molecular cloning and characterization of yeast rho gdp

0 1994 by The American Society for Biochemistry and Molecular Biology, Inc. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 269, No. 31, Issue of August 5, pp. 19713-19718, 1994

Prmted in U.S.A.

Molecular Cloning and Characterization of Yeast rho GDP Dissociation Inhibitor*

(Received for publication, March 21, 1994, and in revised form, May 17, 1994)

Tadayuki MasudaSO, Kazuma TanakaSn, Hidetaro NonakaSn, Wataru YamochiSII, Akio MaedaS**, and Yoshimi TakaiSnSSOO From the Wepartment of Biochemistry, Kobe University School of Medicine, Kobe 650, the tWepartrnent of Cell Physiology, National Institute for Physiological Sciences, Okazaki 444, and the IDepartment of Molecular Biology and Biochemistry, Osaka University Medical School, Suita 565, Japan

We have previously isolated rho GDP dissociation inhibitor (rho GDI) from bovine brain and characterized it. Bovine rho GDI is a protein of a M, of 23,421 with 204 amino acids. rho GDI inhibits the GDPlGTP exchange reaction of post-translationally lipid-modified small GTP-binding proteins (G proteins) of the rho family, including the rho, rac, and cdc42 subfamilies, and keeps them in the GDP-bound inactive form. In the present study, we first purified rho GDI from the cytosol fraction of the yeast Saccharomyces cerevisiae and isolated its gene. Yeast rho GDI gene had an open reading frame without introns encoding a protein of a M, of 23,138 with 202 amino acids. Yeast rho GDI protein was 36% identical with bovine rho GDI. Yeast rho GDI expressed in Escherichia coli was active not only on yeast rho1 but also on mammalian rho family members which were post-translationally modified. Disruption of rho GDI did not induce apparent phenotypes, whereas overexpression of yeast or bovine rho GDI resulted in the inhibition of cell growth. These results indicate that rho GDI exists and regulates the function of the rho family members in yeast.

The rho family belongs to the small G protein superfamily and consists of the rho, rac, and cdc42 subfamilies (1, 2). The rho subfamily is composed of three highly homologous members, rhoA, -B, and -C. All of these members have a unique C-terminal structure of Cys-A-A-L (A: aliphatic amino acid; L: leucine) which undergoes post-translational modifications including the geranylgeranylation or farnesylation of the cys-

search and for Cancer Research from the Ministry of Education, Sci- * This investigation was supported by grants-in-aid for Scientific Re-

ence, and Culture, Japan (19931, by grants-in-aid for Abnormalities in

Ministry of Health and Welfare, Japan (1993), and by grants from the Hormone Receptor Mechanisms and for Aging and Health from the

Yamanouchi Foundation for Research on Metabolic Disease (1993), from the Research Program on Cell Calcium Signal in the Cardiovascular System (19931, and from Setsuro Fujii Memorial, the Osaka Foundation for Promotion of Fundamental Medical Research (1993). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

to the GenBankmIEMBLData Bank with accession number(s)D31630. The nucleotide sequence(s1 reported in this paper has been submitted

Medicine, Kobe 650, Japan. 5 Present address: Dept. of Physiology, Kobe University School of

University School of Medicine, Kobe 650, Japan. I1 Present address: Dept. of Internal Medicine (1st Division), Kobe

** Present address: Dept. of Internal Medicine (3rd Division), Kobe University School of Medicine, Kobe 650, Japan.

Biology and Biochemistry, Osaka University Medical School, 2-2 $5 To whom correspondence should be addressed: Dept. of Molecular

Yamada-oka, Suita 565, Japan. Tel.: 81-6-879-3410; Fax: 81-6-879-3419.

teine residue followed by proteolytic removal of the A-A-L por- tion and the subsequent carboxyl methylation of the exposed cysteine residue (3, 4). The rho subfamily members are ADP- ribosylated at located in the putative effector domain by Clostridium botulinum exoenzyme C3 (51, and the ADP-ribosy- lation impairs their functions (6-17). By use of this C3 and a dominant active mutant rho&l“, rho has been shown to regu- late various actomyosin-dependent cell functions, such as cell morphology of various fibroblasts (&lo), vascular smooth mus- cle contraction (11), platelet aggregation (121, cell motility of Swiss 3T3 cells and 308R keratinocytes (13, 141, cytokinesis of Xenopus oocytes ( X ) , lymphocyte toxicity (X), and lymphocyte aggregation (17).

