275

(2000) 2859928606.

[32] J. Settleman, Rac n Rho: the music that shapes a developing

embryo, Dev. Cell 1 (2001) 321331.

[33] G.D. Moore, T. Ayabe, P.E. Visconti, R.M. Schultz, G. Kopf,

Roles of heterotrimeric and monomeric G proteins in sperm-

induced activation of mouse eggs, Development 120 (1994) 3313

3323.

[34] S. Toratani, T. Katada, H. Yosawa, Botulinum ADP-ribosyl-

transferase C3 induces elevation of the vitelline coat of Ascidian

eggs, Biochem. Biophys. Res. Commun. 193 (1993) 13111317.


A Rho GTPase Controls the Rate of Protein Synthesis

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