Different Regulations of ROM2 and LRG1 Expression by Ccr4, Pop2, and Dhh1 in the Saccharomyces cerevisiae Cell Wall Integrity Pathway

We find here that Ccr4, Pop2, and Dhh1 modulate the levels of mRNAs for specific Rho1 regulators, Rom2 and Lrg1. In budding yeast, Rho1 activity is tightly regulated both temporally and spatially. It is anticipated that Ccr4, Pop2, and Dhh1 may contribute to the precise spatiotemporal control of Rho1 activity by regulating expression of its regulators temporally and spatially. Our finding on the roles of the components of the Ccr4-Not complex in yeast would give important information for understanding the roles of the evolutionary conserved Ccr4-Not complex.

G ene expression can be regulated at many of the steps in the pathway from DNA to protein. In these regulations, posttranscriptional regulation includes the control of mRNA degradation and translation. Both 5=-cap and 3= poly(A) tail structures of mRNAs have important roles in the control of mRNA degradation and translation. In eukaryotes, there are two general mechanisms of cytoplasmic degradation of mRNAs, 5=-to-3= degradation and 3=-to-5= degradation (1). Both degradations are initiated by shortening of the 3= poly(A) tail in a process referred to as deadenylation. This deadenylation is carried out by the Pan2-Pan3 complex as well as by the Ccr4-Not complex. In the 5=-to-3= degradation pathway, the deadenylated mRNAs are decapped by the Dcp1/Dcp2 decapping enzyme and then subjected to 5=-to-3= degradation by Xrn1 exonuclease. Several decapping activators, such as Dhh1, Pat1, Edc3, and Scd6, stimulate the activity of decapping enzyme. In the 3=-to-5= degradation pathway, the deadenylated mRNAs are subjected to 3=-to-5= degradation by the exosome complex. Translation initiation is promoted by binding of the translation initiation complex eIF4F (eukaryotic initiation factor 4F) to the 5=-cap structure. This eIF4F complex contains eIF4E that directly binds to the 5=-cap structure, eIF4A that acts as an RNA helicase, and eIF4G that serves as a scaffold for the complex. Binding of the eIF4F complex to the 5=-cap structure recruits the 43S preinitiation complex, which includes the small ribosomal subunit, the initiator tRNA, and additional initiation factors (2). Translation initiation is also enhanced by the 3= poly(A) tail and the poly(A) binding protein that interacts with eIF4G. In most cases, control of mRNA degradation and translational initiation is mediated by the 3= untranslated regions (3= UTR) of the regulated mRNAs where RNA binding proteins such as Puf family RNA binding proteins bind (3,4).
The cell wall of the budding yeast is required to maintain cell shape and integrity (12). Yeast cells must remodel the rigid structure of the cell wall during vegetative growth and during pheromone-induced morphogenesis. The cell wall remodeling is monitored and regulated by the cell wall integrity (CWI) signaling pathway (12). In the CWI signaling pathway, signals are initiated at the plasma membrane through the cell surface sensors, Wsc1, Wsc2, Wsc3, Mid2, and Mtl1. Together with phosphatidylinositol 4,5-bisphosphate (PI4,5P 2 ), which recruits Rom1/2 guanine nucleotide exchange factors (GEFs) to the plasma membrane, the cell wall sensors stimulate nucleotide exchange on a small GTPase Rho1 through the activation of Rom1/2. The activated Rho1, Rho1-GTP, then activates several effectors, including protein kinase C (Pkc1), ␤1,3-glucan synthase, Bni1 formin protein, exocyst component Sec3, and Skn7 transcription factor. Pkc1 activates downstream mitogen-activated protein (MAP) kinase cascade, which is comprised of Bck1, Mkk1/2, and Mpk1. Mpk1 phosphorylates and activates two transcription factors, Rlm1 and the SBF complex (Swi4/Swi6), which induce gene expression. Rho1-GTP is inactivated by GTPase-activating proteins (GAPs), including Bem2, Sac7, Bag7, and Lrg1.
We have previously found that Ccr4 negatively regulates expression of the LRG1 mRNA encoding one of the Rho1-GAPs in the CWI pathway (11). Loss of LRG1 suppressed the cell lysis of the ccr4⌬ mutant. Ccr4, together with RNA binding protein Khd1, also positively regulates expression of ROM2 mRNA encoding Rho1-GEF (11). The ccr4⌬ khd1⌬ double mutant shows more severe cell lysis.
