Dhh1p, a Putative RNA Helicase, Associates with the General Transcription Factors Pop2p and Ccr4p from Saccharomyces cerevisiae

Corresponding author: Akira Sakai, Cellular Signaling Group, Mitsubishi Kasei Institute of Life Sciences, 11 Minamiooya, Machida-shi, Tokyo 194, Japan, sakai{at}libra.ls.m-kagaku.co.jp (E-mail).

Communicating editor: M. JOHNSTON

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The POP2 (Caf1) protein in Saccharomyces cerevisiae affects a variety of transcriptional processes and is a component of the Ccr4p complex. We have isolated five multicopy suppressor genes of a pop2 deletion mutation: CCR4, DHH1 (a putative RNA helicase), PKC1, STM1, and MPT5 (multicopy suppressor of pop two). Overexpression of either the CCR4 or DHH1 genes effectively suppressed phenotypes associated with pop2 mutant cells; overexpression of PKC1, STM1, or MPT5 genes produced only partial suppression. Disruption of the CCR4 or DHH1 genes resulted in phenotypes similar to those observed for pop2 cells. In addition, overexpression of the DHH1 gene also suppressed the ccr4 mutation, suggesting a close relationship between the POP2, CCR4, and DHH1 genes. Two-hybrid analysis and coimmunoprecipitation experiments revealed that Pop2p and Dhh1p interact physically, and these and other data suggest that Dhh1p is also a component of the Ccr4p complex. Finally, we investigated the genetic interaction between factors associated with POP2 and the PKC1 pathway. The temperature-sensitive growth defect of dhh1 or mpt5 cells was suppressed by overexpression of PKC1, and the defect of mpk1 cells was suppressed by overexpression of MPT5. These results and phenotypic analysis of double mutants from the POP2 and PKC1 pathways suggested that the POP2 and the PKC1 pathways are independent but have some overlapping functions.

THE POP2 (CAF1) gene is required for glucose derepression of gene expression in Saccharomyces cerevisiae (SAKAI et al. 1992 ; DRAPER et al. 1995 ). The pop2 mutant cells exhibit many defects, including reduced levels of reserve carbohydrates, resistance to glucose derepression, temperature sensitivity for growth, increased PGK1 transcription during stationary phase, and reduced levels of alcohol dehydrogenase II, isocitrate lyase, and invertase (SAKAI et al. 1992 ; DRAPER et al. 1995 ). Pop2p has been shown to be part of the Ccr4p complex (DRAPER et al. 1995 ; LIU et al. 1997 ). Ccr4p, a general transcription factor, is required for the transcription of many genes, including glucose-repressible ADH2, and the pleiotropic nature of defects in CCR4 is similar to that in POP2 (DENIS and MALVAR 1990 ; MALVAR et al. 1992 ; SAKAI et al. 1992 ). Homologs of the POP2 gene have been identified from humans, mice, Caenorhabditis elegans, and Arabidopsis thaliana (DRAPER et al. 1995 ). The high degree of evolutionary conservation and their functional interchangeability suggest that the POP2 gene plays an important functional role in cells.

