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Vol. 18, Issue 9, 3568-3581, September 2007
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*Department of Biology, Graduate School of Science, Osaka City University, Sumiyoshi-ku, Osaka 558-8585, Japan; Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Kashiwa, Chiba 277-8562, Japan; and Department of Applied Bioscience and Biotechnology, Faculty of Life and Environmental Science, Shimane University, Matsue 690-8504, Japan
Submitted February 8, 2007;
Revised May 23, 2007;
Accepted June 20, 2007
Monitoring Editor: Akihiko Nakano
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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cells. Electron microscopy revealed significant accumulation membrane vesicles in spo9 cells. We suggest that lack of GGPS activity in a spo9 mutant results in impaired protein prenylation in certain proteins responsible for secretory function, thereby inhibiting forespore membrane formation. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Szkopinska and Plochocka, 2005). FPS catalyzes the sequential condensation of dimethylallyl diphosphate (DMAPP) and geranyl diphosphate (GPP) with isopentenyl diphosphate (IPP). The product, farnesyl diphosphate (FPP), is the precursor for several important metabolites, including sterols, dolichols, ubiquinones, and heme a (for review, see Kawamukai, 2002). GGPS catalyzes a similar condensation reaction as FPS. It uses DMAPP, GPP, and FPP as allylic substrates to generate an amphipathic molecule containing four isoprene units, GGPP, which is a precursor of carotenoids and chlorophylls. FPP and GGPP are also substrates for protein prenylation mediated by farnesyl transferase and geranylgeranyl transferase. Protein prenylation is a posttranslational modification by which isoprenoid compounds are covalently attached to cysteine residues at or near the C terminus of proteins (Casey 1992, 1994; Schafer and Rine, 1992; Omer and Gibbs, 1994). Prenylated proteins, which include small GTP-binding proteins such as Ras and Rab, account for 
2% of total cellular proteins (Epstein et al., 1991). Prenylation has generally been found to be essential for proper localization of membrane proteins.
Sporulation in the fission yeast Schizosaccharomyces pombe is equivalent to gametogenesis in higher eukaryotes. This unique process includes two overlapping processes, meiosis and spore formation. Four haploid nuclei produced by meiosis are packaged into individual spores. During meiosis II, membrane vesicles accumulate in the vicinity of the spindle pole body, a structure equivalent to the centrosome in animal cells, and they fuse to generate a double unit membrane, termed the forespore membrane. The forespore membrane expands by fusing with membrane vesicles, and eventually it encapsulates each of the four nuclei produced by meiosis. Spore wall material is deposited in the lumenal space of the forespore membrane, and its inner membrane becomes the plasma membrane of the spores. Assembly of forespore membranes provides a model system for studying the de novo biogenesis of membrane compartments within the cytoplasm (Yoo et al., 1973; Tanaka and Hirata, 1982; Hirata and Shimoda, 1992, 1994). Many sporulation-deficient S. pombe mutants have been isolated (Bresch et al., 1968; Kishida and Shimoda, 1986). Thus far, we have analyzed spo3+, spo4+, spo6+, spo14+, spo15+, spo19+, and spo20+ (Ikemoto et al., 2000; Nakamura et al., 2000, 2001, 2002; Nakase et al., 2001; Nakamura-Kubo et al., 2003). spo3+, spo14+, and spo20+ have been shown to be involved in expansion of the forespore membrane. spo3+ encodes a membrane protein. Spo3 is expressed exclusively during meiosis and localizes to the forespore membrane (Nakamura et al., 2001). Both spo14+ and spo20+ encode components of a vesicle-trafficking pathway (Nakase et al., 2001; Nakamura-Kubo et al., 2003). In addition to these spo+ genes, several other genes are necessary for expansion of the forespore membrane. psy1+ and sec9+, both of which encode components of the plasma membrane target membrane-associated soluble N-ethylmaleimide-sensitive factor-attachment protein receptor complex, are required for both vegetative growth and sporulation (Nakamura et al., 2001, 2005). vps genes, which are involved in vacuolar protein sorting, are also responsible for expansion of the forespore membrane (Iwaki et al., 2003; Onishi et al., 2003; Koga et al., 2004; Kashiwazaki et al., 2005).