In this study, we examined the roles of Pop2 and Dhh1 in the CWI signaling pathway. The LRG1 mRNA level was increased in pop2⌬ and dhh1⌬ mutants as well as ccr4⌬ mutant and the increased LRG1 mRNA level contributes to the growth defect of pop2⌬ and dhh1⌬ mutants. On the other hand, ROM2 expression or Rom2 function was not impaired in pop2⌬ and dhh1⌬ mutants. Our results indicate that, in addition to the involvement of Ccr4 in the CWI signaling pathway, Dhh1 and Pop2 take a part in the regulation of Rho1 activity through the Rho1-GAP Lrg1.

RESULTS
The ccr4⌬ and pop2⌬ mutants, but not the dhh1⌬ mutant, display a synthetic growth defect with the khd1⌬ mutation. We have shown that ccr4⌬ and pop2⌬ mutants displayed a synthetic growth defect with the khd1⌬ mutation (11). Tetrad analysis revealed that ccr4⌬ and pop2⌬ mutant cells grew slower than wild-type cells, while khd1⌬ ccr4⌬ and khd1⌬ pop2⌬ double mutant cells grew much more slowly than either khd1⌬, ccr4⌬, or pop2⌬ single mutant cells ( Fig. 1A and B). To examine whether the dhh1⌬ mutant shows a synthetic growth defect with the khd1⌬ mutation, we performed tetrad analysis using a diploid strain that was heterozygous for khd1⌬ and dhh1⌬ alleles. The dhh1⌬ mutant cells grew slower than wild-type cells, and khd1⌬ dhh1⌬ double mutant cells and dhh1⌬ single mutant cells grew similarly (Fig. 1C). Therefore, unlike ccr4⌬ and pop2⌬ mutants, the dhh1⌬ mutant does not display a synthetic growth defect with the khd1⌬ mutation.
ROM2 mRNA level was not decreased in the pop2⌬ and dhh1⌬ mutants. We have previously shown that the level of ROM2 mRNA (encodes Rho1 GEF) was slightly decreased in the ccr4⌬ mutant, and this reduction was enhanced by the khd1⌬ mutation (11) ( Fig. 2A). Rom2 and Rom1 comprise a redundant pair of GEF for Rho1 (13). Loss of ROM2 function results in temperature-sensitive growth, whereas loss of both ROM2 and ROM1 is lethal. Using a mutation of ROM1, we have obtained the genetic evidence indicating that Rom2 function was indeed impaired in ccr4⌬ mutant and khd1⌬ ccr4⌬ double mutant cells (11) (Fig. 3A). If ROM2 function were impaired in a strain harboring a given mutation, the mutant would show a synthetic growth defect with the rom1⌬ mutation. Consistent with the fact that the ROM2 mRNA level is decreased in ccr4⌬ mutant and khd1⌬ ccr4⌬ double mutant cells, ccr4⌬ rom1⌬ double mutant cells showed much slower growth than ccr4⌬ single mutant cells, and khd1⌬ ccr4⌬ rom1⌬ triple mutant cells showed much slower growth than khd1⌬ ccr4⌬ double mutant cells (Fig. 3A). This is also consistent with the observation that overexpression of ROM2 from a multicopy plasmid can suppress the growth defects of khd1⌬ ccr4⌬ double mutant, ccr4⌬ rom1⌬ double mutant, and khd1⌬ ccr4⌬ rom1⌬ triple mutant cells (11) (see Fig. 12A) (data not shown).
We next applied this approach to examine whether the Rom2 function is impaired in pop2⌬ and dhh1⌬ mutants. Tetrad analysis using the diploid strain that was heterozygous for pop2Δ, rom1Δ, and khd1⌬ alleles showed that pop2⌬ rom1⌬ double mutant cells and pop2⌬ single mutant cells grew similarly (Fig. 3B). The khd1⌬ pop2⌬ rom1⌬ triple mutant cells and khd1⌬ pop2⌬ double mutant cells also grew similarly (Fig. 3B). Tetrad analysis using the diploid strain that was heterozygous for dhh1⌬ and rom1⌬ alleles showed that dhh1⌬ rom1⌬ double mutant cells and dhh1⌬ single mutant cells also grew similarly (Fig. 3C). These results suggest that Rom2 normally operates in pop2⌬ and dhh1⌬ mutant cells. The ROM2 mRNA level was consistently not altered in pop2⌬ and khd1⌬ pop2⌬ mutant cells compared to wild-type cells (Fig. 2B). Rather, ROM2 mRNA level was marginally increased in dhh1⌬ single mutant and khd1⌬ dhh1⌬ double mutant cells (Fig. 2C). Thus, Rom2 function and ROM2 expression were impaired in ccr4⌬ mutant and khd1⌬ ccr4⌬ double mutant cells, but not in pop2⌬ and dhh1⌬ mutant cells. These results indicate that only Ccr4 functions in regulation of the expression level of ROM2 mRNA.
