characterization of hetero trim eric g protein complexes in kato et al plant journal(2004)

8/3/2019 Characterization of Hetero Trim Eric G Protein Complexes in Kato Et Al Plant Journal(2004)

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Characterization of heterotrimeric G protein complexes inrice plasma membrane

Chiyuki Kato1, Tomohiro Mizutani2, Hisanori Tamaki2, Hidehiko Kumagai2, Takehiro Kamiya1, Ayumi Hirobe1, Yukiko

Fujisawa1, Hisaharu Kato1 and Yukimoto Iwasaki1,

1

Department of Bioscience, Fukui Prefectural University, 4-1-1 Kenjyojima, Matsuoka-cho, Yoshida-gun, Fukui 910-1195,Japan, and2Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Japan

Received 24 October 2003; revised 13 January 2004; accepted 20 January 2004.For correspondence (fax 81 776 61 6015; e-mail [email protected]).

Summary

Two genes in the rice genome were identied as those encoding the g subunits, g1 and g2, of

heterotrimeric G proteins. Using antibodies against the recombinant proteins for the a, b, g1, and g2

subunits of the G protein complexes, all of the subunits were proven to be localized in the plasma

membrane in rice. Gel ltration of solubilized plasma membrane proteins showed that all of the a

subunits were present in large protein complexes (about 400 kDa) containing the other subunits, b, g1,

and g2, and probably also some other proteins, whereas large amounts of the b and g (g1 and g2) subunits

were freed from the large complexes and took a 60-kDa form. A yeast two-hybrid assay and co-

immunoprecipitation experiments showed that the b subunit interacted tightly with the g1 and g2

subunits, and so the b and g subunits appeared to form dimers in rice cells. Some dimers were

associated with the a subunit, because few b, g1, and g2 subunits were present in the 400-kDa

complexes in a rice mutant, d1, which was lacking in the a subunit. When a constitutively active form of

the a subunit was prepared by the exchange of one amino acid residue and introduced into d1, the

mutagenized subunit was localized in the plasma membrane of the transformants and took a free, and

not the 400-kDa, form.

Keywords: heterotrimeric G protein, rice, subunit composition.

Introduction

Heterotrimeric G proteins are well known as consisting of

three subunit species, a, b, and g subunits, and functioning

as signal mediators in the transduction of numerous exter-

nal signals that interact with receptors on the cell surfaces

in mammals and yeast (Kaziro et al., 1991). In addition,

much evidence has shown that the subunits in mammalian

genomes have multigene families for the subunits (more

than 20 genes for the a subunit, 5 for the b subunit, and 11for the g subunit). The presence of the multiform molecules

of the G protein complexes in mammals has also been

demonstrated (Offermanns, 2000). Only limited informa-

tion is available, however, as to the structure of hetero-

trimeric G proteins in higher plants, although candidates

for cDNAs or genes for the three subunits have been iso-

lated from several higher plant species (Assmann, 2002;

Fujisawa et al., 2001; Ma, 2001). Each of the a- and b-

subunit genes is considered to be a single-copy gene, at

least in rice and Arabidopsis, unlike the genes in mammals.

The a and b subunits encoded by the genes are designated

as rice a subunit RGA1 (Ishikawa et al., 1995) and rice b

subunit RGB1 (Ishikawa et al., 1996), respectively, for rice

and Arabidopsis a subunit, GPA1 (Ma et al., 1990) and

Arabidopsisb subunit, AGB1, respectively, for Arabidopsis

(Weiss et al., 1994). In contrast, two g-subunit genes were

recently isolated from Arabidopsis, and the encoded sub-units were named Arabidopsis g1 subunit, AGG1 (Mason

and Botella, 2000) and Arabidopsis g2 subunit, AGG2

(Mason and Botella, 2001). The present paper also shows

the presence of two g-subunit genes in rice (the encoded

subunits were designated as rice G protein g1 subunit,

RGG1 and g2 subunit, RGG2. In the present work, we have

studied the structure of rice heterotrimeric G protein com-

plexes, giving special attention to the subunitsubunit

interaction and the intracellular localization of the subunits

The Plant Journal (2004) 38, 320331

320 2004 Blackwell Publishing Ltd

doi: 10.1111/j.1365-313X.2004.02046.x


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and complexes. We also prepared a constitutively active

form of the a subunit by exchanging one amino acid

residue and have characterized the active subunit, which

would be helpful for understanding the structural and

functional properties of the G protein complexes.

Reports are being accumulated concerning the functions

of the G proteins in higher plants. The main research

approach used to elucidate the functions was to utilize

mutants of the a- and b-subunit genes. Mutants of the

a-subunit gene isolated from rice (Ashikari et al., 1999;

Fujisawa et al., 1999) and Arabidopsis (Ullah et al., 2001)

weredesignated as d1 and gpa1, respectively. Studies on the

a-subunit mutant of rice d1 have suggested that the G

proteins may be involved in more than three signaling

pathways, including the gibberellin (Ueguchi-Tanaka et al.,

2000), pathogen infection-response (Suharsono et al., 2002),

and light (Iwasaki et al., 2002) signaling pathways. Studies

on gpa1 raised the possibility that the G proteins seem to be

involved in more than seven signaling pathways, including

the auxin, abscisic acid, gibberellin, brassinolide, sugar, andlight signaling pathways (Ullah et al., 2001). The research on

the mutants has suggested that the a subunit may be

involved in many signaling pathways, although each a

subunit takes a single molecular form in rice and Arabidop-

sis. A mutant for the b-subunit gene was isolated from

Arabidopsis and named agb1 (Lease et al., 2001). Studies

of the mutant have suggested that the b subunit seems to

play important roles in the body plan independently of the a

subunit in some developmental stages and in co-ordination

with the a subunit in other developmental stages. Another

approach used in thestudy of the functions of the G proteins

in higher plants involves the use of transgenic plants over-

expressing a constitutively active form of the a subunit

(Okamoto et al., 2001). All research in the elds of both

genetic analysis with mutants and forward genetics with

transgenic plants has revealed that the G protein-mediated

signaling is important to the body plan in theplant kingdom.

