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Genes to Cells (2009) 14, 759-771. doi:10.1111/j.1365-2443.2009.01308.x
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Pleiotropic phenotypes caused by an opal nonsense mutation in an essential gene encoding HMG-CoA reductase in fission yeast

 Yue Fang1,2,*, Kiwamu Imagawa1,{dagger}, Xin Zhou1, Ayako Kita3, Reiko Sugiura3, Wurentuya Jaiseng1 and Takayoshi Kuno1

1 Division of Molecular Pharmacology and Pharmacogenomics, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan
2 Department of Pharmacology, China Medical University, 92 North 2nd Road, Heping District, Shenyang 110001, China
3 Laboratory of Molecular Pharmacogenomics, School of Pharmaceutical Sciences, Kinki University, Kowakae 3-4-1, Higashi-Osaka 577-8502, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Schizosaccharomyces pombe genome contains an essential gene hmg1+ encoding the sterol biosynthetic enzyme, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR). Here, we isolated an allele of the hmg1+ gene, hmg1-1/its12, as a mutant that showed sensitivities to high temperature and to FK506, a calcineurin inhibitor. The hmg1-1 allele contained an opal nonsense mutation in its N-terminal transmembrane domain, yet in spite of the mutation a full-length protein was produced, suggesting a read-through termination codon. Consistently, overexpression of the hmg1-1 mutant gene suppressed the mutant phenotypes. The hmg1-1 mutant showed hypersensitivity to pravastatin, an HMGR inhibitor, suggesting a defective HMGR activity. The mutant treated with FK506 caused dramatic morphological changes and showed defects in cell wall integrity, as well as displayed synthetic growth phenotypes with the mutant alleles of genes involved in cytokinesis and cell wall integrity. The mutant exhibited different phenotypes from those of the disruption mutants of ergosterol biosynthesis genes, and it showed normal filipin staining as well as showed normal subcellular localization of small GTPases. These data suggest that the pleiotropic phenotypes reflect the integrated effects of the reduced availability of ergosterol and various intermediates of the mevalonate pathway.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
The conversion of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) to mevalonic acid, a reaction catalyzed by HMG-CoA reductase (HMGR), is the first step in the mevalonate pathway in eukaryotes that is important in the synthesis of many essential cellular molecules including cholesterol, coenzyme Q, prenyl groups, and dolichol (Goldstein & Brown 1990). Statins that specifically and competitively inhibit HMGR are used clinically for the treatment of hypercholesterolemia. In fungi including yeasts, ergosterol instead of cholesterol is generated and several antifungal inhibitors target this pathway.

The mammalian genome contains a single gene encoding HMGR that has a COOH-terminal catalytic region connected by a linker to an NH2-terminal hydrophobic region that is responsible for endoplasmic reticulum (ER) localization (Liscum et al. 1985; Luskey & Stevens 1985) and is required for regulated ER degradation of the native enzyme (Nakanishi et al. 1988). In budding yeast Saccharomyces cerevisiae, two structural genes (HMG1 and HMG2) encode two isozymes of HMGR, Hmg1p and Hmg2p, respectively (Basson et al. 1986) that are integral ER membrane proteins containing a COOH-terminal catalytic domain and an NH2-terminal hydrophobic region (Basson et al. 1988; Wright et al. 1988). Hmg1p contributes most of the activity found in wild-type cells, and a single deletion mutant of HMG1 or HMG2 has only a subtle growth defect, but cells containing hmgl hmg2 double mutation are inviable (Basson et al. 1986, 1987) and can be rescued by the expression of the mammalian HMGR gene (Basson et al. 1988), suggesting a functional conservation. Hmg1p is quite stable, whereas Hmg2p is rapidly degraded in a regulated manner similar to that in the mammalian HMGR (Hampton & Rine 1994). Overexpression of Hmg1p causes the proliferation of the ER membrane, resulting in the stacked perinuclear structures named "karmellae" (Wright et al. 1988).

In fission yeast Schizosaccharomyces pombe, there is a single gene, hmg1+, that encodes HMGR and was isolated on the basis of its ability to confer resistance to lovastatin, a competitive inhibitor of HMGR. Gene knockout analysis showed that hmg1+ is an essential gene (Lum et al. 1996). The expression in fission yeast of increased levels of the S. cerevisiae HMGR isozyme encoded by HMG1 induced "karmellae" formation (Lum & Wright 1995). The regulatory mechanism of a family of ER membrane bound transcription factors called sterol regulatory element binding proteins has been studied extensively (Hughes et al. 2005; Burg et al. 2008). However, limited information is available regarding the phenotypes caused by mutations in the hmg1+ gene.