rho has two interconvertible forms: GDP-bound inactive and GTP-bound active forms. The GDP-bound form is converted to the GTP-bound form by the GDP/GTP exchange reaction which is regulated by GEP.’ The GTP-bound form is converted to the GDP-bound form by the GTPase reaction which is regulated by GAP. Three GEPs, smg GDS (18, 191, dbl(20,21), and rho GDI (22, 231, and two GAPS, rho GAP (24-26) and p190 (27) asso- ciated with ras GAP, have thus far been identified for the rho family members. Of these three GEPs, smg GDS and dbl acti- vate rho, whereas rho GDI inhibits the activation. Moreover, smg GDS and dbl do not exert their full actions in the presence of rho GDI (28,29). By use of this unique property of rho GDI, we have clarified the functions of its substrate small G proteins: microinjection of rho GDI changes morphology of Swiss 3T3 cells, and the comicroinjection with the GTPyS-bound form of rhoA prevents this rho GDI action (10); microinjection of rho GDI inhibits the serum-induced motility of Swiss 3T3 cells, the cytokinesis of Xenopus oocytes, the hepatocyte growth factor- induced motility of 308R keratinocytes, and the hepatocyte growth factor-induced membrane ruffling of KB cells, and the comicroinjection with the GTPyS-bound form of rhoA prevents these rho GDI actions (13-15, 30); and microinjection of rho GDI inhibits the insulin-induced membrane ruffling of KB cells, and the comicroinjection with the GTPyS-bound form of racl prevents this rho GDI action (30).

rho GDI has originally been purified from bovine brain cytosol as a protein with a M, of about 27,000 on SDS-PAGE (22). The rho GDI cDNA has been cloned from a bovine brain cDNA library and its primary structure has been determined (23). Bovine rho GDI is a protein of a calculated M , of 23,421 with

The abbreviations used are: GEP, GDP/GTP exchange protein; GAP, GTPase-activating protein; GDS, GDP dissociation stimulator; GST, glutathione S-transferase; DTT, dithiothreitol;APMSF, (p-amidinophen- y1)methanesulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis; MES, 4-morpholineethanesulfonic acid; GTPyS, guanosine 5’-(3-0- thi0)triphosphate.

19713

19714 Yeast rho GDI

204 amino acids. Northern and Western blot analyses indicate that rho GDI is expressed in most rat tissues. Kinetically, rho GDI interacts with the GDP-bound form of its substrate small G proteins more preferentially than with the GTP-bound form and forms a stable ternary complex of GDP-rho-rho GDI (22, 31). Moreover, rho GDI interacts with the post-translationally modified form but does not interact with the unmodified form (32). Recently, a rho GDI homolog named LyD4 GDI with 78% amino acid identity with bovine rho GDI has been cloned from a human hematopoietic cell cDNA library (33, 34). This LyD4 GDI is specifically expressed in hematopoietic cell types.

In the yeast Saccharomyces cereuisiae, the rho family members including RHOl, -2, -3, and -4, and CDC42 have been isolated (35-37). Of these members, RHOl and CDC42 have been shown to be essential genes (35, 37). CDC42 has been shown to initiate budding site assembly (37), whereas we have recently shown that RHOl regulates bud growth (38). The RH03 and RH04 have also been shown to be involved in bud growth (39). As to the regulatory proteins for the rho family members, cdc24 has recently been shown to be a stimulatory GEP for cdc42 (401, but the existence of rho GDI in the yeast S. cerevisiae has not been investigated.

To understand the mode of activation of the rho family members in yeast, we have attempted here to isolate and charac- terize rho GDI from S. cerevisiae. In this paper, we have purified yeast rho GDI, determined its genomic structure, and characterized yeast rho GDI in comparison with mammalian rho GDI.

EXPERIMENTAL PROCEDURES Materials and Chemicals-The lipid-modified and -unmodified forms

of rhoA were purified from the membrane and cytosol fractions of Spo- doptera frugiperdu cells, respectively, which were infected with the baculovirus carrying the rhoA cDNA (3). The lipid-modified form of rhol, racl, and Ha-ras were similarly purified from the membrane fraction of the insect cells. The lipid-modified form of rab3A was purified from bovine brain membranes (41). Recombinant bovine rho GDI was purified from overexpressing Escherichia coli as a glutathione S-transferase (GST) fusion protein using a glutathione-Sepharose 4B column as described (28). Achromobacter protease I was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). T7 DNA polymerase (Sequenase Version 2.0) was purchased from U. S. Biochemical Corp.