We have identified several new phenotypes of pop2 mutants. First, pop2 cells are sensitive to staurosporine, a potent inhibitor of protein kinase C that is encoded by PKC1. Second, pop2 cells are also sensitive to caffeine (LIU et al. 1997 ). In addition, the temperature-sensitive phenotype of pop2 is suppressed by the addition of 1 M sorbitol to the medium. These phenotypes are characteristic of mutants involved in the PKC1-MPK1 pathway (THEVELEIN 1994 ), which controls cell wall integrity. Cells carrying pkc1 or mpk1 cannot use glycerol as a sole carbon source (COSTIGAN et al. 1994 ), and this suggests the involvement of the PKC1-MPK1 pathway in the transcription of genes required for glycerol utilization. To understand the functions of Pop2p, we have searched for multicopy suppressor genes of a pop2 deletion mutation. This approach has proved to be quite useful for analyzing many cellular processes such as the SNF1 kinase pathway (ESTRUCH and CARLSON 1993 ; HUBBARD et al. 1994 ) and the PKC1-MPK1 protein kinase pathway (LEE et al. 1993A ; NICKAS and YAFFE 1996 ). Using this approach, we hoped to obtain genes encoding proteins with functional relationships to POP2: negative regulators of an antagonistic pathway or positive regulators of a downstream pathway. We report here the isolation of five multicopy suppressor genes of the pop2 deletion mutation in S. cerevisiae. We also present genetic and biochemical evidence indicating that Pop2p, Ccr4p, and Dhh1p are part of the same complex of proteins. A possible interaction between the POP2 and PKC1-MPK1 pathways is discussed.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Strains and genetic methods:
The strains of S. cerevisiae used in this study are listed in Table 1. Crossing, sporulation, and tetrad analyses were carried out by standard genetic methods (SHERMAN et al. 1986 ). Unless otherwise specified, the permissive and restrictive temperatures were 24° and 36°, respectively. The transformation of yeast was performed by the LiOAc method (ITO et al. 1983 ). Escherichia coli strains HB101 and JM109 were used as hosts for constructing and propagating plasmids. The transformation of E. coli was performed as described (HANAHAN 1983 ). The basic culture medium used for S. cerevisiae was YPD medium containing 1% Bacto-yeast extract, 2% Bacto-peptone, and 2% dextrose (SHERMAN et al. 1986 ). The synthetic medium was CSM medium containing 0.67% yeast nitrogen base without amino acids, 2% dextrose, and amino acids as required (SHERMAN et al. 1986 ). The media were solidified with 2% Bacto-agar for plates. To test the sensitivity of various mutants to staurosporine (Kyowa Medix, Tokyo, Japan), the drug was dissolved in dimethyl sulfoxide and added to YPD media to a final concentration of up to 5 µg/ml (TAMAOKI et al. 1986 ; YOSHIDA et al. 1992 ). Tetrad dissection of heterozygous diploids carrying pkc1 was performed on a YPD plate containing 1 M sorbitol. Luria broth was supplemented with ampicillin for selection of the E. coli transformants as described (MANIATIS et al. 1982 ).

Preparation of DNA and RNA:
Preparation of E. coli DNA, Southern hybridization, and Northern hybridization were performed as described (MANIATIS et al. 1982 ). The preparation of yeast DNAs and Northern analysis were performed as described (SHERMAN et al. 1986 ).

ADH II assay:
Yeast cells were grown in YEP medium supplemented with either 8% glucose or 3% ethanol, and the activity of ADH II was measured as described previously (COOK et al. 1994 ).

Isolation of multicopy suppressors of pop2-4:
A yeast genomic DNA library (in YEp13, LEU2 marker) was transformed to an A880 strain carrying the pop2-4 mutation (SAKAI et al. 1992 ), and transformants were selected on CSM lacking leucine at 24° for 5 days. To isolate suppressors for the temperature-sensitive phenotype of pop2 cells, transformants were replica plated on YPD, and the plates were further incubated at 36°. To isolate suppressors for the staurosporine-sensitive phenotype of pop2 cells, the transformants were replica plated on YPD containing 1 µg/ml staurosporine. Of 25,000 transformants, 85 colonies were selected. The 85 suppressor plasmids were recovered, analyzed by Southern hybridization and restriction mapping, and divided into six classes. The largest class (containing 65 plasmids) was identified as POP2 itself. The remaining five classes were tentatively called MPTs (multicopy suppressor of pop two). The classes of MPT1, MPT2, MPT3, MPT4, and MPT5 contained 13, 3, 2, 1, and 1 plasmids, respectively. To localize the suppressor activities for these plasmids, individual restriction fragments were subcloned into the YEp213 vector and tested for their abilities to grow on YPD plates at the restriction temperature or YPD plates containing staurosporine. These MPT genes were further subcloned into pUC18, and the nucleotide sequences were determined by the Sanger method using a Sequenase Kit (United States Biochemical, Cleveland, OH). Nucleotide sequence analysis revealed that three genes had been identified previously. MPT1 is DHH1, which encodes a putative RNA helicase (STRAHL-BOLSINGER and TANNER 1993 ), MPT2 is identical to CCR4, which is required for the full expression of the glucose-repressible ADH2 gene (DENIS and MALVAR 1990 ; MALVAR et al. 1992 ), and MPT3 turned out to be PKC1, a yeast homolog of mammalian protein kinase C (LEVIN et al. 1990 ). MPT4 is STM1, a guanine quartet–binding protein (FRANTZ and GILBERT 1995 ). MPT5 is identical to HTR1. The name HTR1 had been used for a different gene (OZCAN et al. 1993 ); we refer to this gene as MPT5.