In this study, we report the isolation of an allele of the hmg1+ gene, hmg1-1/its12, as a mutant that showed sensitivities to high temperature and to FK506, a calcineurin inhibitor. In the hmg1-1 mutant, tryptophan at the 234th position was mutated to an opal termination codon that was read through to result in the mistranslated full-length protein. To our knowledge, this is the first demonstration of clear phenotypes arising from a nonsense mutation of an essential gene in fission yeast.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Isolation of its12/hmg1-1 mutant

We isolated a new its mutant (its for immunosuppressant- and temperature-sensitive; Zhang et al. 2000) that was sensitive to temperature and to the calcineurin inhibitor FK506, and named it its12 mutant. As shown in Fig. 1A, the its12 mutant grew slightly more slowly than the wild-type cells at 27 °C. The its12 mutant cells failed to grow at 36 °C and they grew poorly or barely grew on YPD plate containing FK506 at 27 °C, whereas wild-type cells grew normally (Fig. 1A). Tetrad analysis was performed by crossing the its12 mutant with calcineurin deletion ({Delta}ppb1) strain. Results showed that no double mutant was obtained, indicating that its12 mutation and calcineurin deletion are synthetically lethal (data not shown).


Figure 1
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  		Figure 1  Mutation in the its12+/hmg1+ gene causes immunosuppressant- and temperature-sensitive phenotypes. (A) The immunosuppressant and temperature sensitivities of the its12/hmg1-1 mutant cells. Wild-type (wt) and hmg1-1 mutant (hmg1-1) cells transformed with the multicopy vector alone or the vector containing the hmg1+ gene were streaked onto each plate containing YPD or YPD plus 0.5 µg/mL FK506, and then incubated for 4 days at 27 °C or for 3 days at 36 °C, respectively. (B) Alignment of protein sequence of S. pombe Hmg1 with related proteins from human and S. cerevisiae. Upper panel, sequence alignment was performed using the ClustalW program. Arrowhead points to tryptophan 234, which was mutated to termination codon in KP4127 cells by a G-to-A transition. Lower panel, schematic representation of the mutation site of hmg1-1. Arrowhead points to tryptophan 234, which is located at transmembrane domain. (C) The hmg1-1 mutant cells showed hypersensitivity to pravastatin. Cells were streaked onto each plate containing YPD, or YPD plus 0.2 mg/mL pravastatin and then incubated at 27 °C for 4 days.

 
The hmg1-1/its12 strain is a mutant allele of the hmg1+ gene encoding the sterol biosynthetic enzyme, HMGR

The mutated gene in its12 mutant was cloned by complementation of the temperature-sensitive growth (Fig. 1A). Nucleotide sequencing of the cloned DNA fragment revealed that in its12 mutant, the mutated gene with the systematic identification SPCC162.09c is hmg1+ that encodes HMGR consisting of a 1053-amino acid sequence. To investigate the genetic relationship between the mutated gene and the hmg1+ gene, linkage analysis was performed as follows. The hmg1+ gene was subcloned into the pUC-derived plasmid containing budding yeast LEU2 gene. Using this construct, the LEU2 gene was integrated by homologous recombination into the hmg1+ gene locus of the genome of KP207 (Table 1). The integrant was mated with its12 strain, and the resultant diploid was sporulated. When the tetrads were dissected, only parental ditype tetrads were found, indicating that the mutated gene was tightly linked to the hmg1+ gene locus (data not shown). Therefore we renamed the its12 mutant as hmg1-1 mutant.


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  		Table 1  Strains used in the present study
 
To characterize the mutation site in the hmg1-1 mutant, the genomic DNA was isolated and the full-length coding region was sequenced. Sequence analysis of the genomic DNA from the hmg1-1 mutant revealed that tryptophan at 234 was mutated to an opal termination codon by a G-to-A transition (TGG to TGA). The tryptophan is conserved between fission yeast and budding yeast (Fig. 1B, upper panel). This nonsense mutation is located in the transmembrane domain (Fig. 1B, lower panel).

The hmg1-1 mutant cells showed hypersensitivity to pravastatin

As the results described above suggest that the sterol biosynthesis is defective in the hmg1-1 mutant cells, we then examined the effect of pravastatin, a competitive inhibitor of HMGR. As shown in Fig. 1C, the growth of the hmg1-1 mutant was significantly inhibited by pravastatin as compared with that of wild-type cells. Studies have reported that the budding yeast cells containing a mutant allele of either HMG1 or HMG2 are viable but are more sensitive to compactin, an inhibitor of HMGR, as compared with that of the wild-type cells. The above results suggest that the hmg1-1 cells contained less HMGR activity as compared with that of wild-type cells.

Intracellular localization of Hmg1-GFP

To examine the intracellular localization of Hmg1 in wild-type cells, the gene was tagged at its C terminus with GFP carrying the S65T mutation. The construct was functional as its thiamine-repressed expression complemented the phenotypes of the hmg1-1 mutant (Fig. 2A). Chromosome-borne Hmg1-GFP was primarily localized in the nuclear envelope and was also observed in the peripheral ER when its expression from the nmt1 promoter was repressed by the addition of thiamine (Fig. 2B).