Growth Conditions for Yeast Cells-For purification of rho GDI, a Saccharomyces cerevisiae strain (20B-12 MATa pep4-3 t rp l ga12) was cultured in YPDAU medium containing 1% Bacto-yeast extract, 2% Bacto-peptone, 2% glucose, 0.04% adenine, and 0.02% uracil with con- tinuous agitation at 30 "C until late logarithmic phase, and cells were harvested and stored at -80 "C. A wild type strain OHNY (MATalMATor ura3lura3 leu2fleu2 his3lhis3 trplltrpl ade2/ade2) was used for genetic study. Yeast transformations, selection of transformants, and tetrad dissections were performed as described previously (42).

Assay for rho GDI Activity-rho GDI activity was assayed by measuring the dissociation of C3HIGDP from rhoA using the Schleicher & Schuell BA-85 nitrocellulose filter as described (22). Briefly, the [3H]GDP-bound form of rhoA was first made by incubating rhoA (2 pmol) in a mixture (24 p1) containing 1 p~ L3H]GDP (7,500 cpdpmol), 20 mM TridHCl at pH 7.5 ,5 mM MgCl,, 10 mM EDTA, 1 mM DTT, and 1 m~ L-a-dimyristoylphosphatidylcholine. The dissociation of l3H1GDP was initiated by addition of the sample to be tested in a mixture (100 pl) containing 0.5 mM GTP, 25 mM TrisMCl at pH 7.5, 8 mM MgCl,, 13 lll~ EDTA, 1 mM DTT, and 0.75 mM L-or-dimyristoylphosphatidylcholine. After incubation for the indicated period of time a t 30 "C, the reaction was stopped by rapid filtration on a nitrocellulose filter.

Preparation of the Cytosol Fraction from E a s t Cells-All manipula- tions were carried out at 0-4 "C. Yeast cells of a strain 20B-12 (40 g wet weight) were suspended in 250 ml of 25 mM TrisMCl at pH 7.5 containing 5 mM EGTA, 0.5 mM EDTA, 1 mM DTT, 0.3 M sorbitol, 10 pdml of leupeptin, 20 pglml of aprotinin, 10 APMSF, and 1 pg/ml of pepstatin A. The cell suspension was homogenized for 5 min using a Bead-Beater (Biospec Products, Bartlesville, OK) with 200 g of glass beads (0.45-mm diameter). The homogenate was centrifuged at 1,700 x g for 10 min. The resulting supernatant was further centrifuged at 100,000 x g for 1 h

twice. The supernatant (180 ml, 800 mg of protein) was used as the cytosol fraction.

Molecular Biological Zkchniques-Standard molecular biological techniques were performed for construction of plasmids, polymerase chain reaction, plaque hybridization, and DNA sequencing (43). For construction of the disruption plasmid of the yeast rho GDI gene (RDZl), the 1,579-base pair SacI-Hind111 fragment containing it was subcloned into pUC18. The 327-base pair HincII fragment corresponding to amino acid positions 29-137 of yeast rho GDI protein (Rdil protein) was then replaced with the 1.8-kilobase pair EamHI-EarnHI fragment containing the HIS3 gene. For construction of overexpression plasmids of yeast and bovine rho GDIs, the 612- and 618-base pair KpnI-KpnI fragments amplified by polymerase chain reaction, respectively, were cloned into the expression vector pKT10-GAL, a URA3- bearing episomal plasmid.

Determination of Protein Concentration-Protein concentrations were determined by the dye-binding method with bovine serum albumin as a standard (44).

RESULTS

Detection of rho GDI Activity in Yeast Cytosol-The cytosol fraction was prepared from a yeast strain 20B-12, and rho GDI activity was assayed by measuring the dissociation of [3H]GDP from bovine rhoA. The rho GDI activity was detected in the yeast cytosol. An aliquot of the cytosol fraction (3.5 ml, 16 mg of protein) was applied to a Mono Q HR5/5 column (0.5 x 5 cm) equilibrated with Buffer A (25 mM Tris/HCl at pH 7.5 containing 0.5 mM EDTA, 1 m~ DTT, and 10 J.IM APMSF). After the column was washed with 8 ml of Buffer A, elution was performed with a 15-ml linear gradient of NaCl(O-O.5 M) in Buffer A. Fractions of 0.5 ml each were collected. When an aliquot of each fraction was assayed for the rho GDI activity, it appeared in Fractions 35-37 as a single peak. The substrate specificity of yeast rho GDI in Fraction 36 was compared with that of bovine rho GDI. Yeast rho GDI was active on mammalian rhoA and racl, but was inactive on mammalian Ha-ras and rab3A. Yeast rho GDI was active on the lipid-modified form of rhoA, but was inactive on the lipid-unmodified form of rhoA. These results indicate that the yeast cytosol contains rho GDI with a substrate specificity similar to that of bovine rho GDI (32, 45).