Disruption of DHH1, CCR4, PKC1, MPT4, and MPT5:
The dhh1::URA3, ccr4::HIS3, pkc1::HIS3, stm1::ADE8, and mpt5::HIS3 alleles were constructed by the standard method and transformed into diploid (D40) cells to replace the chromosomal loci by the one-step gene disruption method (ROTHSTEIN 1983 ).

Construction of dhh1::URA3:
A 2.5-kbp DNA fragment (PmaCI-ApaLI) bearing the DHH1 gene (ORF: 1518 bp) was filled in with Klenow enzyme and subcloned into the SmaI site of pBluescript II. The resulting plasmid DNA was digested with BstEII and Bgl II. This step deleted 115 bp within the coding region (+438 to + 552). After the ends were filled in with Klenow enzyme, a 1.2-kbp URA3 fragment, isolated by digestion of YEp24 plasmid DNA with HindIII and filled in, was inserted. The resulting plasmid was digested with PvuI and KpnI and used for transformation. After heterozygous disruption was confirmed by Southern blotting, the transformants were subjected to sporulation followed by micromanipulation to generate haploid cells carrying the dhh1 deletion mutation.

Construction of ccr4::HIS3:
The ccr4::HIS3 allele was introduced in our background and L40 strain using the TM1 plasmid as described (MALVAR et al. 1992 ).

Construction of pkc1::HIS3:
A 4.3-kbp SphI-SphI fragment bearing the PKC1 gene was recovered from the original MPT3 plasmid. Disruption of the PKC1 gene was performed by the replacement of a 0.6-kbp BamHI fragment of the PKC1 gene with the HIS3 gene from pJJ216 as described (YOSHIDA et al. 1992 ).

Construction of stm1::ADE8:
A 1600-bp DNA fragment (PmaCI-ClaI) bearing the MPT4 gene (ORF: 819 bp) was filled in with Klenow enzyme and subcloned into the SmaI site of the pUC18 plasmid whose PstI site was disrupted. The resulting plasmid was digested with XbaI and HpaI to remove 33 bp within the MPT4 gene (+283 to +316) and a 1152 bp ADE8 DNA whose ends were XbaI and SmaI. The resulting plasmid was digested with EcoRV and Afl II, and was used for transformation.

Construction of mpt5::HIS3:
A 5-kbp AatI-AatI DNA fragment bearing the MPT5 gene was isolated from the original MPT5 plasmid DNA, whose ends were changed to Sal I by linker ligation, and subcloned into the Sal I sited of pUC18. The plasmid DNA was digested with NspV to remove -420 to +3700 of the MPT5 DNA. After the ends were filled in with Klenow enzyme, a 1.8-kbp HIS3 DNA whose ends were filled in with Klenow enzyme was inserted. The resulting plasmid was digested with EcoRI and EcoRV, and was used for transformation.

All disruptions were introduced into D40 cells, and the disruptions were confirmed by Southern blot analysis. Haploid cells carrying the disruption were recovered by standard microdissection.

Two-hybrid analysis:
Plasmid pBTM116 (kindly provided by KATSUNORI TANAKA, Shimane University) was used to construct LexA-Pop2p. BamHI sites were introduced into the DNA encoding the POP2 protein by PCR, and the resulting DNA fragment was subcloned into the BamHI site of pBMT116 in-frame to LexA. Plasmid pGADGH was purchased from Clontech (Palo Alto, CA) and was used to construct Gal4 activation domain fusion plasmids (Gal4AD-Ccr4 and Gal4AD-Dhh1). Yeast strain L40 was used as the two-hybrid host (Table 1). The activity of ß-galacto-sidase was determined as described (MILLER 1972 ). The LexA-Vpu plasmid contains the hydrophilic segment of Vpu (residues 33–81) fused to full-length LexA (CHIANG et al. 1996 ). B42-Pop2p contains residues 149–441 of Pop2p fused in frame to the E. coli–derived B42 transcriptional activator (ZERVOS et al. 1993 ). This segment of Pop2p interacts with Ccr4p in the two-hybrid assay and coimmunoprecipitates with Ccr4p (DRAPER et al. 1995 ; data not shown). B42-Sip1p contains residues 243–500 of Sip1p (YANG et al. 1992 ) fused to B42. LexA-Dhh1 was constructed by removing the complete DHH1 sequence from the Gal4AD-Dhh1AD fusion using BamHI and Sal I and cloning into the BamHI and Sal I sites of LexA-202-1 (COOK et al. 1994 ).