Figure 2
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  		Figure 2  Intracellular localization of Hmg1-GFP. (A) hmg1+-GFP is fully functional. Wild-type and hmg1-1 mutant cells transformed with pREP1-hmg1+-GFP, pREP1-hmg1-1-GFP or the empty vector were streaked onto each plate containing EMM, or EMM plus 4 µM thiamine, then incubated for 4 days at 27 °C or 3 days at 36 °C, respectively. (B) Subcellular localization of wild-type or mutant Hmg1 tagged with GFP in wild-type cells. The fused gene encoding Hmg1-GFP was integrated into the chromosome of wild-type cells under the control of the nmt1 promoter of pREP1 (see Experimental procedures). Cells were grown to early log phase in EMM containing 4 µM thiamine. (C) Overexpressed Hmg1-GFP was concentrated in the nuclear envelope, frequently in an asymmetric pattern. Overexpression of Hmg1-GFP was induced by the removal of thiamine from the media for 24 h. Arrows point to asymmetrically concentrated Hmg1-GFP. Bar, 10 µm. (D) Electron microscopic analysis of Hmg1-GFP overexpressed cells grown at 27 °C for 24 h. Nuclear-associated membranes known as karmellae were produced in cells that overexpressed Hmg1-GFP. Arrows point to proliferated membranes. Bar, 1 µm. (E) Expression of the mutant Hmg1 tagged with GFP in wild-type cells. The wild-type or mutant Hmg1 tagged with GFP was expressed under the control of the nmt1 promoter. Cells were cultured in the presence (+) or absence (–) of thiamine for 20h at 27 °C. Cell lysates were subjected to SDS-PAGE, and immunoblotted using anti-GFP antibody.

 
Overexpression of Hmg1-GFP induced by the removal of thiamine from the media caused a distinct growth defect as shown in Fig. 2A. Overexpressed Hmg1-GFP was concentrated in the nuclear envelope, frequently in an asymmetric pattern and a considerably lesser intensity of staining of the peripheral ER was observed (Fig. 2C). These patterns were very similar to those obtained with Hmg1p-GFP overexpression in S. cerevisiae (Koning et al. 2002). As shown in Fig. 2D, electron microscopic observation revealed that "karmellae", a proliferation of stacked membrane pairs surrounding the yeast nucleus structures was induced as in S. cerevisiae (Wright et al. 1988). These findings were almost identical with the data obtained with overexpression of wild-type hmg1+ without tagging (Lum et al. 1996), further supporting the notion that Hmg1-GFP is fully functional.

Expression of the hmg1-1 mutant gene bearing TGG to TGA nonsense mutation

In addition to the chromosome-borne Hmg1-GFP, the hmg1-1 mutant gene tagged with GFP was also subcloned into pREP1 vector and expressed under the control of the nmt1 promoter. When the gene expression was attenuated by the addition of thiamine to the medium, the suppression of the temperature sensitive phenotype of hmg1-1 mutant by the mutant gene was significant although weaker as compared with the strong suppression by the wild-type gene (Fig. 2A, +thiamine at 36 °C). In contrast, when it was overexpressed in the absence of thiamine, the growth defect was more severe than that observed with the wild-type gene (Fig. 2A, –thiamine at 27 °C). To our surprise, the localization of both the mutant Hmg1 protein tagged with GFP and the wild-type protein was identical (Fig. 2B). Then we analyzed the mutant Hmg1 protein expressed in wild-type cells by immunoblotting (Fig. 2E). The mutant Hmg1 protein tagged with GFP was found to be the same size as that of the wild-type protein. The amount of the mutant protein was lower (approximately 30%–50%) than that of the wild-type protein in both the attenuated and induced conditions.

Filipin staining of the hmg1-1 mutant cells

We next examined the cell morphology and the localization of the sterol using the fluorescent probe filipin, a polyene antibiotic that forms specific complexes with free 3-β-hydroxysterols. In the wild-type cells (Fig. 3A left panel), the filipin fluorescence was enriched in the plasma membrane at the growing cell tips and at the site of cytokinesis similar to that reported by Wachtler et al. (2003). In the hmg1-1 mutant (Fig. 3A right panel), the cells were slightly larger than the wild-type cells, however, their filipin fluorescence pattern was indistinguishable from that obtained with the wild-type cells at the permissive temperature of 27 °C. When the hmg1-1 mutant cells were shifted to the restrictive temperature of 36 °C for 4 h, a significant portion of the cells showed weaker filipin fluorescence suggesting decreased ergosterol levels in the mutant cells (Fig. 3B, right panel), whereas wild-type cells remained essentially unchanged in their staining pattern (Fig. 3B, left panel).


Figure 3
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  		Figure 3  Intracellular localization of filipin in the hmg1-1 mutant cells. Sterol localization detected by filipin staining (see Experimental procedures) in wild-type cells and hmg1-1 mutant cells (A) at 27 °C, or (B) at 36 °C for 4h. DIC, differential interference contrast. Bar, 10 µm.

 
The hmg1-1 mutant cells are defective in cytokinesis

As reported in our previous studies, in wild-type cells cultured at 27 °C in the absence of FK506, the septation index was < 15% and increased to 27% at 6 h after the addition of FK506 (Zhang et al. 2000). In hmg1-1 mutant cells cultured at 27 °C in the absence of FK506, the septation index was 16%–20% which was only slightly higher than that of the wild-type cells (Fig. 4A) and remained unchanged upon temperature upshift (data not shown). Upon the addition of FK506, however, the hmg1-1 mutant cells showed a dramatic morphological change as compared with the wild-type cells and the septation index reached nearly 100% 6 h after the addition of FK506 (Fig. 4B). Also, it was noted that FK506-treated hmg1-1 mutant cells showed a markedly inhibited growth (Fig. 1A).