regulation, we utilized the pGAL-HA-ROM2 construct harboring the ROM2 3= UTR ( Fig. 4B and C). While the HA-ROM2 mRNA levels from the GAL1 promoter were not altered in ccr4⌬ and khd1⌬ ccr4⌬ mutant cells compared to wild-type cells (Fig. 4B), the hemagglutinin-tagged Rom2 (HA-Rom2) protein levels were clearly decreased in ccr4⌬ and khd1⌬ ccr4⌬ mutant cells compared to wild-type cells (Fig. 4C). Together with the observation that ROM2 mRNA levels from the endogenous ROM2 promoter were slightly decreased in ccr4⌬ mutant and khd1⌬ ccr4⌬ double mutant cells ( Fig. 2A), Rom2 expression is likely to be regulated at the both mRNA and protein levels.
We have previously shown that the ccr4⌬ single mutant shows weak cell lysis and that the khd1⌬ ccr4⌬ double mutant shows more severe cell lysis (11). Due to the cell lysis, Mpk1 is constitutively activated in ccr4⌬ and khd1⌬ ccr4⌬ mutants (data not shown). Since it has been reported that Mpk1 downregulates Rom2 (14), we speculated that Mpk1 might be involved in ROM2 expression. We found that the decreased Rom2myc protein levels in ccr4⌬ and khd1⌬ ccr4⌬ mutants were partially suppressed by the mpk1⌬ mutation (Fig. 5). Thus, the decreased Rom2myc protein levels in ccr4⌬ and khd1⌬ ccr4⌬ mutants are partly due to the constitutive activation of Mpk1. 10BD-c163r1 that was heterozygous for khd1⌬, ccr4⌬, and rom1⌬ alleles was sporulated, and tetrads were dissected onto YPD containing 10% sorbitol. Growth after 6 days at 25°C is shown. Genotypes are indicated on both sides of the blots. More than 20 tetrads were dissected, and representative data are shown. (B) Strain 10BD-p163r1 that was heterozygous for khd1⌬, pop2⌬, and rom1⌬ alleles was sporulated, and tetrads were dissected onto YPD containing 10% sorbitol. Growth after 6 days at 25°C is shown. Genotypes are indicated on both sides of the blots. More than 20 tetrads were dissected, and representative data are shown. (C) Strain 10BD-d1r1 that was heterozygous for dhh1⌬ and rom1⌬ alleles was sporulated, and tetrads were dissected onto YPD containing 10% sorbitol. Growth after 6 days at 25°C is shown. Genotypes are indicated to the left of the image. More than 20 tetrads were dissected, and representative data are shown.
LRG1 expression is negatively regulated by Pop2 and Dhh1. We have previously shown that the level of LRG1 mRNA encoding Rho1 GAP was increased in the ccr4⌬ single mutant and khd1⌬ ccr4⌬ double mutant cells (11) (Fig. 6A). Therefore, we quantified LRG1 mRNA levels in pop2⌬ single mutant and khd1⌬ pop2⌬ double mutant  khd1⌬ ccr4⌬ (c1H-1B), khd1⌬ (c1H-1C), and ccr4⌬ (c1H-1D) cells harboring pGAL-HA-ROM2 plasmid were cultured to mid-logarithmic phase in SG؊Ura medium and collected, and total RNA was prepared. The HA-ROM2 transcripts were quantified by Northern blotting as described in Materials and Methods. ACT1 mRNA was included as a quantity control. The mRNA levels are indicated as percentages of wild-type levels and represent the means ؎ standard deviations from three independent experiments. (C) HA-Rom2 protein levels in wild-type, khd1⌬ ccr4⌬, khd1⌬, and ccr4⌬ cells. Wild-type (c1H-1A), khd1⌬ ccr4⌬ (c1H-1B), khd1⌬ (c1H-1C), and ccr4⌬ (c1H-1D) cells harboring pGAL-HA-ROM2 plasmid were cultured to mid-logarithmic phase in SG؊Ura medium and collected, and total protein was prepared. The HA-Rom2 proteins were quantified by Western blotting as described in Materials and Methods. Mcm2 protein was included as a quantity control. The protein levels are indicated as percentages of wild-type levels and represent the means ؎ standard deviations from three independent experiments. Fig. 6B, LRG1 mRNA levels were increased in pop2⌬ and khd1⌬ pop2⌬ mutant cells than in wild-type cells. In addition, we found that LRG1 mRNA levels were increased in dhh1⌬ and khd1⌬ dhh1⌬ mutant cells than in wild-type cells (Fig. 6C). Therefore, LRG1 expression is downregulated by Pop2 and Dhh1.