Immunoprecipitation:
Preparation of protein extracts and immunoprecipitation were performed as described (DRAPER et al. 1995 ). The polyclonal antibody against the Gal4 activation domain was a kind gift from KOUICHI ISHIGURO, Mitsubishi Kasei Institute of Life Sciences. The Ccr4p antibody was described previously (DRAPER et al. 1995 ). Antibodies against the HA1 epitope were commercially obtained. The sequence data presented in this paper have been submitted to the GenBank Data Libraries under the following accession numbers: MPT4, D26183; MPT5, D26184.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Isolation of multicopy suppressors of pop2:
The temperature-sensitive phenotype of pop2 mutants suppressed by the addition of 1 M sorbitol in the medium (Figure 1A). At the restrictive temperature, the pop2 cells become enlarged and swollen, showing a cell lysis phenotype (data not shown). Furthermore, the pop2 cells are also sensitive to staurosporine (1 µg/ml; Figure 1B), a potent inhibitor of the yeast protein kinase C homolog (PKC1; TAMAOKI et al. 1986 ; LEVIN et al. 1990 ; YOSHIDA et al. 1992 ). Finally, the growth of pop2 cells is inhibited by low levels of caffeine (8 mM; Table 2). pop2 cells also exhibited a weak cold-sensitive growth phenotype that is suppressible by 1 M sorbitol (LIU et al. 1997 ). These phenotypes are similar to those caused by mutations in the PKC1 pathway, and they suggest that POP2 plays a role in maintenance of cell wall integrity in addition to its other known phenotypic defects. Since the pop2 mutant affects such varied processes, it was of interest to us to know the multifunctional roles of Pop2p in cells.

View larger version (53K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Newly found phenotypes of pop2 cells. (a) Sorbitol-suppressible, temperature-sensitive growth defect of pop2 deletion cells. (1) A1123 (wild-type), (2) A1152 (stt1-1/pkc1ts), (3) A475 (rgr1-2::URA3), (4) A880 (pop2-4::URA3). Cells were streaked on YPD or YPD+1 M sorbitol plates and were incubated at 24° and 37° for 4 days. (b) Staurosporine sensitivity. A1123 (wild-type) and A880 (pop2-4::URA3) cells were resuspended with YPD media, spread on the YPD plate containing staurosporine, and incubated at 24° for 4 days.

We searched for multicopy suppressors of the temperature- and staurosporine-sensitive growth defects of pop2 mutation (see MATERIALS AND METHODS). We identified five gene products that suppressed the pop2 phenotypes either completely or partially (Table 3). Nucleotide sequence analysis revealed that these genes are DHH1, which encodes a putative RNA helicase (STRAHL-BOLSINGER and TANNER 1993 ), CCR4, which is required for the full expression of the glucose-repressible ADH2 gene (DENIS and MALVAR 1990 ; MALVAR et al. 1992 ), PKC1, a yeast homolog of mammalian protein kinase C (LEVIN et al. 1990 ), STM1, a guanine quartet–binding protein (FRANTZ and GILBERT 1995 ), and MPT5 (KIKUCHI et al. 1994 ; KENNEDY et al. 1997 ). Mpt5p contains eight tandem copies of an ~38 amino acid–repeat, including a strong consensus sequence of 13 amino acids (LxxDxFGxxFLQK). This repeat is also found in YGL023 (CHEN et al. 1991 ) and in the Drosophila pumilio gene (BARKER et al. 1992 ; MACDONALD 1992 ). The biological function of this region remains to be identified.

Overexpression of either CCR4 or DHH1 suppressed all the pop2 phenotypes. In contrast, overexpression of MPT5, PKC1, and STM only suppressed some of these phenotypes (Table 3). These results suggested a close genetic relationship among POP2, CCR4, and DHH1, while the interaction between POP2 and PKC1, STM1, or MPT5 might be relatively weak.

Northern analysis:
It is possible that the overexpression of these genes in pop2 cells suppressed the phenotypic defects if their transcription were being positively regulated by the Pop2. To examine this possibility, we measured the mRNA levels of each suppressor gene in wild-type and pop2 deletion cells. No significant differences in the mRNA amounts were observed between wild-type and pop2 deletion cells for any of the suppressor genes (data not shown).