Figure 4
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  		Figure 4  The hmg1-1 mutant cells are defective in cytokinesis. Wild-type and hmg1-1 mutant cells grown to log phase at 27 °C in liquid YPD medium. Cells were further incubated at 27 °C for 6 h in the absence (A) or presence (B) of FK506 (0.5 µg/mL) and then stained with calcofluor (CF) to visualize cell wall and septum. Bar, 10 µm. DIC, differential interference contrast microscopy. (C) The hmg1-1cdc7-24 or hmg1-1cdc11-136 double mutant showed more marked temperature sensitivity than that of the single mutant. Wild-type, hmg1-1, cdc7-24, hmg1-1cdc7-24, cdc11-136 and hmg1-1cdc11-136 cells were spotted onto YPD plates, then incubated for 4 days at the temperatures as indicated. (D) Fluorescence micrographs of cdc7-24 mutant and hmg1-1cdc7-24 double mutant cells growing at 27 °C stained with DAPI. Bar, 10 µm.

 
We previously isolated cdc7 mutant as an FK506- and temperature-sensitive mutant (Lu et al. 2002) and the roles of Cdc7 kinase in cell septation have been established (Krapp et al. 2004). To examine the importance of Hmg1 in cytokinesis hmg1-1cdc7-24 double mutants were constructed. Results showed that the hmg1-1cdc7-24 double mutants were more sensitive to high temperature than their respective pairs of single mutants (Fig. 4C). Another septation initiation mutant cdc11-136 (Krapp et al. 2004) was also examined for the synthetic growth defect with the hmg1-1 mutation. As shown in Fig. 4C, the hmg1-1cdc11-136 double mutants were more sensitive to high temperature than their respective pairs of single mutants. Microscopic observation of the hmg1-1cdc7-24 double mutant cells stained with DAPI revealed that nearly 50% of the double mutant cells were enlarged unseptated cells with multiple nuclei even at the permissive temperature 27 °C, whereas < 5% of the single cdc7-24 mutant cells showed such morphology (Fig. 4D).

The hmg1+ gene is involved in cell wall integrity

Upon examination of the phenotypes of hmg1-1 mutant, it was observed that its temperature sensitivity was osmoremedial, that is, the mutant cells were capable of forming colonies at 36 °C on rich YPD plates containing 1.2 M sorbitol (Fig. 5A), suggesting a defect in cell wall integrity in hmg1-1 mutant. Then β-glucanase treatments on the hmg1-1 mutant, wild-type and {Delta}pmk1 cells were performed (Toda et al. 1996). Results showed that the hmg1-1 mutant cells lysed faster than wild-type cells, although the sensitivity of hmg1-1 mutant cells to β-glucanase was not as severe as that of the {Delta}pmk1 cells (Fig. 5B), which lack a MAP kinase regulating the cell wall integrity of fission yeast (Toda et al. 1996). These results suggest that hmg1-1 mutant cells are defective in cell wall integrity.


Figure 5
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  		Figure 5  The hmg1-1 mutant showed defective cell wall integrity. (A) High osmolarity suppressed the temperature-sensitive phenotype of the hmg1-1 mutant. Wild-type and hmg1-1 mutant cells were streaked onto each plate as indicated. (B) Cell wall digestion of hmg1-1 mutant and wild-type cells by β-glucanase. Cells exponentially growing in YPD medium were harvested and incubated with β-glucanase (Zymolyase) at 27 °C with vigorous shaking. Cell lysis was monitored by the measurement of optical density at 660 nm (the value before adding the enzyme was taken as 100%). (C) The hmg1-1{Delta}pck1 or hmg1-1{Delta}pck2 double mutants showed more marked temperature sensitivity than the single mutants. Wild-type, hmg1-1, {Delta}pck1, hmg1-1{Delta}pck1, {Delta}pck2 and hmg1-1{Delta}pck2 cells were spotted onto YPD plates and then incubated for 4 days at the temperatures as indicated.

 
To obtain further genetic evidence that hmg1+ is involved in the regulation of cell wall integrity, we crossed the hmg1-1 mutant with either {Delta}pck1 or {Delta}pck2, the deletion mutants of protein kinase C. These protein kinase C homologues are targets of Rho GTPases and differentially regulate cell integrity in fission yeast (Arellano et al. 1999). As shown in Fig. 5C, the hmg1-1{Delta}pck1 or hmg1-1{Delta}pck2 double mutants were more sensitive to high temperature than the hmg1-1 single mutants. The {Delta}pck2 mutant displayed more severe synthetic growth defects as compared with that of the {Delta}pck1 mutant.