We have conducted such an approach and generated rice

transformants carrying the gene for the constitutively active

a subunit described above but not that for the wild type of

the subunit. Studies on the intracellular destination of the

constitutively active subunit formed in the transformants

have given some information on the structural characteris-

tics of rice heterotrimeric G protein complexes, so we pre-

sent the results of such studies in the present paper.

Results

Isolation of two cDNA species for putativeg subunits of G

protein complexes in rice

Two candidate sequences for the g subunit were found in

the DNA database of rice. An open-reading frame, which

encodes a polypeptide consisting of 93 amino acid resi-

dues, was amplied by reverse transcription-polymerase

chain reaction (RT-PCR).The cDNA washighlyhomologous

to that for the AGG1 (Mason and Botella, 2000), and the

encoded protein was designated as the RGG1 (Figure 1).

Another open-reading frame homologous to the coding

regions of the cDNAs for RGG1, AGG1, and AGG2 was

found in a DNA database deposited by the full-length cDNA

sequencing project in National Institute of Agrobiological

Sciences (NIAS). The encoded protein consisted of 150

amino acid residues and contained an extra sequence with

57 amino acid residues at the N-terminal part and a

sequence with 93 amino acid residues homologous to that

of RGG1. In this study, a cDNA for the region encoding the

93-amino-acid sequence,which contained no extra part and

showed homology to theotherg subunits, wasamplied by

PCR, and the encoded protein was designated as RGG2.

This region with the 93 amino acid residues was used for

the production of recombinant protein and the two-hybrid

assay.Three short regions in the sequences of RGG1 and RGG2

exhibit signicanthomology to highly conserved regions in

mammalian g subunits (shadowed sequences in Figure 1).

There are plant-specic insertions or deletions in the

sequences of putative plant g subunits, RGG1, RGG2,

AGG1, and AGG2, as compared with those of mammalian

g subunits. A prenyl-group-binding site (CAAX box) was

observed at the C-terminals of RGG1, AGG1, and AGG2, but

not of RGG2.

Immuno-cross-reactivity between RGG1 and RGG2

Antibodies against g1 subunit with thyoredoxin (Trx), Trx-g1

and g2 subunit with Trx, Trx-g2 were produced in rabbits.

Antibodies against Trx-g1 and Trx-g2 were afnity puried

with lters immobilized with g1 subunit with glutathione

S-transferase (GST), GST-g1 and g2 subunit with GST,

GST-g2, respectively, in order to eliminate antibodies that

recognize the Trx region. Anti-g1 and anti-g2 antibodies

specically recognized recombinant g1s and g2s, respec-

tively (Figure 2). This result shows that the immunoreactivity

ofg1 differs from that ofg2.

Intracellular localization of subunits of G protein

complexes in rice

The antibodies against the a and b subunits of putative

heterotrimeric G proteins in rice were prepared as described

previously by Iwasaki et al. (1997a,b). In the present study,

the intracellular localization of the four subunits, a, b, g1,

and g2, was analyzed using the specic antibodies against

these four subunits. All the subunits were found to be

localized in the plasma membrane fraction (Figure 3), show-

ing that they satisfy one of the necessary conditions for the

Rice heterotrimeric G protein complexes 321

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Figure 1. Comparisons of the deduced amino acid sequences of the two rice g subunits with those of the g subunits from other sources.

RGG1(g1 subunit ofrice),RGG2(g2 subunitof rice), AGG1(g1 subunit ofArabidopsis),AGG2(g2 subunit ofArabidopsis), Gg1 (g1 ofhuman),Gg2 (g2 of human),

Gg3 (g3 ofhuman), Gg4 (g4 ofhuman), Gg5 (g5 ofhuman), Gg7 (g7 ofhuman), Gg8 (g8 ofhuman), Gg9 (g9 ofhuman),Gg10 (g10of human), Gg11 (g11 of human),

Gg12 (g12 of human), and Gg13 (g13 of human). The sequences were aligned to give maximal homology by the introduction of gaps that were indicated by

spaces. Numbers on the right side indicate the numbers of amino acid residues.

Figure 2. Recombinant proteins for rice g1 and g2 subunits and their immuno-cross-reactivity.

(a) Protein staining of puried recombinant proteins. Lane 1, molecular weight markers; lane 2, Trx protein from pET32a; lane 3, GST from pGEX-4T-2; lane 4,

puried Trx-g1; lane 5, puried GST-g1; lane 6, puried Trx-g2; lane 7, puried GST-g2.

(b) Western blot analysis using anti-g1 antibody. Lanes are the same as in (a).

(c) Western blot analysis using anti-g2 antibody. Lanes are the same as in (a).

RGG1 andRGG2 (93 amino acid residues without theextraN-terminal part) cDNAs were subclonedinto pET32a, which contained Trx andhistidine (His) tagwith

22 kDa. The resultant recombinant proteins for RGG1 and RGG2 were designated as Trx-g1 and Trx-g2, respectively. These cDNAs were also subcloned into

pGEX-4T-2,which containedGST tagwith28 kDa.The resultantrecombinant proteins forRGG1and RGG2 were designatedas GST-g1 and GST-g2, respectively.


