YEAST

VOL.

8: 735-741 (1992)

RHO Gene Products, Putative Small GTP-binding Proteins, are Important for Activation of the CALI/CDC43Gene Product, a Protein Geranylgeranyltransferase in Saccharornyces cerevisiae HIROSHI QADOTA, ISAO ISHII, ASAO FUJIYAMA*, YOSHIKAZU OHYAt A N D YASUHIRO ANRAKUS Department of Biology, Faculty of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan *National Institute of Genetics. Yata I I I I , Mishima-shi, Shizuokaken 41 I , Japan

Received 30 December 1991; accepted 3 April 1992

Two multicopy suppressors of the call-I mutation in the yeast Saccharomyces cerevisiae have been isolated and characterized. They are identical to the yeast RHO1 and RHO2 genes, which encode putative small GTP-binding proteins. Multiple copies of either RHO gene suppressed temperature-sensitivegrowth of the call-I mutant but did not suppress the call null mutant. Genetic analysis suggests that overproduction of either RHO gene product acts for activation of the CALI gene product. KEY WORDS

~

CALI/CDC43;Geranylgeranylation; RHOI; R H 0 2 ; putative small GTP-binding proteins; cell cycle.

RAM2 (Finegold et al., 1991; Goodman et al., 1990) is required for both types of prenylation and Post-translational protein prenylation, including encodes the a subunit of both prenyltransferases farnesylation and geranylgeranylation, plays an (Kohl et al., 1991). BET2/ORF2 is essential for important role in helping to anchor many proteins geranlygeranylation of the SEC4 and YPTl gene in the membrane (Glomset et al., 1990). The products having a -Cys-Cys-type carboxyl terminus enzymes, protein farnesyltransferase (Goodman et (Petersen-bjmn et al., 1990; Rossi et al., 1991; Kohl al., 1990; Reiss et al., 1990) and protein geranyl- et al., 1991). geranyltransferase (Ohya et al., 1991a; Seabra et al., The call-1 mutant of Saccharomyces cerevisiae, 1991) have been identified and characterized from which grows well at 37°C in Ca2+-rich medium diverse eukaryotic organisms. Both enzymes have (100 mM-CaC1,) but stops growing in YPD (200 PMbeen purified from rat, and found to form hetero- CaCl,) at 37°C (Ohya et al., 1984), was originally dimers and to share a common a subunit (Seabra et isolated as a Ca2+-dependent mutant (Ohya et al., 1991). Genetic and biochemical studies with al., 1984). Under non-permissive conditions, the yeast cells have shown that several genes are arrested cells have a condensed G2/M nucleus with involved in protein prenylation. DPRl (also called a small bud, showing a typical Cdc phenotype. RAMI; Goodman et al., 1988, 1991; Chen et al., This suggests that geranylgeranylation of unknown 1991; Powers et al., 1986; Schafer et al., 1990) target proteins by the CALl gene product is importand CALI (also called CDC43; Finegold et al., ant for Ca2+-modulated development of the bud 1991; Ohya et al., 1984, 1991a) are essential for (Miyamoto et al., 1991) and for nuclear division farnesylation and geranylgeranylation, respectively, (Ohya et al., 1984). of specifictarget proteins having a -Cxy-X-X-X-type In this study, we isolated and analysed multicopy carboxyl terminus and each encodes the p subunit of suppressors of the call-1 mutation in order to the respective prenyltransferase (Kohl et al., 1991). identify genes required for CALl function. Further tPresent address: Department of Genetics, Stanford University genetic analysis suggests that these multicopy suppressor gene products function for activation of the School of Medicine, Stanford, CA 94305, U.S.A. $Corresponding author. CALl gene product. INTRODUCTION