Identification of multicopy suppressors of the hmg1-1 mutant

To gain further insights into the mechanisms underlying the cause for the phenotypes observed in the hmg1-1 mutant, we screened for multicopy suppressors of the temperature sensitivity of the mutant. The hmg1-1 mutant was transformed with the multicopy genomic library as described for the cloning of the mutated gene. Approximately 30 000 transformants were obtained and replica-plated onto YPD and EMM plates. When the mutated gene in the hmg1-1 mutant was cloned by complementation of the temperature-sensitive growth, results showed that cells that grew at 36 °C were those cells with plasmids containing the hmg1+ gene. Then, the cells with plasmids that grew on EMM at 34 °C but not at 36 °C were isolated and studied further.

We isolated four genes as multicopy suppressors namely, erg12+, spo9+, pck1+, and an open reading frame with the systematic identification SPBC15C4.03, respectively (Fig. 6A). First, the erg12+ gene encodes mevalonate kinase that is located one step downstream from HMGR in the sterol biosynthesis pathway and catalyzes the ATP-dependent phosphorylation of mevalonic acid to form mevalonate 5-phosphate. Second, the spo9+ gene encodes geranylgeranyl pyrophosphate synthase (Ye et al. 2007) that is also located downstream of Hmg1 and Erg12. Third, the pck1+ gene encodes a homologue of protein kinase C that is regulated by Rho GTPases as described above. Pck1 suppressed the FK506-sensitive phenotype more efficiently than the other suppressors. Finally, SPBC15C4.03 encodes a homologue of Rab escort protein. It significantly suppressed the temperature-sensitive phenotype of the hmg1-1 mutant on YPD plate. We named the gene rep1+ for Rab escort protein.


Figure 6
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  		Figure 6  Multicopy suppressors, genetic interaction and intracellular localization of small GTPases. (A) Effect of high copy suppressor on the growth of hmg1-1 mutant. The hmg1-1 mutant was transformed with plasmids containing genes identified by a high copy suppressor screen. Cells were spotted onto each plate as indicated. (B) Genetic interaction between hmg1-1 and ypt3-i5 mutant. Wild-type, hmg1-1, ypt3-i5 and hmg1-1ypt3-i5 cells were spotted onto YPD plates and then incubated for 4 days at the temperatures as indicated. (C) The localization of GFP-Rho2 and GFP-Ypt3 in hmg1-1 cells. The wild-type and hmg1-1 mutant cells expressing GFP-tagged small GTPases were incubated at 36 °C for 4 h, fixed by 0.1% formalin, and were observed under fluorescent microscope. Bar, 10 µm.

 
Rab escort protein serves as a chaperone to introduce the prenyltransferase to its Rab protein substrate (Anant et al. 1998). Then, we suggested that its overexpression may enhance the efficacy of defective prenylation of Rab proteins caused by the hmg1-1 mutation and thus affect membrane trafficking. Then, we crossed the hmg1-1 mutant with the ypt3-i5 mutant. Ypt3 is a member of the Rab11 GTPase family that is required for the formation of trans-Golgi derived vesicles (Cheng et al. 2002). Results showed that the hmg1-1ypt3-i5 double mutants were more sensitive to high temperature than the respective single mutants (Fig. 6B).

The hmg1-1 mutant cells showed normal subcellular localization of small GTPases

In our previous published article, we showed that Rho2 is a target of the farnesyltransferase Cpp1 and cpp1 mutation or deletion resulted in the loss of plasma membrane-associated GFP-Rho2 (Ma et al. 2006). This prompted us to examine the localization of GFP-Rho2 and GFP-Ypt3 in hmg1-1 cells. The wild-type and hmg1-1 mutant cells expressing GFP-tagged small GTPases were incubated at 36 °C for 4 h, fixed by 0.1% formalin, and were observed under fluorescent microscope. In the hmg1-1 mutant cells, GFP-Rho2 localized at the plasma membrane and GFP-Ypt3 localized at the cell tips and at the intracellular dot-like structures in the same pattern as that in wild-type cells (Fig. 6C). These results suggest that reduced prenylation in hmg1-1 mutant alone is not sufficient to cause the mutant phenotypes.

Effects of pravastatin on the Schizosaccharomyces pombe mutants that showed genetic interaction with the hmg1-1 mutant

As described above, the hmg1-1 mutant showed synthetic growth defects with the mutant alleles of five genes that encode Cdc7, Cdc11, Pck1, Pck2 and Ypt3, respectively. Then we examined the effects of pravastatin, a competitive inhibitor of HMGR, on these mutants. The calcineurin deletion, {Delta}ppb1, was also examined for its sensitivity to pravastatin. Among the five mutants tested, only the ypt3-i5 mutant cells exhibited drug sensitivity. The ypt3-i5 mutant failed to grow in the presence of 0.3 mg/mL of pravastatin, whereas cdc7-24, cdc11-136, {Delta}pck1, {Delta}pck2 or {Delta}ppb1 cells grew (Fig. 7A). It was noted that none of the mutants tested showed higher drug sensitivity than that of the hmg1-1 mutant.