322 Chiyuki Kato et al.


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subunits of heterotrimeric G proteins. The apparent mole-

cular masses estimated by SDSpolyacrylamide gel electro-

phoresis (PAGE) for a, b, g1, and g2 were 45, 38, 10, and

18 kDa, respectively. The mass of the g2 subunit was larger

than that of theg1 subunit, as expected from their predicted

amino acid sequences shown in Figure 1.

Solubilization of G protein complexes from plasma

membrane in rice

Plasma membrane proteins were solubilized with cholate,

according to the purication protocol of mammalian het-

erotrimeric G proteins, and the solubilized proteins werefractionated by a Superdex 200 PC3.2/30 column (protein

distribution after gelltration is shown in Figure 4a). Eluted

fractions from numbers 1 to 20 were then subjected to SDS

PAGE and Western blots using specic antibodies against

the G-protein subunits (Figure 4b). Almost all the a sub-

units, together with the b, g1, and g2 subunits, were eluted

in a broad fraction between the eluted positions of two

calibration proteins with molecular masses of 669 and

232 kDa, and thus almost all of the a subunits seemed to

associate with the other subunits to form large complexes,

which were named 400-kDa complexes. As the apparent

molecular mass of the abg trimer should be 100 kDa, rice

heterotrimeric G proteins would not be simple hetero-

trimers such as abg1 and abg2 and may be much larger

complexes with other proteins (other proteins were indi-

cated as `n' in Figure 4b,c). Most of the b, g1, and g2

subunits were present in fractions with a molecular mass

of 60 kDa, showing that large amount of bg1 and bg2

dimers (associated without the a subunit) were present

in rice plasma membrane.

The plasma membrane fraction was incubated with

100 mM GTPgS for 30 min, and the proteins in the fractionwere solubilized with cholate and fractionated with a

Superdex 200 PC3.2/30 column in the presence of

10 mM GTPgS. The distribution of proteins after gel

ltration was the same as that in the prole shown in

Figure 4a. In this case, the a subunit was present in a

fraction with a molecular mass of 60 kDa and not in the

fraction of the 400-kDa complexes (Figure 4c). Thus, the a

subunit containing GTPgS, the active form of the a subunit,

appears to be in a free form. Almost all of the b and g

Figure 3. Intracellular localization of the subunits of the G protein complexes in normal cultivar rice.

(a) Protein staining for the subcellular fractions.

(b) Western blot using anti-a antibody.

(c) Western blot using anti-b antibody.

(d) Western blot using anti-g1 antibody.

(e) Western blot using anti-g2 antibody.

Proteins in Soluble, MT, MS, and PM from normal cultivar were separated by SDSPAGE.




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subunits were present in the fraction with a molecular mass

of 60 kDa. The results suggest that the addition of GTPgS

caused the dissociation of the 400-kDa complexes into the

active form of the a subunit and the bg1 and bg2 dimers.

Yeast two-hybrid assay among subunits of G protein

complexes in rice

In order to analyze the interaction among the four subunits,

a, b, g1, and g2, a yeast two-hybrid analysis was conducted

(Table 1). The four cDNAs for RGA1 (a subunit), Q223L

(constitutively active form of the a subunit), RGG1 (g1

subunit), and RGG2 (g2 subunit) were fused with the activa-

tion domain of GAL4 in pACT2 plasmid, and the cDNA for

RGB1 (b subunit) was fused with the binding domain of

GAL4 in pAS2-1 plasmid. The a subunit was observed to

interact weekly with the b subunit, which supports the gel

ltrationdata showingthat thea subunitassociates with the

bg1 and bg2 subunits to form the 400-kDa complexes in the

rice plasma membrane. A constitutively activeform of thea

subunit (Q223L) did not interact with the b subunit, accord-

ing to the general consideration that active forms of the a

subunit are unable to associate with the b subunit. This

resultalso coincides with theabove resultthat thea subunit

was released from the 400-kDa complexes in the presenceof GTPgS (Figure 4c). The g1 and g2 subunits interacted

tightly with theb subunits, which coincides with the nding

that the bg1 and bg2 dimers always co-fractionated in gel

ltration (Figure 4).

In vitro co-immunoprecipitation among subunits of G

protein complexes in rice

The ve cDNAs for HA-RGA1 (a subunit with HA tag), HA-

Q223L (a constitutively active form of the a subunit with

HA tag), myc-RGB1 (b subunit with myc tag), HA-RGG1

(g1 subunit with HA tag), and HA-RGG2 (g2 subunit with

HA tag) were in vitro transcribed, and the resultant

mRNAs were in vitro translated. Immunoanalysis of the

translation products showed that the a subunit and its

constitutively active form did not co-immunoprecipitate

with the b subunit (Figure 5a, lanes 13). They also did

not co-immunoprecipitate with the b subunit in the pre-

sence of the g1 and g2 subunits (Figure 5a, lanes 47). Theb

subunit co-immunoprecipitated with theg1and g2 subunits

(Figure 5a, lanes 47), and both the g1 and g2 subunits co-

immunoprecipitated with the b subunit (Figure 5b, lanes

14). The results indicate that the interaction between the b

and g (g1 and g2) subunits is very strong, whereas that

between the a subunit and the others is very weak, anindication that coincides with the results of the yeast

two-hybrid assay.

Accumulation profile of subunits of G protein complexes

in d1, a rice mutant defective in the a-subunit gene

Rice dwarf mutant d1 (DK22), an alleleofd1, has a one-point

mutation at the eighth exon of the a-subunit gene (Fujisawa

et al., 1999); the exchange of G for T (position 598 in RGA1

Figure 4. Gel ltration of solubilized plasma membrane proteins from

normal cultivar rice.