cells. As shown in
Pop2 and Dhh1 encode a cytoplasmic deadenylase and a DExD/H box RNA helicase known as mRNA decapping activator, respectively, and they are important factors acting in mRNA degradation (1). Therefore, we speculate that Pop2 and Dhh1 are involved in the degradation of LRG1 mRNA. To analyze the decay rates of LRG1 mRNA, we employed the controllable GAL1 promoter to express LRG1 mRNA. As shown in Fig. 7A and B, LRG1 mRNA were stabilized in pop2⌬ and dhh1⌬ mutant cells. Notably, in pop2⌬ and dhh1⌬ mutant cells, LRG1 mRNA has a twofold-longer half-life than in wild-type cells. These results indicate that Pop2 and Dhh1 are involved in the degradation of LRG1 mRNA.
Loss of LRG1 suppresses the growth defect of the pop2⌬ and dhh1⌬ mutations. We have shown that LRG1 mRNA expression is increased in the khd1⌬ ccr4⌬ mutant and that deletion of LRG1 suppressed the growth defect of the khd1⌬ ccr4⌬ mutant (11). At high temperature, the severe growth defect was observed even in the ccr4⌬ single mutant (Fig. 8A). The defect associated with the ccr4⌬ single mutation was effectively suppressed by deletion of LRG1 (Fig. 8A), indicating that the increased level of LRG1 contributes to the growth defect of ccr4⌬ mutant cells. Since LRG1 mRNA levels were also increased in pop2⌬ and dhh1⌬ mutant cells, we examined whether deletion of LRG1 can also suppress the growth defect caused by pop2⌬ and dhh1⌬ mutations. The pop2⌬ and dhh1⌬ mutant cells failed to grow at elevated temperature (37°C) (Fig. 8B and C). Their growth defects are due to cell lysis, since addition of osmotic stabilizer sorbitol to medium improved their growth at 37°C (data not shown). The pop2⌬ lrg1⌬ and dhh1⌬ lrg1⌬ double mutant cells could grow at 37°C, although their growth was slightly slower than that of wild-type cells ( Fig. 8B and C). These results indicate that the increased LRG1 mRNA level contributes to the growth defect of pop2⌬ and dhh1⌬ mutant cells.
We have previously shown that ccr4⌬ rom2⌬ double mutants and khd1⌬ ccr4⌬ rom2⌬ triple mutants were inviable (11) (Fig. 9A). This raised the possibility that the lethality of the ccr4⌬ rom2⌬ mutant was attributed to the increased LRG1 mRNA level. To test this, we examined whether the lrg1Δ mutation suppresses the growth defect of the ccr4⌬ rom2⌬ mutant. Indeed, the lrg1Δ mutation suppressed the growth defect of the ccr4⌬ rom2⌬ mutant (Fig. 9A). We then examined growth of pop2⌬ rom2⌬ and dhh1⌬ rom2⌬ double mutant cells and found that both mutants were also inviable ( Fig. 9B and C). The lrg1⌬ mutation also suppressed the growth defect of pop2⌬ rom2Δ and dhh1⌬ rom2⌬ mutants ( Fig. 9B and C), indicating that the increased LRG1 mRNA level causes the lethality in the pop2⌬ rom2Δ and dhh1⌬ rom2Δ mutants. These results suggest that LRG1 mRNA is a target mRNA for Ccr4, Pop2, and Dhh1 and that regulation of the LRG1 mRNA stability mediated by Ccr4, Pop2, and Dhh1 is important for yeast cells to grow at high temperature.
To clarify whether Ccr4 and Dhh1 function in the linear pathway, we examined the growth of ccr4⌬ dhh1⌬ double mutant cells. Surprisingly, ccr4⌬ dhh1⌬ double mutant cells are inviable (Fig. 11A). This result was inconsistent with a previous observation of Hata et al. (7), in which ccr4⌬ and dhh1⌬ mutations do not have any additive phenotypes. Deletion of LRG1 failed to suppress the growth defect of ccr4⌬ dhh1⌬ double mutant cells (Fig. 11). Furthermore, the addition of sorbitol, an active allele of RHO1 (Q-to-L change at position 68 encoded by RHO1 [RHO1-Q68L]), or an active allele of PKC1 (PKC1-R398P) failed to suppress the growth defect of the ccr4⌬ dhh1⌬ double mutant ( Fig. 11B and data not shown). These results suggest that, in addition to the CWI pathway, Ccr4 and Dhh1 cooperatively regulate another biological process.