Genetic interactions among POP2, CCR4, and DHH1:
Disruptions of POP2, CCR4, and DHH1 were constructed (see MATERIALS AND METHODS), and the resulting phenotypes were examined. In addition to the phenotypes previously observed for a CCR4 disruption (MALVAR et al. 1992 ; DRAPER et al. 1995 ; Table 2), we found that in-strain backgrounds other than those of the originally reported, ccr4 cells are also temperature sensitive for growth, which is suppressed by addition of 1 M sorbitol to the medium. Furthermore, the ccr4 cells are sensitive to staurosporine and caffeine. While it has been reported that the disruption of DHH1 does not cause any phenotype (STRAHL-BOLSINGER and TANNER 1993 ), we found that dhh1 cells in our genetic background show a temperature-sensitive cell lysis phenotype that is suppressed by 1 M sorbitol (Table 2). Proteins with RNA helicase activity are often involved in mRNA transport or RNA processing (MARGOSSIAN and BUTOW 1996 ; DALBADIE-MCFARLAND and ABELSON 1990 ). To examine dhh1 effects on mRNA transport, dhh1 cells were shifted to 37° for 1 hr, and mRNA accumulation in the nucleus was measured by in situ hybridization with an oligo dT probe. No significant difference was detected between dhh1 and wild-type cells (data not shown). We also failed to detect pre-mRNA accumulation of the intron-containing CYH2 gene in dhh1 cells (T. TANI, personal communication). These observations suggest that the dhh1 mutation did not affect RNA processing. Cells carrying the dhh1 mutation also showed cold-sensitive growth at 15°, do not grow on a CSMGlycerol plate, and grow slowly on a YPGlycerol plate. Furthermore, dhh1 cells are sensitive to staurosporine and caffeine (Table 2, data not shown). Table 4 also shows that DHH1 is required for the full expression of the ADH2 gene, a phenotype previously observed with ccr4 (DENIS 1984 ) or pop2/caf1–deleted cells (DRAPER et al. 1995 ). These phenotypic analyses confirm a close functional link between POP2, CCR4, and DHH1. In contrast, disruption of PKC1, STM1, and MPT5 displays only some phenotypic similarities to pop2, ccr4, and dhh1 (Table 2) and their mutant phenotypes were consistent with those observed previously (LEVIN and BARTLETT-HEUBSH 1992 ; FRANTZ and GILBERT 1995 ; KIKUCHI et al. 1994 ; CHEN and KURJAN 1997 ). It is noteworthy that temperature-sensitive growth of mpt5 cells was suppressed by 1 M sorbitol.

Multicopy plasmids carrying POP2, CCR4, and DHH1 were introduced into each mutant in different combinations, and the effects on their growth defects were tested. The pop2 mutation was suppressed by CCR4 or DHH1, and the ccr4 mutation was also suppressed by DHH1 (Table 3); however, the growth defects in dhh1 cells were not suppressed by either POP2 or CCR4. To further test the epistasis among these genes, cells carrying double mutations were constructed and the phenotypic additivity was tested. We did not find any additive phenotypes in the double-mutant cells, suggesting that POP2, CCR4, and DHH1 function in the same pathway, and that CCR4 and DHH1 genetically function downstream of POP2.

Dhh1p physically interacts with Pop2p:
The physical association of Dhh1p with Pop2p and Ccr4p was analyzed by two-hybrid analysis and coimmunoprecipitation experiments. The combination of LexA-Pop2 and Gal4AD-Dhh1AD in the two-hybrid system resulted in a significant increase in ß-galactosidase activity beyond that obtained with LexA-Pop2p alone (Figure 2, Table 5). LexA-Dhh1p also was observed to interact specifically with a B42-Pop2 fusion, resulting in 18 U/mg of ß-galactosidase activity as compared to 2.5 U/mg activity for the interaction of LexA-Dhh1 with B42 alone. Figure 2 shows that residues 147–171 of Pop2p are necessary for the interaction. This region is similar to that which was observed to be necessary for the Pop2p and Ccr4p interaction (DRAPER et al. 1995 ). Also, disruption of CCR4 did not abrogate the interaction between LexA-Pop2 and Gal4AD-Dhh1 (Table 5). It should be noted that a ccr4 disruption reduced the transcriptional activity of LexA-Pop2, which is a general effect that ccr4 has on LexA activators (DRAPER et al. 1995 ). We further tested the possibility of an interaction in the two-hybrid system between LexA-Ccr4 and Gal4AD-Dhh1 or B42-Dhh1, and between LexA-Dhh1 and B42-Ccr4, but we were unable to detect any interaction between Ccr4p and Dhh1p, although each of these proteins was expressed in yeast (data not shown). These results suggest that Ccr4p and Dhh1p do not interact directly.