0749--503X/92/090735-07 $08.50 0 1992 by John Wiley & Sons Ltd

736 MATERIALS AND METHODS

Strains and plasmids Saccharomyces cerevisiae strains used were YPH499 (MATa ade2 his3 leu2 Iys2 trpl ura3) (Sikorski and Hieter, 1989), YOTl59-2D (MATa leu2 call-l), YOTl59-3C (MATa ade2 leu2 trpl ura3 call-l), YOT159-1 IOU4 (MATaIMATa ade21ade2 hisjhis leu21leu2 trplltrpl ura3/ura3 CALllAcall:: URA3) and YOD501L-8B (MATa ade2 his3 leu2 lys2 trpl ura3 Adprl::LEU2) (Ohya et al., 1991a). Yeast media and genetic analyses were as described by Sherman et al. (1986) and Ohya et al. (1984). pY0323, pY0324, pY0325 and pSQ326 are multicopy shuttle vectors containing the HZS3, T R P l , LEU2 and URA3 genes, respectively. These vector plasmids were constructed from integration vectors of pRS plasmids (Sikorski and Hieter, 1989) and the 2-pm plasmid origin of replication of YEpl3. pRS314 (Sikorski and Hieter, 1989) was used as single copy shuttle vector containing the TRPl gene. Genomic DNA bank was constructed in YEpl3 multicopy shuttle vector (Yoshihisa and Anraku, 1989). YEpL-CALI was isolated from this DNA bank. The constructions of YEpT-CALI, YCpT-CALI and YCpT-DPRI were described in Ohyaetal. (1991a). YEpT-RHO1 andYEpT-RHO2 were constructed from pY0324, and YEpU-RHO1 and YEpU-RHO2 were from pSQ326. pY0323 was used for construction of YEpH-RHO1 and YEpHRH02. pKS87 plasmid contains the URA3 gene and the RAS2 gene under control of the galactoseinducible GAL1 promoter. Standard recombinant techniques were carried out according to Sambrook et al. (1989). Nucleotide sequence was determined by the dideoxy chain termination method (Sanger et al., 1977) with the Sequenase system (U.S. Biochemical).

H. QADOTA ET AL.

dodecyl sulfate polyacrylamide gels using the buffer system of Laemmli (1970) and immunoblotted as described by Towbin et al. (1979) with some modifications (Ohya et al., 1991b). The immunoblot was analysed with ljl000 diluted anti-ras monoclonal antibody Y13-259 as primary antibody and 1/5000 diluted alkaline phosphatase-conjugated rabbit anti-rat IgG (Jackson Immunoresearch Laboratory) as second antibody. The rainbow markers (Amersham, Tokyo, Japan) were used as molecular weight standards. RESULTS AND DISCUSSION

Two multicopy suppressors of the call-1 mutation, MSCl and MSC2, were isolated (Figure 1A) by screening a DNA bank cloned in YEpl3 (Yoshihisa and Anraku, 1989) in a call-1 strain (YOT159-2D) and selecting for growth on YPD at 37°C. The resultant genomic 10 kb DNA fragments were subcloned into pY0325. We identified a 1.7 kb fragment of MSCl and a 2.7 kb fragment of MSC2 as minimal essential regions for suppression of the call-1 mutation (Figure 1B). The DNA sequences (data not shown) of these regions revealed that the two multicopy suppressors, MSCl and MSC2, are identical to the yeast RHO1 and RHO2 genes (Madaule et al., 1987), respectively, which encode putative small GTP-binding proteins (Bourne et al., 1990, 1991). We analysed interactions between the RHO and CALl gene products by investigating whether or not multiple copies of RHO genes suppressed the call null mutation. The dipoloid strain YOTl59110U4, which contains the wild-type allele and the C A L l disrupted allele (call::URA3), was transformed with multicopy plasmids containing either RHO gene. These diploid strains were sporulated and subjected to tetrad analysis. Table 1 summarizes the tetrad data. As all viable spores Biochemical assay of Ras2 protein show ura+ phenotype, these data must show that Yeast cells were grown in synthetic medium multiple copies of either RHO gene cannot suppress containing 2% glucose without supplement of the call null mutation and that the call-1 gene appropriate amino acids, harvested, suspended in product itself must be present for multicopy synthetic medium containing 2% galactose and suppression by the RHO genes. CALl and DPRl are both involved in protein 0.1% sucrose, and incubated for 4 h at 23°C for overproduction of Ras2 protein. The cells were prenylation, and their gene products exhibit 32% collected and resuspended at a density of 1 x lo9 identity witheach other. Multiplecopies ofthe CALl cellslml in a buffer containing 50 mM-Tris-HC1, gene weakly suppress the temperature-sensitive 10 mM-EDTA and 1 mM-phenylmethylsulfonyl growth of the Adprl-containing strain in Ca2+-rich fluoride. The cells were then broken by glass beads medium, indicating functional similarity between and boiled in a heat block for 5 min. The resultant CALl and DPRl (Ohya et al., 1991a). We took supernatants were run o n 12.5% (w/v) sodium advantage of this weak suppression to test whether

INTERACTION BETWEEN THE CALl CDC4.j GENE AND THE RHOGENES

737

A

B

Figure I . Two multicopy suppressors ofthe call-l mutation. (A) The strain YOTl59-2D was transformed with plasmids containing the C A L l gene (YEpL-CALI) and the two multicopy suppressor genes (pYECIOI, pYEC104), which were selected from the Y E p l 3 genomic DNA bank. (B) Single-copy and multicopy plasmids were constructed from pRS3 14 and pY0324, respectively. Transformants were streaked on both Y P D plates and Y P D plates containing 100 mM-CaClz. After 3 days' incubation at non-permissive temperature (37°C). colony formation was analysed.

the RHO products act as activators of the CALI product or not. Multiple copies of RHO1 or R H O 2 by themselves have no effect on the growth of the

Adprl mutant. However, multiple copies of the RHO2 gene enhance the extent of multicopy suppression by the CALI gene in Ca2+-rich medium

738

H. QADOTA E T A L .

Table I .