Figure 7
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  		Figure 7  Ergosterol deficiency might not be the primary cause for the pleiotropic defects of the hmg1-1 mutant. (A) The ypt3-i5 mutant showed hypersensitivity to pravastatin. Wild-type, hmg1-1, cdc7-24, cdc11-136, {Delta}pck1, {Delta}pck2, ypt3-i5 and {Delta}ppb1 cells were spotted onto each plate containing YPD or YPD plus pravastatin as indicated, and then incubated at 27 °C for 4 days. (B) Effects of various drugs on the hmg1-1 mutant and disruption mutants of sterol biosynthesis genes. Cells were spotted onto each plate as indicated, and then incubated at 27 °C for 4 days or at 36 °C for 3 days.

 
Effects of various drugs on the hmg1-1 mutant and disruption mutants of sterol biosynthesis genes

Recently, Iwaki et al. reported that disruption mutants of sterol biosynthesis genes were deficient in ergosterol and were resistant to polyene drugs such as amphotericin B (AmB) (Iwaki et al. 2008). The polyene drugs bind ergosterol, creating pores and causing cell lysis. Therefore, the tolerance to these drugs reflects the ergosterol deficiency of the mutants. Then we compared the tolerance to these drugs between the hmg1-1 mutant and disruption mutants of sterol biosynthesis genes, namely erg31+, erg32+, erg5+ and sts1+. The erg31+ gene and the erg32+ gene with the systematic identification SPAC1687.16c and SPBC27B12.03c, respectively, encode C-5 sterol desaturase; the erg5+ gene encodes C-22 sterol desaturase Erg5; and the sts1+ gene encodes C-24 (28) sterol reductase Sts1 (Iwaki et al. 2008). All of them were responsible for catalyzing a sequence of reactions from zymosterol to ergosterol. As regards the effect of AmB (Fig. 7B), the hmg1-1 mutant was sensitive, whereas {Delta}erg31{Delta}erg32 ({Delta}erg31&32), {Delta}erg5 and {Delta}sts1 disruption mutants were resistant. Notably, the hmg1-1 mutant was slightly but significantly more resistant to AmB compared with the wild-type cells. These results are consistent with the data obtained with filipin staining of the mutant cells which was indistinguishable from that obtained with the wild-type cells at the permissive temperature of 27 °C (Fig. 3A). Taken together, these observations suggest that ergosterol synthesis is attenuated, but its ergosterol level is not so affected in the hmg1-1 mutant as that of the disruption mutants of sterol biosynthesis genes. As regards the effect of high temperature and the effect of pravastatin (Fig. 7B), the hmg1-1 mutant was very sensitive while the disruption mutants were not sensitive. Iwaki et al. also reported that disruption mutants of sterol biosynthesis genes deficient in ergosterol were found to be hypersensitive to cycloheximide (Iwaki et al. 2008). As regards the effect of cycloheximide (CHX) (Fig. 7B), the ergosterol-deficient mutants especially {Delta}erg31{Delta}erg32 and {Delta}sts1 were hypersensitive to cycloheximide while the hmg1-1 mutant was not hypersensitive. As regards the effect of FK506 (Fig. 7B), however, all the ergosterol-deficient mutants were not hypersensitive to FK506 while the hmg1-1 mutant was hypersensitive. Taken together, drug-sensitivity characteristics between the hmg1-1 mutant and the disruption mutants of sterol biosynthesis genes were distinct suggesting that ergosterol deficiency alone is not sufficient to cause the mutant phenotypes of the hmg1-1 mutant.


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
HMGR, which catalyzes the conversion of HMG-CoA to mevalonate and constitutes the rate-limiting step in the biosynthesis of cholesterol, and inhibition of HMGR is the target of the most widely prescribed drug, statins, that is used to lower serum cholesterol (Shepherd et al. 1995; Futterman & Lemberg 2005). In addition to the cholesterol lowering effect, there are reports on the "pleiotropic" effects of statins such as those involved in improving the endothelial function, enhancing the stability of atherosclerotic plaques, decreasing oxidative stress and inflammation, and inhibiting the thrombogenic response (Liao & Laufs 2005), and the clinical significance of the pleiotropic effects of statins have been well-discussed (Davidson 2005; Wang et al. 2008). Many potential pleiotropic effects of HMGR inhibitors have been reported by statin administration in rodents because statins do not lower circulating cholesterol concentrations in these animals (Davidson 2005). Statin-induced depletion of isoprenoids has been considered to be the most likely cause of these pleiotropic effects, because in the mevalonate pathway, the downstream products such as farnesylpyrophosphate and geranylgeranylpyrophosphate are essential isoprenoids for the function of small GTPases including Ras, Rho, Rac and Rab, which are essential key regulators of many biological processes (Takai et al. 2001; Liao & Laufs 2005). In man, the adverse effects of statins on the skeletal muscles including myalgia, myositis and rhabdomyolysis are also possibly caused by the depletion of isoprenoids (Rosenson 2004). However, it is still difficult to confirm the cholesterol-independency of these pleiotropic effects due to the complexity of the mammalian system (Li et al. 2003).