Different roles of Ccr4 and Pop2 in the CWI pathway. Both ccr4⌬ and pop2⌬
mutants displayed a synthetic growth defect with the khd1⌬ mutation (11). In the khd1⌬ ccr4⌬ double mutant, Rom2 function is decreased and Lrg1 function is increased. These results suggest that, in the khd1⌬ ccr4⌬ double mutant, Rho1 activity is severely decreased, which results in its growth defect. This idea is supported by the findings that the growth defect of the khd1⌬ ccr4⌬ double mutant could be suppressed by ROM2 overexpression and expression of RHO1-Q68L (11) (Fig. 12A). On the other hand, in the khd1⌬ pop2⌬ double mutant, Lrg1 function is increased, but Rom2 function is normal. Therefore, it is anticipated that a reduction of Rho1 activity is less severe in khd1⌬ pop2⌬ double mutant cells than in khd1⌬ ccr4⌬ double mutant cells. ROM2 overexpression and RHO1-Q68L failed to suppress the growth defect of the khd1⌬ pop2⌬ double mutant (Fig. 12B), indicating that decreased Rho1 activity caused by the increased Lrg1 level cannot account for the growth defect of the khd1⌬ pop2⌬ double mutant.
Rho1 acts as an activator of five effectors, including Pkc1, Fks1, Bni1, Sec3, and Skn7 (12). The growth defect of the khd1⌬ ccr4⌬ double mutant can be suppressed by PKC1-R398P (11) (Fig. 12A), suggesting that reduction of Pkc1 activity is responsible for the growth defect of the khd1⌬ ccr4⌬ double mutant. We unexpectedly found that PKC1-R398P also suppressed the growth defect of the khd1⌬ pop2⌬ double mutant

d1H-1A cells carrying the pGAL-LRG1 plasmid and dhh1⌬ cells (d1H-1D cells) carrying pGAL-LRG1 plasmid. (B) WT cells (p1H-2A) carrying pGAL-LRG1 plasmid and pop2⌬ cells (p1H-2B) carrying
pGAL-LRG1 plasmid. Cells harboring the pGAL-LRG1 plasmid were grown in SG؊Ura, and the medium was changed to SC؊Ura to inhibit transcription from the GAL1 promoter. Cells were harvested at the times indicated above the lanes, and total RNA was isolated. Samples were analyzed by Northern blotting with specific probes, and the half-lives (t 1/2 ) (in minutes) were determined as the means from three independent experiments. ACT1 mRNA was used as a reference for quantification. (Fig. 12B). This result suggests that the signaling from Rho1 to Pkc1 requires a cooperative function of Pop2 and Khd1. Taken together, these results suggest that Pop2 and Ccr4 not only destabilize a common target, LRG1 mRNA, but also function upstream of Pkc1 in the CWI pathway in a manner independent of each other.

DISCUSSION
In this study, we found that the LRG1 mRNA level was increased in pop2⌬ and dhh1⌬ mutants and the ccr4⌬ mutant than in the wild type. The growth defect of pop2⌬ and dhh1⌬ mutants at high temperature and the lethality of pop2⌬ rom2⌬ and dhh1⌬ rom2 double mutants are suppressed by the lrg1⌬ mutation. Thus, the increased LRG1 mRNA level does contribute to the growth defect of pop2⌬ and dhh1⌬ mutants. The ccr4⌬, pop2⌬, and dhh1⌬ mutants show more severe growth defects at high temperature, suggesting that the negative regulation of LRG1 expression is more important at high temperature. Since it is well-known that the CWI pathway is activated at high temperature (12,15), the negative regulation of LRG1 expression is more important at high temperature to ensure the proper activation of Rho1 at high temperature. Besides Lrg1, there are three other Rho1-GAPs, Bem2, Sac7, and Bag7 (12). The level of expression of BEM2, SAC7, or BAG7 mRNA was not altered significantly in ccr4⌬ and pop2⌬ mutants (data not shown). In these GAPs, Lrg1 has been reported to participate in the regulation of ␤-1,3-glucan synthase (16). Bem2 and Sac7 are involved in the downregulation of the Pkc1-activated mitogen-activated protein kinase (MAPK) pathway (17,18). Thus, it is possible that the negative regulation of LRG1 expression by Ccr4, Pop2, and Dhh1 is important for Rho1 to activate ␤-1,3-glucan synthase properly. Data from Candida albicans also support this idea, as ccr4 and pop2 mutants showed relatively lower glucan in the cell wall (19).