Among many sporulation-deficient mutants, spo9 has not been well characterized. In the present study, we isolated and analyzed the spo9+ gene, which encodes a protein with significant homology to a typical FPS. We also identified another gene, fps1+, which encodes FPS. Although fps1+ was found to be essential for growth, spo9+ was required for proper assembly of the forespore membrane. Despite its high sequence homology with FPS, Spo9 was largely responsible for GGPS activity. We report the novel finding that S. pombe GGPS consists of a heteromer made up of a typical FPS protein, Fps1, and an FPS-like protein, Spo9.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Leu+ transformants on SSA (7500 colonies) were then exposed to iodine vapor (Gutz et al., 1974). The few colonies that turned brown were taken as candidates for sporulation-proficient transformants. Two plasmids were isolated from these candidates and their nucleotide sequences were determined. One of these plasmids, pYF1, was further analyzed (Table 2).
Cloning of spo9+ and fps1+
The spo9-B261 mutant YF2 was transformed with the S. pombe genomic library, pTN-L1 (Nakamura et al., 2001). Leu+ transformants on SSA (7500 colonies) were then exposed to iodine vapor (Gutz et al., 1974). The few colonies that turned brown were taken as candidates for sporulation-proficient transformants. Two plasmids were isolated from these candidates and their nucleotide sequences were determined. One of these plasmids, pYF1, was further analyzed (Table 2).
The fps1+ gene was cloned as follows. Polymerase chain reaction (PCR) primers 5'-ATATTTTTCTCCTCAACCTCTAGTAGCTTT-3' and 5'-GCGCGGTCGAC(SalI)ATTATTAATGGATTTGCTG-3' were used to amplify a 3.5-kb fragment encompassing the entire fps1+ gene, and the resulting BamHI–SalI fragment was subcloned into pBluescript II-KS+ (Stratagene, La Jolla, CA) to generate pBS(fps1).
Disruption of spo9+ and fps1+
Disruptions of the spo9+ and fps1+ genes by using ura4+ were constructed as follows. PCR primers 5'-AGAGAGCGGCCGC(NotI)AAACGGATCCAGATGGTTA-3' and 5'-AGAGACTCGAG(XhoI)TTCGGAGCGCTTTCGCGTAGAATT-3' were used to amplify a 2.7-kb fragment encompassing the entire spo9+ gene. The resulting NotI–XhoI fragment was then subcloned into pGEM-T easy vector (Promega, Madison, WI) to generate pGEM(spo9). After digestion with HindIII and XbaI, pGEM(spo9) was filled in and ligated to a HindIII linker. The resulting plasmid was digested with HindIII, and a 1.7-kb ura4+ fragment was inserted at the HindIII site. The resulting plasmid was digested with BglII and BamHI and used to transform strain TN75.
To disrupt fps1+, pBS(fps1) was digested with PstI and EcoRI to remove a 1.1-kb fragment of the open reading frame (ORF). A HindIII linker was then inserted into at these restriction sites. A 1.7-kb ura4+ fragment was inserted at the HindIII site. The resulting plasmid was digested with ClaI and BamHI and used to transform strain TN75. Disruptions were confirmed by Southern hybridization of genomic DNA (data not shown).
Southern and Northern Blot Analyses
Genomic DNA was digested, separated in a 1% agarose gel, and then transferred onto nylon membranes for Southern blotting. Total RNA was isolated from cells harvested at specific times. The isolated RNA was separated on a 1% agarose gel, transferred to a nylon membrane, and subjected to Northern blot analysis (Thomas, 1980).
Nucleotide Sequence Analysis of the spo9-B261 Mutant Allele
The entire spo9+ ORF and promoter region were amplified by PCR with primers 5'-CTCCACTCGAG(XhoI)CATATAGCCATGGGTTCTC-3' and 5'-AGAGACTCGAG(XhoI)TTCGGAGCGCTTTCGCGTAGAATT-3', by using genomic DNA from the spo9-B261 mutant as template. The amplified DNA fragment was cloned into pGEM-T easy vector and sequenced.
Ubiquinone Extraction and Measurement
Ubiquinone was extracted as described previously (Kainou et al., 1999). The extracted crude ubiquinone was analyzed by normal phase thin layer chromatography (TLC) with an authentic standard UQ6. Normal phase TLC was carried out on a Kiesel gel 60 F254 plate (Merck, Frankfurt, Germany) by using benzene:acetone (97:3, vol/vol). The UV-visualized band containing ubiquinone was collected from the TLC plate and extracted with chloroform:methanol (1:1, vol/vol). Samples were dried, and the pellets were redissolved in ethanol. The purified ubiquinone was further analyzed by high-performance liquid chromatography using ethanol as the solvent.