(a) Protein distribution after gel ltration of the solubilized plasma mem-

brane proteins. The plasma membrane proteins were solubilized with

cholate as described in Experimental procedures. After centrifugation, the

solubilizedproteins (0.25 mg) werefractionated by a Superdex 200 PC3.2/30

column. The positions of marker proteins are indicated with arrows.

(b) Fractions from numbers 1 to 20 in (a) were subjected to SDSPAGE and

Western blots using antibodies against the a, b, g1, and g2 subunits.

(c) The plasma membrane fraction was incubated with 100 mM GTPgS for

30 min and then proteins in the fraction were solubilized under the sameconditions as those in (a). The solubilized proteins (0.25 mg) were fractio-

nated by a Superdex 200 PC3.2/30 column with a buffer containing 10 mM

GTPgS. Fractions from numbers 1 to 20 were subjected to SDSPAGE and





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cDNA sequence) had occurred, resulting in the generation

of an ocher mutant. If the mutated a subunit was accumu-

lated in d1, it would consist of 170 amino acid residues.There was, however, no polypeptide immuno-cross-

reacted with anti-a-subunit antibody in any subcellular

fraction (Figure 6a). This result shows that d1 is a null

mutant for the a subunit. The b, g1, and g2 subunits were

present in the plasma membrane fraction from d1 at levels

similar to those for the normal cultivar (Figure 6bd).

The plasma membrane fraction from d1 was solubilized

with cholate, and the solubilized proteins were gel ltrated.

The prole of protein distribution after gel ltration of the

solubilized membrane proteins from d1 was the same as

that from normal cultivar shown in Figure 4 (data notshown). When gel-ltrated fractions were analyzed with

Western blots, almost all of the b, g1, and g2 subunits were

recovered in the fraction with a molecular mass of 60 kDa

(Figure 6e), suggesting that the subunits are present in the

forms ofbg1 and bg2 dimers in d1, as in the normal cultivar.

The results show that the b and g subunits are able to

accumulate in the plasma membrane fraction, even if the

a subunit is missing.

Figure 5. In vitro co-immunoprecipitation among the subunits of the G protein complexes.

(a) Co-immunoprecipitation of thea, b, g1,and g2 subunits. Lanes 19, immunoprecipitates; lanes1012, total translation products. The mRNAsused for in vitro

translation were indicated under the photograph: a, b, g1, g2, and Q; transcripts of the cDNAs for HA-RGA1, myc-RGB1, HA-RGG1, HA-RGG2, and HA-Q223L,

respectively. Antibodies used for immunoprecipitation were shown above the photograph: Anti-HA, antibody against the HA-tag; anti-b, antibody against the b

subunit.

(b) Co-immunoprecipitation of the b, g1, and g2 subunits. Lanes 17, immunoprecipitates; and lanes 8 and 9, total translation products. The mRNAs and

antibodies are described for (a).

Theve cDNAs forHA-RGA1 (a subunit with HA tag), HA-Q223L (constitutively activeform of thea subunit with HA tag), myc-RGB1 (b subunit with myctag),HA-

RGG1(g1 subunit with HAtag),and HA-RGG2 (g2 subunit with HA tag) were in vitrotranscribed andthe resulted mRNAs were in vitrotranslated.The translation

products were subjected to SDSPAGE before or after immnoprecipitation.

Table 1 Two-hybrid assay of the subunits of the rice G protein complexes

Combination

Activation

domain

Binding

domain

b-galactosidase

activity (103 U)

Relative domain

(U mg1 protein)

1 RGA1 RGB1 25.7 2.8

2 Q223L RGB1 4.2 0.45

3 RGG1 RGB1 13200 1400

4 RGG2 RGB1 4280 460

5 RGA1 9.5 1.0

6 Q223L 4.2 0.45

7 RGB1 4.2 0.45

8 RGG1 6.9 0.80

9 RGG2 5.1 0.55

10 9.3 1.0

MATCHMAKER Two-Hybrid System 2 was used in this study. The four cDNAs for RGA1, Q223L, RGG1, and RGG2 were fused with the

activation domain of GAL4 and, RGB1 with the binding domain of GAL4. RGA1, rice a subunit; Q223L, constitutively active form of the a

subunit; RGG1, rice g1 subunit; RGG2, rice g2 subunit; and RGB1, rice b subunit.




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Accumulation profile of subunits of G protein complexes

in rice transformant, QL/d1

Itis known thatthea subunits of mammalian heterotrimeric

G proteins become constitutively active forms through the

exchange of one amino acid residue. We have introduced a

mutation into the RGA1 by site-directed mutagenesis of

RGA1 cDNA on the basis of the information concerning the

mutagenized a subunits in mammals (Kaziro et al., 1991). A

mutagenized RGA1, in which glutamate(Q) wassubstituted

for leucine (L) at position 223 of RGA1, has been demon-

strated to be a constitutively active form of the a subunit;

namely, the recombinant protein for the mutagenized sub-unit had the ability to bind GTP and lost GTPase activity,

and rice transformants introduced with the gene for the

mutagenized subunit into d1 through the Agrobacterium-

mediated transformation exhibited normal like pheno-

types, recovering abnormal phenotypes of d1 such as

dwarsm (unpublished data). The constitutively active a

subunit and the transformants were named Q223L and QL/

d1, respectively. In the present work, we explored the

subcellular localization of Q223L in QL/d1 transformants.