How is LRG1 mRNA specifically recognized by Ccr4, Pop2, and Dhh1 as a target mRNA? Stewart et al. (20) have reported that an RNA binding protein Puf5/Mpt5 negatively regulates the LRG1 mRNA level and that the lrg1⌬ mutation suppresses the growth defect of the puf5⌬ mutant. Puf5 was originally isolated as a multicopy suppressor of the pop2 mutation (7). Puf5 directly binds to the 3= UTR of LRG1 mRNA (21,22) and physically interacts with Ccr4, Pop2, and Dhh1 (4,7). Previous studies showed that Puf5 does not bind to the 3= UTR of BEM2, SAC7, or BAG7 mRNA encoding other Rho1-GAPs (21,22). Thus, Ccr4, Pop2, and Dhh1 may specifically regulate LRG1 mRNA via the ability of Puf5 to recruit them to the 3= UTR of LRG1 mRNA.
Ccr4 and Pop2 shorten the poly(A) tail of LRG1 mRNA, and Dhh1 stimulates decapping by Dcp1/2. The ccr4Δ, pop2⌬, and dhh1⌬ mutants show severe growth defect at high temperature, and their growth defects are suppressed by lrg1Δ mutation, suggesting that rapid degradation of LRG1 mRNA is important for cell growth, especially at high temperature. We have shown here that overexpression of Dhh1 suppressed the growth defect of the khd1⌬ ccr4⌬ mutant at 37°C and that the elevated LRG1 mRNA level in the khd1⌬ ccr4⌬ mutant was reduced by Dhh1 overexpression .   FIG 9 Loss of LRG1 suppresses the lethality of the ccr4⌬ rom2⌬, pop2⌬ rom2⌬, and dhh1⌬ rom2⌬ 10BD-c163r2l1 that was heterozygous for khd1⌬, ccr4⌬, rom2⌬, and lrg1⌬ alleles was sporulated, and tetrads were dissected onto YPD containing 10% sorbitol. Growth after 6 days at 25°C is shown. Genotypes are indicated on both sides of the blots. More than 20 tetrads were dissected, and representative data are shown. (B) Strain 10BD-pr1l1 that was heterozygous for pop2⌬, rom2⌬, and lrg1⌬ alleles was sporulated, and tetrads were dissected onto YPD containing 10% sorbitol. Growth after 6 days at 25°C is shown. Genotypes are indicated on both sides. More than 20 tetrads were dissected, and representative data are shown. (C) Strain 10BD-d1r1l1 that was heterozygous for dhh1⌬, rom2⌬, and lrg1⌬ alleles was sporulated, and tetrads were dissected onto YPD containing 10% sorbitol. Growth after 6 days at 25°C is shown. Genotypes are indicated to the left of the blots. More than 20 tetrads were dissected, and representative data are shown.

mutants. (A) Strain
Dhh1 overexpression could be inducing the deadenylation-independent decapping, and in that way confer a decrease of the LRG1 mRNA level. Thus, Dhh1 acts downstream of Ccr4 in the degradation pathway of LRG1 mRNA. While Ccr4, Pop2, and Dhh1 share LRG1 mRNA as a target, they may act independently on other targets. We found here that the combination of ccr4⌬ dhh1⌬ mutations was lethal and that deletion of LRG1 failed to suppress the lethality of the ccr4⌬ dhh1⌬ double mutant. Thus, the LRG1 mRNA is not the sole target mRNA for Ccr4 and Dhh1. Since Ccr4 and Dhh1 are global regulators acting on practically all mRNAs, the lethality of ccr4Δ dhh1Δ double mutant cells could be caused by more general changes in mRNA degradation and translational repression, rather than control of specific target mRNAs.