HMGR and small GTPases are highly conserved between yeasts and human, and yeast genetic approaches may prove useful in defining the cause of the pleiotropic effects of statins or in examining the effects of HMGR mutation. In budding yeast, however, a single deletion mutant of HMG1 or HMG2 has only a subtle growth defect other than growth on the medium containing the HMGR inhibitor compactin (Basson et al. 1986, 1987). Attempts to isolate temperature-sensitive lethal mutations in genes encoding enzymes of the sterol biosynthetic pathway also failed to identify mutations affecting HMGR activity (Servouse et al. 1984). In our study here, we showed that a mutation in the fission yeast hmg1+ gene caused temperature and FK506 sensitivity, and defects in cytokinesis and cell wall integrity. We compared the drug-sensitivity characteristics between the hmg1-1 mutant and the disruption mutants of sterol biosynthesis genes, and results suggest that these phenotypes are "pleiotropic", that is, the defects are not caused by ergosterol deficiency alone. Probably, these phenotypes are caused by the integrated effects of the reduced availability of ergosterol and various intermediates of the mevalonate pathway.

Previous study reported that the hmg1 null mutant is lethal (Lum et al. 1996). Here we demonstrated that the hmg1-1 allele contains an opal nonsense mutation at codon 234, which might result in a truncated protein lacking at the C-terminal three-fourths of the full-length product including a catalytic domain of Hmg1 that supplies the essential HMGR activity. So how is this mutant strain viable? When the mutated gene was expressed in wild-type cells (Fig. 2E), the immunoblot analysis detected a full-length protein with reduced amount but not a truncated protein. These results suggest that the viability of the hmg1-1 mutant might be the result of translational read-through, inasmuch as TGA is an infrequently (15%) used codon in S. pombe (Forsburg 1994). Consistent with this hypothesis, overexpression of tef1+ gene with the systematic identification SPAC23A1.10 encoding an elongation factor 1-{alpha} that is required during the elongation phase of translation suppressed the phenotypes of the hmg1-1 mutant (data not shown). Also in support of this hypothesis, overexpression of tRNA tryptophan encoded by the gene with the systematic identification SPATRNATRP.01 suppressed the phenotypes of the hmg1-1 mutant, as reported by Hsieh et al. (2004) that read-through of UGA stop codons by tRNATRP expressed (data not shown). Expression of the mutated gene did not fully suppress the mutant phenotype and was more toxic as compared with that of the wild-type gene. This suggests that the introduced termination codon may be mistranslated at some frequency. The proteins produced by the mistranslation might be nonfunctional and might also have the dominant negative effects on the cell growth as shown in Fig. 2A. The dominant negative nature of the mutation is consistent with the insufficient complementation of the hmg1-1 mutation by wild-type hmg1+ as shown in Fig. 1A. The mutation site of the hmg1-1 mutant, tryptophan at 234, is located in the transmembrane domain (Fig. 1B, lower panel). This site is also involved in the sterol-sensing domain where a homologue of Insig, Ins1, binds and regulates the phosphorylation of Hmg1 (Burg et al. 2008). Probably, Hmg1 phosphorylation might be reduced in the hmg1-1 mutant that contains an opal nonsense mutation at codon 234, thereby causing the dominant negative effects of the hmg1-1 mutant.

When the mutated gene tagged with GFP was expressed in wild-type cells, the amount of the mutant protein was lower (approximately 30%–50%) than the wild-type protein in both attenuated and induced conditions (Fig. 2C). Thus, it is suggested that lower ergosterol levels may be the direct cause of the mutant phenotypes observed. However, to our surprise, the hmg1-1 mutant was only slightly more resistant to AmB as compared with that of wild-type cells, suggesting that ergosterol level in the mutant is not very low as might be expected. This is in good agreement with the normal filipin staining pattern observed in the hmg1-1 mutant at 27 °C (Fig. 3A). In addition, the phenotype of the hmg1-1 mutant was not similar to that of any of the disruption mutants of sterol biosynthesis genes. Furthermore, hmg1-1 mutation showed synthetic growth defects with these disruption mutants (our unpublished observation). These results suggest that ergosterol deficiency alone is not sufficient to cause the mutant phenotypes. As described in the results, other researchers have shown that yeasts are more sensitive to FK506 and to inhibitors of sterol biosynthesis that act downstream of HMGR than to either drug alone (Onyewu et al. 2003). However, in the present study, we showed that {Delta}ppb1 (calcineurin deletion) was not hypersensitive to pravastatin. Although at present we do not know the relationship between sterol biosynthesis and calcineurin-mediated signal transduction, these results suggest that the HMGR inhibitor and the inhibitors of the enzymes that act downstream of HMGR in the ergosterol biosynthesis pathway have different effects on the physiology of yeast cells. These results also suggest that ergosterol deficiency is not the primary cause for the phenotypes of hmg1-1 mutant.