Purification of Yeast rho GDI-Yeast rho GDI was then highly purified from yeast cytosol by successive Q-Sepharose, Mono S, and TSK-GEL HA-1000 column chromatographies. The cytosol (180 ml, 800 mg of protein) prepared from yeast cells (40 g wet weight) was applied to a HiLoad 26/10 Q-Sepha- rose HP column (2.6 x 10 cm) equilibrated with Buffer A. After the column was washed with 150 ml of the same buffer, elution was performed with a 540-ml linear gradient of NaCl(0-0.5 M) in Buffer A. Fractions of 9 ml each were collected. When an aliquot of each fraction was assayed for the rho GDI activity, it appeared in Fractions 64-66 as a single peak (Fig. M). The active fractions of the peaks of the three same column chromatographies were pooled and dialyzed against Buffer B (20 mM MES/NaOH at pH 5.7 containing 0.5 m~ EDTA, 1 mM DTT, and 10 J.IM APMSF). This sample (50 ml, 40 mg of protein) was applied to a Mono S HR10/10 column (1 x 10 cm) equilibrated with Buffer B. After the column was washed with 31 ml of the same buffer, elution was performed with a 240-ml linear gradient of NaCl(0-0.5 M) in Buffer B. Fractions of 4 ml each were collected. When an aliquot of each fraction was assayed for the rho GDI activity, it appeared in Fractions 48-50 as a single peak (Fig. 1B). The active fractions were pooled and directly applied to a TSK-GEL HA-1000 column (0.75 x 7.5 cm) equilibrated with Buffer C (10 m~ KH,PO,&HPO, at pH 7.5 containing 0.5 mM EDTA, 1 mM DTT, and 10 J.IM APMSF). After the column was washed with 6 ml of Buffer C, elution was performed with a 60-1111 linear gradient of KH,PO,/&HPO, (0.01- 0.2 M) in Buffer C. Fractions of 1 ml each were collected. When an aliquot of each fraction was assayed for the rho GDI activity,

Yeast rho GDI 19715

A' I I I I I 1 I

7 15-

x E

Fraction Number

B " " " " ' 9-

f 8 - X

Fraction Number FIG. 1. Q-Sepharose and Mono S column chromatographies. A,

Q-Sepharose column chromatography. A 50-1.11 aliquot of the indicated fractions was assayed for the t3H1GDP dissociation reaction for 20 min. B, Mono S column chromatography. A 20-1.11 aliquot of the indicated fractions was assayed for the c3H1GDP dissociation reaction for 20 min. 0, rho GDI activity; - - - -, absorbance a t 280 nm; -, NaCl concentration. The results shown are representative of three independent experiments.

it appeared in Fractions 42-46 as a single peak (Fig. 2A). Another aliquot of each fraction was subjected to SDS-PAGE followed by staining with Coomassie Brilliant Blue. Only one protein band with a M, of about 25,000 appeared in parallel with the rho GDI activity on SDS-PAGE (Fig. 2 B ) . Judging from the parallelism of this protein staining pattern and the rho GDI activity on the TSK-GEL HA-1000 column chromatography, a protein band with a M, 25,000 was most likely to be a protein corresponding to the rho GDI activity.

Amino Acid Sequencing of Yeast rho GDI-Since a most possible protein molecule for yeast rho GDI was identified on SDS- PAGE, this protein was accumulated to determine the partial amino acid sequences. For this purpose, rho GDI was purified in a manner slightly different from the method described above: the cytosol was first successively subjected to the HiLoad 26/10 Q-Sepharose HP column (2.6 x 10 cm) and Mono S HR10/10 column (1 x 10 cm) chromatographies under the conditions described in Fig. 1, but the active fractions of the Mono S column chromatography were pooled and applied to a AP-802 C, column (0.6 x 10 cm) which was equilibrated with 0.1% trifluoroacetic acid. Elution was performed with a 40-ml linear gradient of acetonitrile containing 0.08% trifluoroacetic acid and 30% 2-propanol (&loo%), and polypeptide peaks were monitored at 215 nm. When an aliquot of each major peak was subjected to SDS-PAGE followed by protein staining, a protein band with a M, of about 25,000 corresponding to rho GDI appeared in the second peak with a retention time of 26-27 min. The peak mainly contained this band with one minor protein on SDS-PAGE. This sample was accumulated from the three C, column chromatographies.