As a result, Q223L was observed to be localized in the

plasma membrane fraction from QL/d1 (Figure 7a). The

protein recognized by anti-a antibody must be Q223L itself

because there is no a subunit in d1. The amount of the

active protein in the plasma membrane fraction from QL/d1

seemed to be about the same as that of the a subunit in the

plasma membrane fraction from the normal cultivar. The b,

g1, and g2 subunits were also detected in the plasma

membrane fraction from QL/d1 as for the normal cultivar

(Figure 7bd).

The prole of protein distribution after gel ltration of

solubilized proteins from the plasma membrane fraction

from QL/d1 was very similar to that for the normal cultivarshown in Figure 4 (data not shown). When gel-ltrated

fractions were analyzed with Western blots, Q223L was

present in a fraction with a molecular mass of 60 kDa

(Figure 7e), and only a small amount of Q223L was present

in the 400-kDa complexes, which suggests that most of the

Q223L is present in a free form. Almost all of the b, g1, and

g2 subunits were present in the fraction with a molecular

mass of 60 kDa (Figure 7e), and only a small amount of the

subunits was detected in the 400-kDa complexes.

Figure 6. Intracellular localization of the subunits of the G protein complexes in d1.

(a) Western blot using anti-a antibody.

(b) Western blot using anti-b antibody.

(c) Western blot using anti-g1 antibody.


(e) The plasma membrane proteins from d1 were solubilized with cholate as described in Experimental procedures. After centrifugation, the solubilized proteins

(0.25 mg) were fractionated by a Superdex 200 PC3.2/30 column. Fractions from numbers 1 to 20 were subjected to SDSPAGE and Western blots using

antibodies against the a, b, g1, and g2 subunits.

Proteins in Soluble, MT, MS, and PM from d1 (DK22) were separated by SDSPAGE. NC, normal cultivar rice; d1, mutant defective in the a-subunit gene.




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Discussion

A number of reports areavailable regarding thefunctions of

heterotrimeric G proteins in higher plants; namely, studies

with mutants lacking in the a or b subunit have revealed

that the G proteins play important roles in the body plans

and the responses to external signals in higher plants. Little

information is available, however, concerning the structure

of the G protein complexes in higher plants. In the present

work, we studied the structural characteristics of rice het-

erotrimeric G protein complexes, such as the intracellular

localization of the complexes and subunits, the interactionamong the subunits, and the biochemical properties of a

constitutively active form of the a subunit.

We rst isolated two cDNA species for the g subunit of the

G protein complexes in rice. Of the subunits (g1 and g2)

encoded by thecDNAs, one, the g1 subunit (RGG1), seems to

be an ortholog to the g subunits in yeast and mammals,

judging from the length of the amino acid sequence and the

presence of a prenyl-group-binding site. The g2 subunit

(RGG2) however, had an extra part with 57 amino acid

residues at the N-terminal and no prenyl-group-binding site.

As RGG2 was localized in the plasma membrane and inter-

acted very strongly with the b subunit, it would also be a

strong candidate for the g subunit. We suppose that RGG2

maybe anchored in theplasmamembrane viasome cysteine

residue (at position 100 because RGG2 contains a cysteine

only at this position), whichmay be modied by lipidsuch as

palmitoyl group. The g subunit of yeast heterotrimeric G

protein (Ste18p) has been shown to be anchored in the

plasma membrane through palmitoylated cysteine residue

at position 106 in addition to farnesylated or geranylgerany-

lated cysteine residue at position 107 in the CAAX box(Manahan et al., 2000). Concerning the extra N-terminal part,

its function should be claried in the future. Large proteins

with sequences homologous to those of the a subunits and

with extra sequences, named XLas (Kehlenbach et al., 1994)

and Arabidopsis thaliana extra large GTP-binding protein,

AtXLG1 (Lee and Assmann, 1999), have been shown to exist

in a mammal and Arabidopsis, but it remains to be investi-

gated whether the proteins function as members of hetero-

trimers and as substitutes for the typical a subunits.

Figure 7. Intracellular localization and gel ltration prole of Q223L from rice transformants, QL/d1.

(a) Western blot using anti-a antibody.

(b) Western blot using anti-b antibody.

(c) Western blot using anti-g1 antibody.


(e) Plasma membrane proteins in rice transformants QL/d1 were solubilized with cholate as described in Experimental procedures. After centrifugation, the

solubilized proteins (0.25 mg) were fractionated with a Superdex 200 PC3.2/30 column. Fractions from numbers 1 to 20 were subjected to SDSPAGE and


Proteins in Soluble, MT, MS, and PM from the rice transformants QL/d1 were separated by SDSPAGE. NC, normal cultivar rice; QL/d1, transformant introduced

with the chimeric gene ProRGA1:QL into d1.




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All the a subunits were found to be associated with the b

and g (g1 and g2) subunits to form large protein complexes

with a molecular mass of 400 kDa in the plasma membrane

in rice. Consequently, rice heterotrimeric G protein com-

plexes may not be simple heterotrimers such as abg1 and

abg2, because the molecular masses of such simple hetero-

trimers would be about 100 kDa when calculated from the

masses of the constituents. Our results suggest that the rice

G protein complexes may contain some other proteins, in

addition to the a, b, and g (g1 and g2) subunits. Some

mammalian G protein complexes have been shown to be

solubilized in large molecules containing G protein-

coupled receptors (GPCRs), such as D2 dopamine, vaso-

pressin, opiate, angiotensin, glucagon, a2-adrenergic,

adenosine, and muscarinic receptors (Poyner, 1990).