The level of ROM2 mRNA encoding Rho1 GEF was slightly decreased in the ccr4⌬ mutant, and this reduction was enhanced by the khd1⌬ mutation (11) (Fig. 2A). We also confirmed genetically that Rom2 function was indeed impaired in the ccr4⌬ single mutant and khd1⌬ ccr4⌬ double mutant using a mutation of the ROM1 gene. Using this genetic approach, we found that Rom2 function was not impaired in pop2⌬ and dhh1⌬ mutants. Consistently, ROM2 mRNA level was not decreased in pop2⌬ and dhh1⌬ mutants. Thus, Ccr4 acts independently of Pop2 and Dhh1 in regulating ROM2 expression. Rom2 protein levels and ROM2 mRNA levels were decreased in ccr4⌬ and khd1⌬ ccr4⌬ mutants than in wild-type cells, and the decreased protein levels were more evident than the decreased mRNA levels. Thus, Rom2 expression level is regulated at both the mRNA and protein levels. How do Khd1 and Ccr4 positively regulate the expression of ROM2? The myc-tagged ROM2 construct used in Fig. 4A and 5 had the ADH1 3= UTR instead of the endogenous ROM2 3= UTR, and the Rom2myc protein levels were decreased in ccr4⌬ single mutant and khd1⌬ ccr4⌬ double mutant cells, implying that the ROM2 3= UTR seems not to be essential for the regulation of ROM2 expression. In the case of the regulation of MTL1 mRNA stability by Khd1, MTL1 mRNA itself bears the multiple CNN repeats involved in destabilization by the decapping enzyme Dcp1/2 and the 5=-to-3= exonuclease Xrn1, and Khd1 stabilizes MTL1 mRNA by binding to this element (23,24). Since ROM2 mRNA contains three CNN repeats in the coding sequence and Khd1 associates with ROM2 mRNA (11,23), Khd1, together with Ccr4, may stabilize ROM2 mRNA by binding to the CNN repeats of the ROM2 mRNA. Since Rom2 expression is regulated at both mRNA and protein levels, the binding to the CNN repeats by Khd1 may also be involved in translational control. Consistently, the HA-Rom2 protein levels expressed from the GAL1 promoter were decreased in ccr4⌬ and khd1⌬ ccr4⌬ mutant cells compared to wild-type cells, while the HA-ROM2 mRNA levels were not altered. Additionally, Mpk1, which is activated in ccr4⌬ and khd1⌬ ccr4⌬ mutants, may be involved in the decrease of the ROM2 mRNA. The decreased Rom2myc protein levels in ccr4⌬ and khd1⌬ ccr4⌬ mutant cells were partially suppressed by the mpk1⌬ mutation, implying the possibility that Mpk1 is also involved in ROM2 expression at the protein level.
While the ccr4⌬ mutant displays a synthetic growth defect with the khd1⌬ mutation, the dhh1⌬ mutant does not. The simple explanation is that in the ccr4⌬ mutant, where Rom2 function is decreased and Lrg1 function is increased, Rho1 activity is severely decreased. Consistently, a constitutively active RHO1 allele is able to suppress the growth defect of the khd1⌬ ccr4⌬ double mutant (11) (Fig. 12A). In the dhh1⌬ mutant, where Rom2 function is normal and Lrg1 function is increased, the decrease in Rho1 activity is lower than that in the ccr4⌬ mutant. This raises the possibility that khd1⌬ mutation would affect cell growth only when Rho1 activity is more severely impaired. However, this explanation is not consistent with the pop2⌬ case. The pop2⌬ mutant displays a synthetic growth defect with the khd1⌬ mutation, but Rom2 function is normal in the pop2⌬ mutant. In the pop2⌬ mutant, where Rom2 function is normal and Lrg1 function is increased, the decrease in Rho1 activity is lower than that in the ccr4⌬ mutant. Since a constitutively active RHO1 allele cannot suppress the growth defect of the khd1⌬ pop2⌬ double mutant (Fig. 12B), the khd1⌬ pop2⌬ double mutant might have an additional defect in the CWI signaling pathway. Intriguingly, the growth defect of the khd1⌬ pop2⌬ double mutant as well as the khd1⌬ ccr4⌬ double mutant could be suppressed by the constitutively active PKC1 allele. These results suggest that Pop2 and Ccr4 not only destabilize a common target, LRG1 mRNA, but also regulate the CWI pathway at different points, and that Ccr4 and Pop2 act at a point upstream of Pkc1 in the CWI pathway. Although Rho1 activity is severely decreased in the khd1⌬ ccr4⌬ double mutant, where Rom2 function is decreased and Lrg1 function is increased, Mpk1 seems to be activated in the khd1⌬ ccr4⌬ double mutant. Since Lrg1 participates in the regulation of ␤-1,3-glucan synthase (16), one possibility is that the decreased Rho1 activity could not activate ␤-1,3-glucan synthase due to the increased Lrg1 but could still activate the Pkc1-Mpk1 branch in the khd1⌬ ccr4⌬ double mutant. The levels of expression of BEM2 and SAC7, which are involved in the downregulation of the Pkc1-activated MAPK pathway (17,18), were not altered significantly in the khd1⌬ ccr4⌬ double mutant (data not shown). Regulation of the levels of different Rho1-GAPs by modulation of mRNAs might ensure Rho1 activation in a target-specific manner.