Then, we suggested that a defective isoprenoids biosynthesis in the hmg1-1 mutant cells may result in insufficient prenylation of Rho and Rab GTPases, which in turn affects cytokinesis and cell wall integrity because of the following observations: (i) the hmg1-1 mutant cells showed defects in cytokinesis and cell wall integrity that are regulated by Rho GTPases in fission yeast (Arellano et al. 1996; Garcia et al. 2006); (ii) genes encoding protein kinase C that are activated by Rho GTPases showed gene dosage-dependent suppression of the phenotype of hmg1-1 mutant; and (iii) overexpression of rep1+ that encodes Rab escort protein suppressed the phenotype of hmg1-1 mutant. In addition, Bialek-Wyrzykowska et al. reported the potential relationship between decreased prenylation as well as temperature sensitivity, and cell wall defects (Bialek-Wyrzykowska et al. 2000). To test whether the degree of prenylation in these small GTPases is affected by the mutation, we examined the localization of two small GTPases, Rho2 and Ypt3, in hmg1-1 mutant. As shown in Fig. 6C, the subcellular localization of GFP-tagged small GTPases appeared to be normal in hmg1-1 mutant cells, suggesting that reduced prenylation is not the primary cause for the phenotypes of hmg1-1 mutant, either.

In our previous study, we suggested that calcineurin is involved in the regulation of the septation initiation network pathway, and is required for the proper formation and maturation of the septum in fission yeast (Lu et al. 2002). Our present study showing the genetic interaction between calcineurin and HMGR suggest that HMGR is also involved in the regulation of cytokinesis.

In summary, our results suggest that an opal nonsense mutation caused the pleiotropic phenotypes of hmg1-1 mutant, and that ergosterol deficiency or reduced prenylation is not the primary cause for the pleiotropic defects. Probably, the defects reflect the integrated effects of the reduced availability of various intermediates of the mevalonate pathway. For example, the function of many membrane-bound or membrane associated proteins can be affected by changes in membrane fluidity or composition caused by decreased ergosterol production. Thus, rather than being a direct effect on prenylation alone, the observed phenotypes may reflect indirect effects on the function or activity of enzymes involved in prenylation.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Strains, media, genetic and molecular biology techniques

Schizosaccharomyces pombe strains used in this study are listed in Table 1. The complete medium, YPD, and the minimal medium, EMM, have been described previously (Toda et al. 1996). Standard methods for S. pombe genetics were performed as described previously (Moreno et al. 1991). Gene disruptions are abbreviated by the gene preceded by {Delta} (for example, {Delta}pck1). Proteins are denoted by roman letters and only the first letter is capitalized (for example, Hmg1). FK506 and pravastatin were obtained from Astellas Pharma (Tokyo, Japan) and Daiichi Sankyo (Tokyo, Japan), respectively. All other chemicals and reagents were from commercial sources.

Gene expression

For ectopic expression of proteins, we used the thiamine-repressible nmt1 promoter (Basi et al. 1993; Maundrell 1993). Expression was repressed by the addition of 4 µg/mL thiamine to EMM and was induced by washing and incubating the cells in EMM lacking thiamine. To express Hmg1 tagged with green fluorescent protein (GFP), the genes from wild-type or hmg1-1 cells were tagged at their C terminus with GFP carrying S65T mutation. To obtain the chromosome-borne gene instead of the plasmid-borne gene, the gene with the nmt1 promoter was subcloned into the vector containing the ura4+ marker and was integrated into the chromosome at the ura4+ gene locus of KP1248 (Table 1). GFP-Rho2 and GFP-Ypt3 were expressed as described previously (He et al. 2006; Ma et al. 2006).

Bioinformatics

Database searches were performed using the National Center for Biotechnology Information BLAST network service (http://www.ncbi.nlm.nih.gov) and the Sanger Center S. pombe data base search service (http://www.sanger.ac.uk). Sequence alignment was performed using protein BLAST and the ClustalW program.

Microscopy and miscellaneous methods

Methods in light microscopy, such as fluorescence microscopy and differential interference contrast (DIC) microscopy, were performed as described (Cheng et al. 2002; Kita et al. 2004). Calcofluor and 4',6-diamidino-2-phenylindole (DAPI) were used to visualize cell wall and DNA, respectively as described previously (Cheng et al. 2002). For staining sterol, we used the fluorescent probe filipin, a polyene antibiotic that forms specific complexes with free 3-β-hydroxysterols (Drabikowski et al. 1973). Cells were grown to exponential phase in YPD medium at 27 °C, fixed in 2% formalin for 5 min, washed with phosphate buffered saline, and filipin was added to the medium and cells were observed immediately. Cell wall digestion by β-glucanase (Zymolyase, Seikagakukogyo, Tokyo, Japan) was performed as described (Toda et al. 1996).


   Acknowledgements
 
We thank Kaoru Takegawa, Takashi Toda and Mitsuhiro Yanagida for providing the strains and plasmids, and Susie O. Sio for critical reading of the manuscript, and Astellas Pharma Inc. for gifts of FK506. This work was supported by 21st Century COE Program, Global COE Program and research grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan.


   Footnotes
 
Communicated by: Yoshimi Takai

{dagger}These authors contributed equally to this article. Back

* Correspondence: fangyue{at}mail.cmu.edu.cn


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Accepted: 24 March 2009





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