The pooled sample (25 pg of protein) was then digested with Achromobacter protease I. The digested sample was applied to a Capcell Pak SG 120 C,, column (0.46 x 25 cm) which was equilibrated with 0.06% trifluoroacetic acid. Elution was performed with a gradient of Buffer D (0.052% trifluoroacetic acid

0 €0

0.03

0.02 3 n a

0.01

4

B Fraction Number

66K- 45K - S K -

20.1 K -

30 39 40 41 42 43 44 45 46 47 48 49

Fraction Number FIG. 2. TSK-GEL HA-lo00 column chromatography. Aliquots (5

and 40 111) of the indicated fractions were used for the rho GDI assay and SDS-PAGE (13% polyacrylamide gel), respectively. A, elution profile of the rho GDI activity. The [3H1GDP dissociation reaction was performed for 5 min. -, KH,PO,&HPO, concentration. Other symbols used here are the same as those used in Fig. 1. B , protein staining patterns visualized with Coomassie Brilliant Blue. The protein markers used were bovine serum albumin (M, = 66,000), ovalbumin (M, = 45,000), carbonic anhydrase (M, = 29,000), and trypsin inhibitor (M, = 20, 100). The arrowhead shows the position of yeast rho GDI.

and 80% acetonitrile) (0-60 min; 2.0-37.5% of Buffer D, 60-90 min; 37.5-75.0% of Buffer D, 90-105 min; 75-98.0% of Buffer D), and peptide peaks were monitored at 215 nm. At least nine major peptide peaks appeared, and six of them were subjected to amino acid sequencing using an automated gas-phase se- quencer (Applied Biosystems, Model 477 A). Their amino acid sequences were: number 1, VWQK, number 2, TVDEYK, number 3, NLDAEDESLA; number 4, IDDHLGSYAPNTK, number 5, VQHEIITGLRYVQYIK, and number 6, PFYEVELPE.

Molecular Cloning of East rho GDI-On the basis of the amino acid sequences, we designed the 5' primer 5"GGCCG- GATCCAA(T/C)TT(G/A)GA(T/C)GC(G/A/T/C)GA(G/A)GA(T/ C)GA(G/A)TC-3' and 3' primer 5'-GGCCGGATCCAC(G/ A)TA(T/C)CT(T/C)AA(G/APT/C)CC (G/A/T/C)GT(G/A/T)AT(G/AI T)AT(T/C)TC(G/A)TG-3' corresponding to the number 3 and number 5 peptide sequences of the purified yeast rho GDI, respectively, taking into consideration the bias of codon usage in yeast (UU(GA) for leucine, UC(UC) for serine, and AG(GA) for arginine). These amino acid sequences were homologous to the parts of already known amino acid sequence of bovine rho GDI. These oligonucleotides were used in a polymerase chain reaction using 100 ng of yeast genomic DNA as a template. One major product (280 base pair) was cloned into pBluescript and sequenced using T7 DNA polymerase and turned out to contain the nucleotide sequence corresponding to one of the amino acid- sequenced peptides, number 1: VWQK. Furthermore, some homology was detected between the amino acid sequence deduced from this product and the amino acid sequence of bovine rho GDI. This cloned fragment was thus used as a probe for screening a yeast genomic DNA library constructed in hZAP2.

19716 Yeast rho GDl -290 aaaaataaactgcaaattgccttgcaaattttcttcttttattattcttcagatatatca

-230 atcaatacgaggctttttttcctccccaacgctttaatcattctgatcattactccgggt

-170 gaaacggttctctaacgcgatcgtttaaatttcgcgatagatcagggaaacgttataaag

-110 attgagtggaaagaacttccattcttcaggctgcttgtattactttgattctttttctag

-50 ccacacgaataactcgagtattgagcttttttattagagaaaaactgaataatggccgaa

M A E

10 gaaagtaccgactttagtcaattcgaagaggagagaaacaacgatcagtataaagtttca E S T D F S Q F E E E R N N D Q Y K V S

70 gccaaaaaaactgttgacgaatacaaaaatctggatgctgaagacgaatccttggcgaaa

A v E y fJ".L..D..A..E..D..~"-~- A 2 3

130 tggaaagagtctttgggtctaagttcagacgtcttgccattggaatttcccggtgacaaa W K E S L G L S S D V L P L E F P G D K

190 aggaaggtggttgttcaaaaaattcaattactagtcaatacagaaccaaatcctatcaca R K Y V V O K I Q L L V N T E P N P I T

1 250 tttgatcttaccaacgaaaagacaatcaaagaactggcatctaaaagatacaaaattaaa F D L T N E K T I K E L A S K R Y K I K

310 gaaaattctatttataagttgaagattgtgttcaaagttcaacatgaaattatcacaggg E N S I Y K L K I V F K Y O H E I I T G