T e t r a d analysis of t h e + / A d / diploid strain containing multiple copies of either R H O gene S p o r e viability

4+:0-

Strain genotype

+ /Ac,ull::U R A 3 +jAc~cdl::U R A 3 [YCpT-CALI] +/A(’t~llU : : R A 3 [YEpT-RHO11 /Ac~ull:: U R A 3 [YEpT-RHO21

+

0 6 0 0

3+:l0 5 0 0

2+:256 6 31 30

1+:38 3 8 6

0+:4-

3 2 2 6

After sporulation. tetrads were dissected on a YPD plate containing 100 mM-CaClz. After 3 days’ incubation at 23 C. colony formation by the spores was analysed.

Y PD YCpT-DPR-

YPD+100 mM CaCI2 pY0324

Figure 2. The A d p l mutation can be suppressed by multiple copies of the C A L l gene in conjugation with multicopy of the RHO/ or RHO2 gene. YOD501L-8B containing the CALI gene on a multicopy plasmid (YEpTC A L I ) was transformed with a multicopy plasmid of either RHO gene (YEpU-RHO1 or YEpU-RHOZ). These transformants were streaked on both YPD plates and Y P D plates containing 100 mM-CaCI,. After 7 days’ incubation at 37”C, colony formation was analysed.

(Figure 2). Although the multicopy suppression of the Adprl mutation by the C A L l geneis not obvious in Ca’+-poor medium (Ohya et al., 1991a), introduction of multiple copies of either RHO gene weakly suppresses Adpr.1 in Ca”-poor medium (Figure 2). These results indicate that functional replacement of D P R l by CALI is dependent on a

dose of R H O genes, and suggest that the R H O gene products act to activate the C A L l product. To confirm this activator function of the R H O gene products, we analysed biochemically the effect of the R H O products on the activity of the CALI product. To measure the C A L l activity, we assayed Ras2 protein processing with and without multiple

739

INTERACTION BETWEEN THE CALlICDc43 GENE AND THE RHOGENES

1

2

3

4

5

6

7

/s

\

f

Figure 3. Western blotting analysis of Ras2 protein processing in Adprl cells with and without multiple copies of the CALI and

RHO genes. In lanes 1 (YPH499 (wild-type)+pKS87), 4 (YOD501L-8B (Adprl)+pKS87), 5 (YODSOlL-8B+pKS87+YEpT-

CALl), 6 (YOD501L-8B +pKS87 + YEpT-CALI +YEpH-RHOI) and 7 (YOD501L-8B +pKS87 +YEpT-CAL1 +YEpHRH02), cell lysate (10 pl) from 1 x lo7cells was loaded on 12.5% (w/v) sodium dodecyl sulfate polyacrylamide gels. In lanes 2 and 3, volumes of 2/5 and l/5of the cell lysate from the wild-type cells, were loaded, respectively. Bands marked s andfcorrespond to the unprocessed and processed forms of the Ras2 protein, respectively.

copies of the CALI and RHO genes in Adrpl cells, in which Ras2 protein is no longer processed by the DPRI product (Goodman et al., 1990; Schafer et al., 1990). Figure 3 shows that the Ras2 protein was detected in two forms by Western blotting analysis. Fujiyama and Tamanoi (1990) reported that a slower-moving band (s-band) and a faster one (f-band) correspond to the unprocessed form and the processed form, respectively. In the wild-type strain (YPH499), we detected the processed form as the major band, while the unprocessed form appeared to be the major band in the Adrpl mutant (YOD501L-8B). Addition of multiple copies of the CALI gene alone had little effect on Ras2 protein processing, since there was almost no change in the ratio of processed to unprocessed forms. Simultaneous expression of multiple copies of the CALI gene and RHO genes leads to a higher ratio of processed to unprocessed forms qualitatively than with the CALI gene only (Figure 3). These data further support the idea that the products of the RHO genes function to activate the CALI gene product. Further genetic analysis has been carried out with a number of call/cdc43 alleles (Y. Ohya, H. Qadota, M. F. Tibbetts, J. R. Pringle, Y. Anraku and D. Botstein, unpublished results). Unlike the result shown in Figure 2, they have found that the cdc432- 43-7 mutations, which are allelic to the call-I mutation, cannot be suppressed by the RHO genes at 37"C, but are weakly suppressed at lower temperature. For example, multiple copies of the RHOI gene suppress the cdc43-5 and cdc43-7 mutations weakly at 30°C, and those of the RHO2 gene suppress the cdc43-4 mutation at 33°C and weakly suppress the cdc43-2, 43-5, 43-6 and 43-7 mutations at 33°C or 30°C. These results show that the multicopy suppression of the call/cdc43