View larger version (19K): [in this window] [in a new window] [Download PPT slide]   Figure 2. Region of Pop2p responsible for the interaction with Dhh1p. ß-Galactosidase assays and interactions with Gal4p-Dhh1p were conducted as described in MATERIALS AND METHODS. Amino acid residues of each segment of Pop2p are indicated. All assays and interactions were done in strain L40. All LexA-Pop2p derivatives were expressed at equivalent levels as assessed by Western analysis (data not shown). Schematic illustration shows location of the characteristic domains. Q1, polyglutamine stretch (residues 81–91); Q2, polyglutamine stretch (residues 112–126); Q3, glutamine-rich region (residues 364–371); P, proline-rich region (residues 139–155); S/T, serine/threonine-rich region (residues 375–391). All the values were the average of at least five independent experiments. Standard errors were <15%.

To confirm the physical interaction between Pop2p and Dhh1p, a coimmunoprecipitation experiment was carried out using transformants carrying LexA-Dhh1 and B42-Pop2 that was tagged with the HA1 epitope (designated HA1-Pop2 in Figure 3). Immunoprecipitating LexA-Dhh1 with anti-LexA antibody resulted in the coimmunoprecipitation of B42-Pop2 (Figure 3, lane 5). The presence of the B42-Pop2 fusion in the immunoprecipitated materials was dependent on LexA-Dhh1 (Figure 3, lane 6). Sip1p, a protein (YANG et al. 1992 ) that is not part of the Ccr4p complex (LIU et al. 1997 ), did not coimmunoprecipitate with the LexA-Dhh1 fusion (Figure 3, lane 4). These results suggest that Dhh1p is physically associated with the CCR4 complex, and that Pop2p can physically interact with both Ccr4p and Dhh1p.

View larger version (40K): [in this window] [in a new window] [Download PPT slide]   Figure 3. LexA-Dhh1p coimmuneprecipitates with Pop2p. Crude extracts (lanes 1–3) were prepared from diploid EGY188/EGY191 containing the LexA and B42 (HA1) fusion proteins as indicated. Immunoprecipitations (lanes 4–6) were conducted from these extracts using an antibody directed against LexA. Extracts and immunoprecipitated samples were separated by SDS-PAGE, and proteins were identified by using an antibody directed against the HA1 epitope. LexA-Dhh1p and LexA-Vpu were expressed to comparable extents (data not shown). The protein that comigrates with HA1-Pop2p in lane 1 is a degradation product of HA1-Sip1p and does not coimmunoprecipitate with LexA-Dhh1p (see lane 4).

Genetic interaction between POP2 and PKC1-MPK1 pathways:
Because PKC1 was isolated as a weak multicopy suppressor of the pop2 mutation, and the phenotypes of pop2 cells and mpt cells are similar to cells carrying mutations involved in the PKC1-MPK1 pathway, we determined the epistatic relationship among these genes. Overexpression of POP2, CCR4, or DHH1 in pkc1, bck1, mkk1 mkk2, and mpk1 mutant cells did not suppress any of their phenotypes, including temperature-sensitive growth and caffeine sensitivity. PKC1 suppressed the temperature-sensitive growth of mpk1 deletion cells at 35° (Figure 4). Overexpression of STM1 suppressed the staurosporine- and caffeine-sensitive growth, not only of pop2 mutants, but also of pkc1, bck1, mkk1 mkk2, or mpk1 mutants. Thus, STM1 may function downstream of these genes. MPT5 overexpression suppressed temperature-sensitive growth defect of mpk1 deletion cells at 35° (Figure 4), but no other phenotype of this mutant. These results suggest an interaction between the POP2 and PKC1-MPK1 pathways, and that Ccr4 and Dhh1 may function downstream of both the POP2 and PKC1-MPK1 pathways.