Previously, we revealed that Ccr4, a component of the Ccr4-Not cytoplasmic deadenylase complex, functions in the CWI pathway (11). In this study, we further identified Pop2 deadenylase and Dhh1 DExD/H box protein as the regulator of the CWI pathway. Ccr4, Pop2, and Dhh1 modulate the levels of mRNAs for specific Rho1 regulators, Rom2 and Lrg1. In budding yeast, Rho1 activity is tightly regulated both temporally and spatially (12). It is anticipated that Ccr4, Pop2, and Dhh1 may contribute to the precise spatiotemporal control of Rho1 activity by regulating expression of its regulators temporally and spatially. Therefore, to further elucidate how Ccr4, Pop2, and Dhh1 regulate ROM2 and LRG1 mRNAs will undoubtedly provide valuable insights into the precise spatiotemporal regulation of this signaling pathway.

MATERIALS AND METHODS
Strains and general methods. Escherichia coli DH5␣ was used for DNA manipulations. S. cerevisiae strains used in this study are described in Table 1. Standard procedures were followed for yeast manipulations (25). The media used in this study included rich medium, synthetic complete medium (SC), and synthetic minimal medium (SD) (25). SC lacking amino acids or other nutrients (e.g., SCϪUra is SC lacking uracil) were used to select transformants. Recombinant DNA procedures were carried out as described previously (26).
Plasmids. Plasmids used in this study are described in Table 2. Plasmids pCgLEU2, pCgHIS3, and pCgTRP1 are pUC19 carrying the Candida glabrata LEU2, HIS3, and TRP1 genes, respectively (27). Plasmid pKlURA3 is pUC19 carrying the Kluyveromyces lactis URA3. Plasmid pGAL-HA-LRG1 expressing HA-LRG1 from the GAL1 promoter was used for the experiment for LRG1 mRNA degradation. Plasmid YCplac33-ROM2myc expressing ROM2myc from the endogenous promoter and plasmid pGAL-HA-ROM2 expressing HA-ROM2 from the GAL1 promoter were used for Western blotting of Rom2 protein.
Gene deletion and protein tagging. Deletions of KHD1, CCR4, POP2, DHH1, ROM1, ROM2, and LRG1 were constructed by PCR-based gene deletion method (27)(28)(29). Primer sets were designed such that 46 bases at the 5= ends of the primers were complementary to those at the corresponding region of the target gene and 20 bases at their 3= ends were complementary to the pUC19 sequence outside the polylinker region in the plasmid pCgLEU2, pCgHIS3, pCgTRP1, or pKlURA3. Primer sets for PCR were designed to delete the open reading frame (ORF) completely. The PCR products were transformed into the wild-type strain and selected for Leu ϩ , His ϩ , Trp ϩ , or Ura ϩ . The ROM2myc strains were prepared by the method of Longtine et al. (30) using pFA6a-13myc-kanMX6.
Northern blot analysis. Total RNA was prepared from cells using Isogen reagent (Nippon Gene) and RNeasy minikit (Qiagen). RNA samples were separated by 1.5% denatured agarose gel electrophoresis and transferred to a nylon membrane. Then, RNA was hybridized using digoxigenin (DIG)-labeled antisense probe. The primer pair j298 (TGACGATATGATGAGCTCCTCCTTACGTCA) and j297 (TTAACCCCA GAAATCTAACGACG) and primer pair j259 (ATGATTCAAAATTCTGCTGGTTA) and j260 (GCCAATATTTATG AATTCCATAAC) were used to detect transcript containing ROM2 and LRG1, respectively. After washing and blocking, the membrane was incubated with alkaline phosphatase-conjugated anti-DIG antibody, and the signal was detected by enhanced chemiluminescence. mRNA degradation was determined from Northern blots as described previously (31,32). Cells were grown in SGϪUra, and the medium was changed to SCϪUra to inhibit transcription from the GAL1 promoter. Cells were harvested at the times indicated in the figures, and total RNA was isolated. Samples were analyzed by Northern blotting with specific probes, and half-lives (t 1/2 ) (in minutes) were determined as the means from three independent experiments.