Accordingly, there is a possibility that the 400-kDa com-

plexes may contain GPCRs. Very little information is avail-

ablefor higher plant GPCRs. A gene for a protein named the

putative G protein-coupled receptor (GCR)1, a putative

GPCR, was isolated from Arabidopsis (Plakidou-Dymocket al., 1998). Transformants overexpressing the GCR1 gene

abolished seed dormancy and had a shortened owering

time (Colucci et al., 2002). The results show that GCR1

regulates the body plans, suggesting its involvement in

G protein-mediated pathways. It remains to be studied,

however, as to whether GCR1 interacts with the G protein

complexes. Recently, a sphingosine-1-phosphate, a signal

molecule for heterotrimeric G protein-mediated signaling

in mammals, has been shown to regulate the K transport

of guard cells in Arabidopsis(Coursol et al., 2003). Evidence

is needed, however, to identify a sphingosine-1-phosphate

receptor as GPCR. Thus, it would be very signicant to

determine whether or not the proteins other than the a,

b, g1, and g2 subunits in the 400-kDa complexes are GPCRs.

A cupin-domain protein, the Arabidopsis cupin-domain

protein (AtPirin1), was shown to interact with the a subunit

in Arabidopsis (Lapik and Kaufman, 2003), and so AtPrin1

may also be a subunit of 400-kDa complexes.

Large amounts of the bg (g1 and g2) dimers (molecular

masses; about 60 kDa) containing no a subunit were pre-

sent in the plasma membrane in rice. The presence of such

free dimers, as well as the heterotrimers, was found in

mammalian brains (Dingus et al., 2002), but the physiolo-

gical meaning of the situation remains unknown. The free

bg (g1 and g2) dimers in rice may transmit signals to someeffectors rather than doing nothing, because the Arabidop-

sisb subunit has been suggested to transmit signals inde-

pendently of the a subunit in some developmental stages

(Ullah et al., 2003).

The yeast two-hybrid analysis and co-immunoprecipita-

tion experiments showed that the b subunit of the rice G

proteins interacted tightly with theg1 and g2 subunits.The

Arabidopsisb subunit was also shown to interact with the

g1 and g2 subunits by the yeast two-hybrid assay (Mason

and Botella, 2000, 2001). On the other hand, the present

study showed that the a subunit interacted only weakly

with the b subunit in the yeast two-hybrid assay and did

not interact in co-immunoprecipitation experiments. The

lipid modication of the a subunit may be necessary for

interacting with the b subunit, because the a subunit is

known to be myristoylated or palmitoylated in the plasma

membrane of mammals. The results regarding the sub-

unitsubunit interaction may support the proposition that

the a subunit is sometimes associated with the other

unknown subunits (when no signals have been received)

but becomes free from the complexes at other times

(when signals are received), whereas the bg (g1 and g2)

dimers may never be dissociated into the constituent

subunits.

The dissociation of the G protein complexes into the a

subunits and the bg dimers through activation of the com-

plexes by signal reception has been well established in

mammals and yeast. The present work shows that such

events also occur in heterotrimeric G proteins in higherplants. All the a subunits in the normal cultivar of rice were

observed to associate with the b subunit or the other

unknown subunits to form large complexes, the 400-kDa

complexes; it is likely that the a subunit in the complexes is

inactive. In contrast, the a subunit was dissociated from the

complexes in the presence of GTPgS; it is well known that

the a subunit is bound with GTPgS and becomes active. In

addition, when the gene for a constitutively active form of

the a subunit (Q223L) generated by in vitro mutagenesis

was introduced into d1, a rice mutant containing no a

subunit, the active form was present in a free form with

a molecular mass of 60 kDa. The results show that the

inactive form of the a subunit is associated with the b

subunit or the other unknown subunits to form the

400-kDa complexes, and the active forms are free from

the b or the other subunits; namely, the RGA1 is dissociated

from thecomplexes when activatedby signalreception, like

the mammalian and yeast subunits. In addition, all of the

400-kDa complexes, the active forms of the a subunit, and

the bg dimers were localized in the plasma membrane in

rice, showing that these operate in the plasma membrane.

This study shows the presence of four kinds of hetero-

trimeric G protein complexes in rice: the 400-kDa complex

with the abg1 trimer, the 400-kDa complex with the abg2

trimer, thebg1 dimer, and the bg2 dimer. In mammals, bothfree a subunits and bg dimers are known to function as the

regulators of the downstream molecules; they work as

synergists in some cases and as antagonists in other cases

(Clapham and Neer, 1993). Thus, the presence of the four

kinds of G protein complexes would make it possible that

multiple signaling pathways are regulated by very limited

species of the heterotrimeric G proteins in rice plants.

Namely, the four kinds of G protein complexes may sepa-

rately regulate different signaling pathways.




10/12

Experimental procedures

Plant materials

Rice normal cultivar (Oryza sativa L. cv. Nipponbare) and DK22,

were used in this study. DK22, one of d1 alleles, has a one-point

mutation at theeighth exon of thea-subunit gene; the exchange of

G forT (position598 in RGA1 cDNA sequence) hadoccurred,whichresults in the generation of an ocher mutant. The recurrent parent

ofd1 (DK22) was Nipponbare. All rice plants, normal cultivar, d1,

and rice transformants, were grown under 14 h light with cool-

white uorescent light at 30 000 lux at 308C and 10 h dark at 258C

cycles.

Isolation of RNA, reverse transcription, PCR and

sequencing

Total RNA from various tissues was directly extracted by using

RNeasy Plant Mini Kits (Qiagen K. K., Japan). The intactness of

isolated RNA was checked with ethidium bromide staining after

agarose gel electrophoresis. First strand cDNA was synthesized

using Super Script First-Strand Synthesis System for RT-PCR(Invitrogen Japan K. K., Japan). Total RNA (0.5 mg) and oligo-dT

were used as a temperate and primer, respectively, for the rst

strand cDNA synthesis.