View larger version (41K): [in this window] [in a new window] [Download PPT slide]   Figure 4. PKC1 and MPT5 suppressed the mpk1 mutation. Homozygous diploid cells carrying the mpk1 deletion mutation (DL456) were transformed with YEp213 bearing various genes indicated. The transformants were suspended in water and spotted on YPD plates and a YPD plate containing 2 µg/ml staurosporine. The plates were incubated at 24° or 35° for 4 days. MKK1-6 encodes a dominant active form of Mkk1p kinase (LEE et al. 1993B ).

POP2 and PKC1 pathways may function independently but have overlapping functions. To examine these possibilities, double-mutants carrying pop2 dhh1, pop2 mpt5, and dhh1 mpt5 were constructed. They all showed temperature-sensitive phenotypes. No phenotypic additivity was observed, except that the temperature-sensitive growth of dhh1 mpt5 double-mutant cells was not suppressed by 1 M sorbitol (data not shown). On the other hand, haploid cells carrying pkc1 pop2, pkc1 dhh1, or pkc1 mpt5 were not recovered. Among the tetrads analyzed (10 for pop2 and pkc1, eight for dhh1 and pkc1, and eight for mpt5 and pkc1), most tetrads contained less than three viable spores, even on the YPD plate containing 1 M sorbitol (data not shown). Judging from the disruption markers, all the dead cells were found to carry either pop2 pkc1, dhh1 pkc1, or mpt5 pkc1. This synthetic lethality displayed phenotypic additivity and suggested that the POP2 pathway and PKC1 function independently. Taken together, the phenotypic similarity and genetic suppression among these mutants suggest that POP2 and PKC1 pathways function independently but have some overlapping functions.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Dhh1p, a putative RNA helicase, associates with Pop2p and Ccr4p physically and functionally:
We identified five multicopy suppressor genes of the pop2 deletion mutation. Two of these, CCR4 and DHH1, appear functionally related to POP2. The overexpression of DHH1 or CCR4 rescues all the pop2 defects tested, including high-temperature–sensitive growth, cold-sensitive growth, the inability to use glycerol, and staurosporine and caffeine sensitivity (Table 2 and Table 3). Overexpression of DHH1 also suppresses the growth defects in ccr4 cells. In addition, dhh1 cells display a spectrum of phenotypes similar to pop2 or ccr4 cells. Importantly, DHH1 was required for the full expression of the ADH2 gene (Table 4), another phenotype shared by pop2 and ccr4 cells. Furthermore, cells carrying the double mutations pop2 ccr4, pop2 dhh1, or ccr4 dhh1 showed no phenotypic additivity (data not shown; DRAPER et al. 1995 ). These results suggest that there exists a close genetic interaction among the POP2, CCR4, and DHH1 genes, and that these three proteins function together to control transcriptional processes. Because Dhh1p is a putative RNA helicase but no defect in RNA processing was apparent in dhh1 cells, we think that Dhh1p is involved in a novel aspect of gene expression.

Pop2p and Dhh1p were found to interact physically (Table 5, Figure 3), suggesting that Dhh1p is associated with the Ccr4p regulatory complex. Because we have no evidence to suggest an interaction between Ccr4p and Dhh1p by two-hybrid analysis, it cannot be excluded that Dhh1p interacts with Pop2p in a complex separate from that which associates with Ccr4p. This seems unlikely, however, since Dhh1p has also been found to coimmunoprecipitate with Dbf2p and Caf17p, two other Ccr4p complex components (unpublished observations; LIU et al. 1997 ). More importantly, nearly all the Ccr4p in the cells copurifed with Pop2p (Caf1p; M. LIU, personal communication), suggesting that there is no separate complex. Because there are multiple components of the Ccr4p complex, it is difficult at this point to clearly determine the order and directness of the interactions between these proteins.

Because overexpression of PKC1, STM1, and MPT5 suppressed some phenotypes of pop2 cells, these genes might function further downstream of POP2 or bypass some Pop2p functions (Table 3). Stm1p has G4 nucleic acid–binding activity (FRANTZ and GILBERT 1995 ), and a null allele of the stm1 mutation does not cause phenotypes, as observed with the other mpt mutations. The relationship between the G4 nucleic acid–binding activity and POP2 function remains to be elucidated. MPT5 is required for growth at high temperatures and for the recovery from mating factor–induced G1 arrest (KIKUCHI et al. 1994 ), and it is involved in yeast cell aging by redistribution of the Sir2p-Sir3p-Sir4p complex from the telomeres to the nucleolus (KENNEDY et al. 1997 ). MPT5 has been shown to interact with Sst2p and Cdc28p, which is involved in the pheromone signaling pathway and the cell cycle control pathway, respectively (CHEN and KURJAN 1997 ). Since the MPT5 gene is also involved in the stress response, it may be a general suppressor of temperature stress effects.