370 ttgcgatatgtccagtatatcaaaaaagcgggtattgccgttgacaagatagacgatcat L R Y V O Y I K K A G I A V D K J D D H

5

"""."""_ 430 ttgggatcgtatgctcctaataccaagaccaaaccattttatgaggtggaactaccggaa

4

L G S Y A P N T K T K P F Y E V E L P E 6

490 agtgaagcacctagtgggtttttagcaagaggtaactacagtgccgtatcaaaattcatt S E A P S G F L A R G N Y S A V S K F I

550 gatgatgataaaactaaccacttgactttaaattggggggtcgaaattgtcaaaaaataa D D D K T N H L T L N W G V E I V K K *

610 taagttcttgaaatgcacaatgtagatatggcctgtataaacaagagcatacctttatct

670 acatatggattaatacgactttcctgttctactacactttttttttcttaatttcaaatt

730 gaagtataggggtagggctcctgactgattatgacgtcggatcctatgacaaagatggca

790 aataaaaatttaatgtagtatattgatttagctagtagatcactaataaaataaattgcc

FIG. 3. Nucleotide and deduced amino acid sequences of yeast rho GDI. Amino acid sequences of peptides from purified yeast rho GDI

respond to the peak numbers in SG 120 C,, column chromatography. are indicated by solid underlines; numbers below these sequences cor-

Amino acid sequences used for designing polymerase chain reaction primers are indicated by dashed underlines.

One-hundred thousand phages were screened, yielding nine positives, which were all considered to share some part with one another, judging from restriction enzyme mapping. The DNA sequence of yeast rho GDI was determined from these overlapping positive clones. The amino acid sequence deduced from the nucleotide sequence contained the amino acid sequences of the peptides from purified yeast rho GDI (Fig. 3).

The yeast rho GDI gene cloned in this way had no introns and an open reading frame encoding a protein of a calculated M, of 23,138 with 202 amino acids. The amino acid sequence of yeast rho GDI was compared with those of mammalian rho GDIs (Fig. 4). Yeast rho GDI was overall, although sparsely, homologous with human, bovine, and LyD4 GDIs. Yeast rho GDI was 36% identical with both human and bovine rho GDIs. LyD4 GDI was recently cloned as a gene encoding a protein 78% identical with human and bovine rho GDIs (33,34). It was also demonstrated that LyD4 GDI possessed rho GDI activity (34). Yeast rho GDI was also 36% identical with LyD4 GDI.

Expression and Characterization of Recombinant Yeast rho GDZ-A DNA fragment encoding the entire open reading frame of yeast rho GDI was amplified from an isolated rho GDI- containing clone by the polymerase chain reaction, which was performed using the 5' primer 5"GCCGGATCCGGTACCATG- GCCGAAGAAAGTACCGACTTTAG-3' and the 3' primer

CC-3' corresponding to the N and C terminus for yeast rho GDI, respectively. The resulting product was inserted into pGEX-2T cut with BamHI to yield pGEX-2T-yeast rho GDI for the pro- duction of a glutathione S-transferase (GST) fusion protein. Yeast GST-rho GDI was purified using a glutathione-Sepharose 4B column as described (28). The purified yeast GST-rho GDI showed a single protein band with a M, of about 52,000 on SDS- PAGE. Since the M, of GST is about 27;000, the M , of the part corresponding to yeast rho GDI of the fusion protein should be estimated to be 25,000, which was identical with the M, of the purified yeast rho GDI shown in Fig. 2 B .

The kinetic properties of yeast GST-rho GDI were compared with those of bovine GST-rho GDI. Both yeast and bovine GST- rho GDIs were active on yeast rho1 and mammalian rhoA and racl, but were inactive on mammalian rab3Aand Ha-ras (Figs. 5 and 6). Both yeast and bovine GST-rho GDIs were also active on the lipid-modified form of rhoA, but were inactive on the lipid-unmodified form of rhoA (Fig. 7). Yeast GST-rho GDI was much more active on rhoA than bovine GST-rho GDI. These results clearly indicate that yeast rho GDI is a homolog of mammalian rho GDI. Therefore, we named the gene RDZl(& GBZ).