In order to isolate RGG1 and RGG2 cDNAs, the primers were

designed on the basis of the database information. The sequence

information of g1 subunit (RGG1) gene was obtained from a

database (http://btn.genomics.org.cn/cgi-bin/blast1.cgi). The se-

quence of cDNA for the g2 subunit (RGG2) was deposited in the

BLAST-enabled database as unknown protein (Accession number

AK060530):

g1-1 primer (5H-CGCACCCGCACCGGATCGAACG-3H),

g 1-4 primer (5HAGCAATCATTGTCTGCCCTCGG-3H),

g 2-b primer (5H-GGCGGCGACCGACGGATGTG-3H),

g 2-d primer (5H-AATCTGCACAAAGCTGGTAG-3H).

The amplied PCR products were subcloned into pBluescriptand sequenced with a THERMO sequence dye terminator cycle

sequencing kit (Amersham Biosciences K. K., Japan) by using a

DNA sequencer Model 377 (Applied Biosystem Ltd., Japan). The

sequence of cDNA for the g1 subunit was deposited in the BLAST

database (Accession number AB120662).

Preparation of recombinant proteins for riceg1 andg2

subunits and antibodies againstg1 andg2 subunits

Rice G protein g1 subunit, RGG1 and g2 subunit, RGG2 cDNAs

were subcloned in pET32a containing Trx and Histidine tags

(Novagen, Merck Ltd., Japan). The resultant clones Trx-RGG1

and Trx-RGG2 were synthesized in Escherichiacoli, and the recom-

binant proteins were designated as Trx-g1 and Trx-g2, respec-tively. These recombinant proteins were puried with Ni2

nitrilotricetic acid metal-afnity chromatography matrices (Qiagen

K. K., Japan). The purication procedures were followed by the

protocols recommended by the manufacture. RGG1 and RGG2

cDNAs were also subcloned in pGEX-4T-2 containing GST tag

(Amersham Biosciences). The resultant clones GST-RGG1 and

GST-RGG2 were synthesized in E. coli and recombinant proteins

were designated as GST-g1 and GST-g2, respectively. These

recombinant proteins were puried with GlutathioneSepharose

(Amersham Biosciences). The procedures for the induction and

purication of recombinant proteins were followed by the proto-

cols recommended by the manufacture.

Antibodies against Trx-g1 and Trx-g2 were produced in rabbits.

The antibodies against Trx-g1 and Trx-g2 were further afnity

puried with ImmobilonTM-P lters (Nihon Millipore Ltd., Japan)

immobilized with GST-g1 and GST-g2, respectively.

Preparation of mitochondrial, microsomal, and plasma

membrane fractions

The procedures for subcellular fractionation were performed as

described previously by Iwasaki et al. (1997a). Briey, the green

leaves of the normal cultivar, d1 or QL/d1 grown under the 14 h

light and 10 h dark at 308C for 7 days were homogenized with four

times volume of the grinding buffer. The homogenate was cen-

trifuged at 10 000 gfor 20 min. The precipitate was designated as

mitochondrial fraction (MT) and the supernatant was further cen-

trifuged at 100 000 g for 60 min. The precipitate and supernatant

were designated as microsomal fraction (MS) and soluble fraction

(Soluble), respectively. Plasma membrane fraction (PM) was

obtained by the aqueous two-phase method from the MS.

Solubilization of plasma membrane proteins

and gel filtration

Allprocedures werecarried out at 48C. Plasma membrane proteins

(500 mg) were incubated with 250 ml of the solubilization buffer

(10 mM TrisHCl, pH 8.0, 10% glycerol, and 1.5% (w/v) cholate-Na)

for 30 min. After centrifugation for 30 min at 80 000 g, the super-

natant was ltrated through 0.45 mm lter (Amicon, Millipore Ltd.,

Japan.). Theltratedfractionwas applied to a gelltration column,

Superdex 200 PC3.2/30, using SMART system with a gel buffer

(50 mM TrisHCl, pH 8.0, 10% glycerol, 100 mM NaCl, and 1%

(w/v) cholate-Na). Gel Filtration LMW and HMW calibration kits

(Amasham) were used for the estimation of apparent molecular

weights. All fractions were subjected to SDSPAGE and Western

blot analysis.

The solubilization buffer containing 100 mM GTPgS and the gelbuffer containing 10 mM GTPgS were used in some experiments.

Yeast two-hybrid assay

The interaction among the ve subunits, a, Q223L, b, g1, and g2,

was studied using MATCHMAKER Two-Hybrid System2 and 3 (BD

Biosciences, Clontech, Japan). The four cDNAs for RGA1 (a sub-

unit), Q223L, RGG1 (g1 subunit), and RGG2 (g2 subunit) were

cloned into pACT2 or pGADT7, which contained the activation

domain of GAL4. The RGB1 cDNA (b subunit) was cloned into

pAS2-1 or pGBKT7, which contained the binding domain of GAL4.

The constructs were introduced into Saccharomyces cerevisiae

strain Y190. All procedures, including the assay ofb-galactosidase

activity, were performed as described in the yeastprotocols' hand-book (BD Biosciences, Clontech).

In vitro co-immunoprecipitation

For in vitro co-immunoprecipitation experiments, the sequences

for T7 promoter, HA and myctags, which werepresent in plasmids

supplied in yeast two-hybrid assay kit, were used. The resultant

clones were called HA-RGA1 (a subunit with HA tag), HA-Q223L

(Q223L with HA tag), HA-RGG1 (g1 subunit with HA tag), HA-RGG2

(g2 subunit with HA tag), and myc-RGB1 (b subunit with myc tag).