mutations by either RHO gene is the call-1 allele specific. This allele-specific multicopy suppression suggests that the activation of the CALI gene product by the RHO gene products may be caused by protein-protein interaction (Y. Ohya, H. Qadota, M. F. Tibbetts, J. R. Pringle, Y. Anraku and D. Botstein, in preparation). Two yeast RHO genes have been identified as counterparts of the Aplysia rho (ras homologous) gene (Madaule and Axel, 1985; Madaule et al., 1987). A recent paper has reported that the RHO1 product is found primarily in secretory organelles (McCaffrey et al., 1991). The function of the RHO genes is not known yet, but the above results suggest that one of the roles of the RHO genes may be activation of the CALI gene product. Since RHO1 shows only 53% similarity to R H 0 2 , there may be subtle functional differences between them. In fact the RHO1 gene has been reported to be essential for cellular growth in yeast, whereas the RHO2 gene is not (Madaule et al., 1987). In mammalian cells, the rhoA protein is reported to regulate actin polymerization (Paterson et al., 1990). Consistent with this, the CALI gene product (Finegold et al., 1991;Ohya et al., 1991a) has been shown to modulate actin cytoskeleton in the budding process (Adams et al., 1990; Drubin, 1991; Ohya et al., 1991a). These recent data suggest that the RHO gene products in yeast may regulate the formation of the actin cytoskeletal network. Besides RHOI and R H 0 2 , another yeast rho-like gene, CDC42, has been identified and is involved in bud emergence (Johnson and Pringle, 1990). These three rho-like gene products share a sequence motif at the carboxyl-terminal end, Cys-X-X-Leu (Finegold et al., 1991), which is proposed to act as a signal for geranylgeranylation (Reiss et al.,

740

H.QADOTA ET AL.

p21IaS farnesyltransferase, the counterpart of yeast 1990; Seabra et al., 1991). The rhoA and G25K proDPRI/RAMl. Cell66, 327-334. teins, which are mammalian counterparts of the RHOl and CDC42 gene products, respectively, Drubin, D. G. (1991). Development of cell polarity in budding yeast. Cell65,1093-1096. have a geranylgeranyl-modified cysteine residue at Finegold, A. A., Johnson, D. I., Farnsworth, C. C., the carboxyl terminus (Katayama et al., 1991; Gelb, M. H., Judd, S. R., Glomset, J. A. and Tamanoi, Yamane et al., 1991). The three rho-related gene F. (1991). Protein geranylgeranyltransferase of products in yeast are therefore possible substrates Saccharomyces cerevisiae is specific for Cys-Xaa-Xaaof the CALI-encoding geranylgeranyltransferase. Leu motif proteins and requires the CDC43 gene This evidence suggests an alternative possiblity that product but not the DPRl gene product. Proc. Natl. either the RHOl or RHO2 gene functions downAcad. Sci. USA 88,44484452. stream of the CALl gene. Phenotypic analysis of the Fujiyama, A. and Tamanoi, F. (1990). RAS2 protein of Saccharomyces cerevisiae undergoes removal of call-I mutation has shown that the CALIICDC43 methionine at N terminus and removal of three amino gene product is essential for bud growth and nuclear acids at C terminus. J. Biol. Chem. 265,3362-3368. division (Ohya et al., 1984). The CDC42 gene is also indispensable for bud emergence (Adams et al., Glomset, J. A., Gelb, M. H. and Farnsworth, C. C . , (1990). Prenyl proteins in eukaryotic cells: a new type of 1990; Johnson and Pringle, 1990). Thus, the defect membrane anchor. TZBS 15,139-142. of bud growth by the call-1 mutation may be due to Goodman, L. E., Perou, C. M., Fujiyama, A. and loss of the Cdc42p function. In a parallel way, we Tamanoi, F. (1988). Structure and expression of can assume that the defect of nuclear division by the yeast D P R l , a gene essential for the processing and call-I mutation may be caused by loss of the Rho Ip intracellular localization of ras proteins. Yeast 4, 27 1-28 1. function. These are the next important questions remaining to be answered and we are studying Goodman, L. E., Judd, S. R., Farnsworth, C. C., Powers, S., Gelb, M. H., Glomset, J. A. andTamanoi, F. (1990). biochemically whether any of the three rho-like Mutants of Saccharomyces cerevisiae defective in the yeast proteins are indeed geranylgeranylated by the farnesylation of Ras proteins. Proc. Natl. Acad. Sci. CALl gene product.

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