Disruption of Yeast rho GDZ-To clarify the function of yeast rho GDI, its gene (RDZ1) was disrupted. A wild type diploid strain OHNY was transformed with a plasmid in which the DNA sequence of RDZl encoding the amino acid positions 29- 137 was replaced with the HIS3 gene. The resulting transfor- mant, OHNY-TM1, was grown, sporulated, and tetrad-ana- lyzed. All of the 12 dissected asci contained four viable spores, and these asci gave 2 His+:2 His- segregation pattern. To verify that RDZl was properly disrupted in these clones, the region corresponding to the RDZl open reading frame was amplified by polymerase chain reaction. OHNY-TM1 had one rd i l allele disrupted with HIS3 and this rdil::HZS3 allele segregated into two His+ spore clones in the four spore clones from an ascus. Therefore, we concluded that yeast rho GDI was not essential for vegetative growth of yeast cells. The rdil::HZS3 mutant was normal in phenotypes so far tested, including growth at 24 "C or at 37 "C, mating potency with wild type cells of an opposite mating type, sporulation, heat shock sensitivity, and budding pattern. No other positive restricted fragments from yeast genomic DNA than those of the yeast rho GDI gene (RDZ1) were detected by low stringency Southern hybridization with whole RDZl coding region as a probe, suggesting that there may be no gene highly homologous with RDZl.

Suppression of Cell Growth by Overexpression of Yeast or Bovine rho GDZ in Yeast-The effect of overexpression of yeast or bovine rho GDI on the growth of yeast cells was investigated. For this purpose, a DNA fragment of yeast or bovine rho GDI

5"CCGGATCCGGTACCTTATTTTTTGACAATTTCGACCCC-

Yeast rho GDI 19717

FIG. 4. Amino acid sequence ho-

man, and Ly/D4 GDIs. y-GDZ, yeast rho mologies among yeast, bovine, hu-

GDI; h-GDI, human rho GDI; b-GDI, bovine rho GDI; ly-GDZ, Lym4 GDI; vertical lines, identical amino acids between yeast and human rho GDIs; colons, conserved amino acids between yeast and human rho GDIs; boldface letters, identical amino acids between yeast rho GDI and human, bovine, or LyD4 GDI; dashes, gaps im- posed to maximize alignment. Conserva- tive amino acids are grouped as follows: (K, R), ( D , E), (N, Q ) , (S, T) , (L, I , VI.

y-GDI

b-GDI h-GDT

ly-GDI

y-GDT

b-GDI h-GDI

ly-GDI

y-GDI

b-GDI h-GDI

ly-GDI

A B

M A E E S T D F S Q F E E E R N N D Q Y R V S A K K T V D E Y K N L D A E D E S L P L E F P G D K R

MAEQEPTAEQLAQIAAENEEDEHSVNYKPPAQKSIQEIQELDKDDESLRKYKEAL-LGRVAVSAD--PNVP MAEQEPTAEQLAQIAAENEEDEHSVNYKPPAQKSIQEIQELDKDDESLRKYKEAL-LGRVAVSAD--PNVP

MTEKAPEPHVEEDDDDELDSKLNYKPPPPQKSLKELQEMDKDDESLIKYKKTL-LG~PWTD--PKAP

1 1 : : I l I I : : I I I : I l l I I / I : I I : :

KVWQKIQLLVNTEPNPITFDLTNEKTIKELASKRYKIKENSIYKLKIVF~QHEITTGLRWQYIKKAGI

NVWTGLTLVCSSAPGPLELDLTGD--LESFKKQSFVLKEGVEYRIKISFRVNREIVSGMKYIQHTYRKGV I l l : I : : I I : I l l : : : I I I : : I I 1 : I : 1 l : : l : l : l : I :

NVWTRLTLVCSTAPGPLELDLTGD--LESFKKQSFVLKEGVEYRIKISFRVNREIVSGMKYIQHTYRKGV NVWTRLTLVCESAPGPITMDLTGD--LEALKKETIVLKEGSEYRVKIHFKVNRDIVSOLKWPHTYR

AMKIDDHLOSYAPNTKTKPFYEVELPESEAPSGFISAVSKFIDDDKTNHLTLNWGVEIVXK

KMKTDYMVGSYGP--RAEE-YEFLTPVEEAPKGMLARGSYSIKSRFTDDDKTDHLSWEIPNLTIKKDWKD I l l I : I l l I : I I I I l l I I I I I I I I : I I I I I I I I : 1 : I I

KIDKTDmVOSYGP--RAEE-YEFLTPMEEAPKGMLARGSYSIKSRPTDDDKTDHLSWEWNLTIKKDWKD KM~TFMTGSYGP--RPEE-YEFLTPVEEAPKGMLARGTYHNKSFFTDDDKQDHLSWE~LSIKKEWTE

Time ( min ) Time ( min )

r3H1GDP dissociation reaction from each small G protein (2 pmol) was FIG. 5. Substrate specificity of yeast and bovine rho GDIs. The

performed for various indicated periods of time in the presence of recombinant rho GDI (20 pmol). A, yeast GST-rho GDI. B , bovine GST-rho GDI. 0, rhol; A, rhoA, M, rucl; V, rub3A +, Ha-rus; 0, A, 0, V, 0 , in the absence of rho GDI; 0, A, M, V, + , in the presence of rho GDI. The results shown are representative of three independent experiments.