11/12

TNT T7 Coupled Wheat Germ Extract System (Promega K. K.,

Japan) was used for in vitro transcription and translation experi-

ments. All RNAs were synthesized by using T7 RNA polymerase

and translated in the presence of35S-methionine and 35S-cysteine

with a wheat germ extract. Labeled proteins were immunopreci-

pitated by following the protocol of MATCHMAKER Co-IP Kit (BD

Biosciences, Clontech) using anti-HA tag antibody and anti-b

subunit antibody. Labeled proteins were electrophoresed on a

12.5% polyacrylamide gel containing 0.1% SDS. The gel was driedand exposed to an X-ray lm at 808C for 24 h.

SDSPAGE and Western blot

Electrophoresis on 12.5 and 15% polyacrylamide gels containing

0.1% SDS, and Western blot were carried out as described earlier

by Iwasaki et al. (1997a).

In vitro mutagenesis ofa-subunit gene (RGA1 gene)

Site-directed mutagenesis was carried out using Mutan-Express

Km kit (Takara Bio Inc., Japan). All procedures were followed by

the protocols of the manufacture. The full-length sequence for the

coding region of RGA1 cDNA was subcloned in pKF19 plasmid forsite-directed mutagenesis and designated as pKF-RGA1. The

Q223L oligonucleotide (5H-CTCATTCCTCAGGCCTCCTAC-3H) was

used in the site-directed mutagenesisexperiment. Oligonucleotide

sequence was indicated in the antisense orientation of RGA1

cDNA. The mutagenized construct was sequenced to conrm

mutagenesis.

Cloning of RGA1 gene and construction of chimeric gene,

ProRGA1:QL

A rice genomic library constructed in the lgt10 vector was a

generous gift of Tanaka (NIAS, Japan). In order to isolate a pro-

moter region of the a-subunit gene, approximately 2 106 recom-

binant plaques were transferred to a nylon membrane (RocheDiagnostics K. K., Japan) and screened by plaque hybridization

with 32P-labeled RGA1 cDNA fragment as a probe. The conditions

forthe plaque hybridizationwere followed by theprotocolsrecom-

mended by the manufactures. A 6.6-kbp fragment, which was

obtained by SalI digestion of a positive plaque, was subcloned

into pBluescript (Stratagene, La Jolla, CA, USA) and sequenced

with a THERMO sequence dye terminator cycle sequencing kit

(Amersham Biosciences) by using a DNA sequencer Model 377

(Applied Biosystems Ltd., Japan). A 2.7-kbp fragment obtained by

digestion of the 6.6-kbp fragment with SalI and KpnI covered from

the promoter region of 1 kbp to the third exon of RGA1 gene and

was designated as ProRGA1 fragment. Part excised with SalI and

KpnI in pKF-Q223L was replaced with ProRGA1 fragment and the

resultant chimeric clone was designated as ProRGA1:QL. Thus, the

region from the third exon to the stop codon in ProRGA1:QL wasderived from RGA1 cDNA, and Q223L mutation was present in the

tenth exon in ProRGA1:QL.

Production of rice transformants

The binary vector pBI101-Hm, expressing b-glucuronidase (GUS)

under control of the cauliower mosaic virus 35S promoter, was

used as the control vector for rice transformation. Part of 35S

promoter and GUS gene in pIG121-Hm was replaced with a chi-

meric gene, ProRGA1:QL. Transgenic rice plants were generated by

using the Agrobacterium-mediated transformation method

described previously by Toki (1997). De-husked d1 seeds were

sterilized and inoculated on the callus-induction plates. After

3 weeks, the proliferated calli derived from the scutella were used

for transformation. Agrobacterium, EHA101 containing the binary

vector was co-cultured with rice d1 calli and transgenic rice plants

were selected in the presence of hygromycin. Transformants

expressing Q223L mRNA were selected by reverse transcription

and PCR using the specic primers for RGA1.

Acknowledgements

We thank Dr TadashiAsahi forhis critical review of this manuscript

and Dr Yoshiyuki Tanaka for the gift of rice genomic library. Part of

the work was carried out at the Biological Resource Research and

Development Center, Fukui Prefectural University. We acknowl-

edge funding from three sources: a Grant-in-aid for Scientic

Research on Priority Areas (no. 15031223), a Grant-in-aid for

Science Research C (no. 13660342) from the Ministry of Education,

Science and Culture, Japan, and a grant from the Ministry of

Agriculture, Forestry and Fisheries of Japan (Rice Genome Project

IP-1002).

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The accession number for the sequences of RGG1 and RGG2 are AB120662 and AK060530, respectively. Accession numbers

for the sequences shown in Figure 1 are as follows: g1 subunit ofArabidopsis, AGG1 (AF283673); g2 subunit ofArabidopsis,

AGG2 (AF347077); g1 subunit of human, Gg1 (S62027); g2 subunit of human, Gg2 (AF493870); g3 subunit of human, Gg3

(NM012202); g4 subunit of human, Gg4 (HSU31382); g5 subunit of human, Gg5 (BT006823); g7 subunit of human, Gg7

(AB010414); g8 subunit of human, Gg8 (NM033258); g9 subunit of human, Gg9 (AF493876); g10 subunit of human, Gg10

(AF493877); g11 subunit of human, Gg11 (NM018841); g12 subunit of human, Gg12 (NM018841); and g13 subunit of human,

Gg13 (NM016541).



characterization of hetero trim eric g protein complexes in kato et al plant journal(2004)

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