Since the pop2 and mpt5 showed no phenotypic additivity, POP2 and MPT5 also appear to function in the same pathway. Based on the facts that overexpression of DHH1 or MPT5 can suppress the ccr4 mutants, whereas overexpression of CCR4 had no effect on dhh1 or mpt5 cells (Table 3), we hypothesize that DHH1 and MPT5 function genetically downstream of CCR4. Overexpression of DHH1, however, could not suppress any phenotypes of mpt5 cells and vice versa. Furthermore, the dhh1mpt5 double-mutant cells showed temperature-sensitive growth, and this defect was not suppressed by 1 M sorbitol (data not shown). These results suggest that DHH1 and MPT5 genetically function in two independent pathways that act after POP2 and CCR4.

Interaction between the POP2 and PKC1-MPK1 pathways:
The protein kinase C pathway, including PKC1, BCK1, MKK1 or MKK2, and MPK1, is important for cell wall integrity in yeast (THEVELEIN 1994 ). Mutants of PKC1 require an osmotic stabilizer (e.g., 1 M sorbitol) for survival. Also, cells carrying the pkc1 or mpk1 mutation cannot use glycerol as a sole carbon source (COSTIGAN et al. 1994 , Table 2). This may suggest that the PKC1-MPK1 pathway activates the transcription of genes that require nonfermentable carbon sources and those that are involved in cell wall integrity. Since PKC1 was isolated as a multicopy suppressor of pop2, and most of the mpt mutants including pop2 itself showed staurosporine-sensitive phenotypes and inability to utilize nonfermentable carbon sources, and the temperature-sensitive cell lysis phenotype of pop2 and the cold sensitivity phenotypes of ccr4, pop2(caf1), and dbf2, were suppressed by 1 M sorbitol (LIU et al. 1997 ), it was of great interest to us to investigate the relationship between the POP2 pathway and PKC1 pathway.

Overexpression of PKC1 suppressed temperature-sensitive growth of dhh1 or mpt5 mutants (Table 3). This suggests that DHH1 and MPT5 function upstream of PKC1. But cells carrying double mutations of pop2pkc1, dhh1 pkc1, or mpt5 pkc1 were synthetically lethal, ruling out a direct and dependent interaction among PKC1 and POP2, DHH1, and MPT5 (data not shown). In addition, the dhh1 and mpt5 mutations were suppressed by overproduction of PKC1 but not by MPK1 (data not shown), and overproduction of MPT5 suppressed the mpk1 mutation (Figure 4). Because epistasis among these genes is unclear, we speculate that the POP2 pathway and the PKC1 pathway are two independent pathways that have overlapping functions. To clarify this, we are currently searching for the common target of the two pathways. It should be noted that a well-accepted model of the PKC1 pathway suggests that MPK1 is epistatic to PKC1 (THEVELEIN 1994 ). Our finding that overproduction of PKC1 suppressed the mpk1 mutation (Figure 4) seems to conflict with this model. It is possible that this might not be a direct effect, or that overproduction of PKC1 might activate another pathway (LEE et al. 1993A ).

	FOOTNOTES

¹ Present address: Fukushima Medical College, 1 Hikarigaoka, Fukushima-shi, Fukushima 960-12, Japan.
² Present address: Institute of Genetic Ecology, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai-shi, Miyagi 980-70, Japan.
³ Present address: Frontier Research Program, The Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako-shi, Saitama 351-01, Japan.
⁴ Present address: Department of Biochemistry and Molecular Biology, Rudman Hall, University of New Hampshire, Durham, NH 03824-3544.

	ACKNOWLEDGMENTS

We thank KOUICHI ISHIGURO for providing the antibody against Gal4AD, and KENJI IRIE, DAVID LEVIN, KUNIHIRO MATSUMOTO, KATSUNORI TANAKA, and SATOSHI YOSHIDA for providing S. cerevisiae strains and plasmids. We also appreciate helpful discussions with FUMIO HISHINUMA. This research was partly supported by National Institutes of Health grant GM41215, National Science Foundation grant MCB-9561832, and Hatch project 291 (C.L.D.).

Manuscript received June 26, 1997; Accepted for publication October 8, 1997.

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