A B

0 20 40 0 20 40 Yeast GST-Rho GDI ( pmol ) Bovine GST-Rho GDI ( pmol )

FIG. 6. Dose-dependent effect of yeast and bovine rho GDIs on the dissociation of ['HIGDP from small G proteins. The r3H1GDP dissociation reaction from each small G protein (2 pmol) was performed for 5 min in the presence of various amounts of rho GDI. A, yeast GST-rho GDI. B , bovine GST-rho GDI. 0, rhol; A, rhoA M, rucl; V, rub3A; + , Ha-rus. The rho GDI activity was expressed as percent inhibition of the dissociation of L3H1GDP. The results shown are representative of three independent experiments.

encoding the entire open reading frame was cloned into the expression vector pKT10-GAL. The resulting plasmid, pKT1OGAL-yeast rho GDI or pKT10-GAL-bovine rho GDI, ex- presses rho GDI under control of the GALl promoter. A wild

A 0 20 40 Yeast GST-Rho GDI ( pmol )

B

- 20 40

Bovine GST-Rho GDI ( pmol )

FIG. 7. Requirement of lipid modifications of rhoA for yeast and bovine rho GDI activity. The r3H1GDP dissociation reaction from each form of rho A (2 pmol) was performed for 5 min in the presence of various amounts of recombinant rho GDI. A, yeast GST-rho GDI. B, bovine GST-rho GDI. 0, lipid-modified rhoA, A, lipid-unmodified rhoA M, lipid-unmodified GST-rhoA. The rho GDI activity was expressed as percent inhibition of the dissociation of r3H1GDP. The results shown are representative of three independent experiments.

type strain OHNY carrying pKT10-GAL-yeast rho GDI or pKT10-GAL-bovine rho GDI did not grow on the medium containing galactose, in which the GALl promoter was induced (Fig. 8). This result suggests that overexpression of these rho GDIs resulted in the inhibition of yeast cell growth.

DISCUSSION We have first purified rho GDI from the cytosol of yeast S.

cerevisiae and determined its partial amino acid sequences. We have then isolated its genomic gene on the basis of the partial amino acid sequences and determined its nucleotide sequence. The yeast rho GDI gene, named here RDZl, has an open reading frame without introns encoding a protein of a calculated M, of 23,138 with 202 amino acids. The yeast rho GDI protein is 36% identical with human, bovine, and LyD4 GDIs. Recombi- nant yeast rho GDI is active not only on yeast rho l but also on mammalian rhoA and racl, whereas recombinant bovine rho GDI is not only active on mammalian rho family members but also on yeast rho l . Recombinant yeast rho GDI is active on only lipid-modified rhoA as recombinant bovine rho GDI is. These structural and biochemical properties of yeast rho GDI clearly indicate that it is a homolog of mammalian rho GDI.

It has been demonstrated that the rho family members regu- late various actomyosin-dependent cell functions. We have shown previously that mammalian rho GDI indeed functions as a negative regulator for the rho family members. Microinjec- tion of rho GDI changes morphology in Swiss 3T3 cells (10) and inhibits the serum-induced motility of Swiss 3T3 cells, the cy-

Yeast rho GDI 19718

vector vector

SGlu-ura

I . . . .

b-GDI

SGal-ura

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We have shown previously that stimulatory GEPs for the rho family members such as smg GDS and dbl do not exert their full actions in the presence of rho GDI in a cell-free system (28, 29). Therefore, it is likely that the rho family members may be overactivated by their stimulatory GEPs in the absence of the yeast rho GDI gene in intact cells. Although disruption of the yeast rho GDI gene does not induce apparent phenotypes, further detailed studies may reveal the subtle change of pheno- type caused by disruption of the yeast rho GDI gene. Southern blot analysis suggests that there may not be an apparent structural homolog of the yeast rho GDI gene (RDZl ) in S. cerevisiae, but there may be a gene functionally redundant with it. Genetic screening to identify such a gene is being undertaken.

Acknowledgments-We thank Y. Matsui (University of Tokyo) for a yeast genomic library and Y. Kaneko (Institute of Fermentation) for a yeast strain.

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molecular cloning and characterization of yeast rho gdp

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