Prenyl Diphosphate Synthase Assay
Prenyl diphosphate synthase activity was measured by detection of [1-14C]IPP incorporated into reaction products as described previously (Kainou et al., 2001; Saiki et al., 2003). E. coli DH5
harboring a plasmid containing the human GGPS1 gene was incubated to late log phase in LB medium containing ampicillin at 37°C. Cells were harvested by centrifugation, suspended in buffer A (100 mM potassium phosphate, pH 7.4, 5 mM EDTA, and 1 mM 2-mercaptoethanol), and ruptured by sonication (six 30-s pulses with 30-s intervals in an ice bath between sonications). After centrifugation of the homogenate, the supernatant was used as a crude enzyme extract. Similarly, S. pombe cells were grown to mid- to late log phase in minimum medium. Cells were harvested by centrifugation and suspended in buffer A. The washed cells were ruptured by vigorous mixing with glass beads 14 times for 30 s with 60-s intervals in an ice bath between mixings. After centrifugation of the homogenate, the supernatant was used as a crude enzyme extract. The assay reaction mixture contained 1.0 mM MgCl2, 0.1% (wt/vol) Triton X-100, 50 mM potassium phosphate buffer, pH 7.5, 10 µM [1-14C]IPP (specific activity 0.92 TBq/mol), 5 µM FPP, and 200 µg of crude extract containing the enzyme in a final volume of 0.4 ml. Sample mixtures were incubated for 60 min at 30°C. Reaction products such as prenyl diphosphates were extracted with 1-butanol–saturated water and hydrolyzed with acid phosphatase. Hydrolysis products were extracted with hexane and analyzed by reversed phase TLC by using acetone:water (19:1, vol/vol). Radioactivity on the TLC plate was detected with an imaging analyzer BAS1500-Mac (Fuji Film, Tokyo, Japan). The plate was exposed to iodine vapor to detect marker prenols.
Yeast Two-Hybrid Analysis
For yeast two-hybrid analysis, PCR primers 5'-GCGCGCCATGG(NcoI)AAATGGTCAACGATTTTAA-3' and 5'-GCGCGCTCGAG(XhoI)CTACCGTGACCTTTTGTAA-3' were used to amplify the spo9+ gene. The amplified fragment was digested with NcoI and XhoI and inserted into the corresponding sites of pGBKT7 (GAL4 DNA binding domain; Takara-Bio, Otsu, Japan) and pGADT7 (GAL4 activation domain; Takara-Bio), yielding pGBK(spo9) and pGAD(spo9), respectively. Similarly, PCR primers 5'-GCGCGCCATGG(NcoI)AGATGTCTGCAGTTGATAA-3' and 5'-GCGCGCTCGAG(XhoI)TTACTTATTTCTTTTGTAA-3' were used to amplify the fps1+ gene. The amplified fragment was digested with NcoI and XhoI, and inserted into the corresponding sites of pGBKT7 and pGADT7 yielding pGBK(fps1) and pGAD(fps1), respectively. These plasmids were introduced into S. cerevisiae AH109 (Takara-Bio), and transformants were replicated onto selective minimal medium supplemented with appropriate amino acids, except for His to assay for protein interactions in vivo.
Coimmunoprecipitation Assays
The wild-type strain (YF76) was transformed with pDS473(spo9) plus pAL(fps1-GFP), or with pDS473(spo9) plus pTN54 to yield YF69 and YF75 strains, respectively. YF69 and YF75 were grown overnight at 28°C in MM. The cells were harvested in log phase, resuspended in TES (20 mM Tris-HCl, pH 7.5, 1 mM EDTA, and 150 mM NaCl) and then ruptured with glass beads. The lysate was centrifuged at 800 x g for 5 min to remove cell debris. The supernatant was then centrifuged at 13,000 x g for 20 min to prepare a soluble fraction. The soluble fraction was incubated with mouse anti-green fluorescent protein (GFP) antibody (Roche Diagnostics, Mannheim, Germany) at 4°C for 1 h, and then mixed with protein G-Sepharose (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom). After further incubation at 4°C for 1 h, the solution was centrifuged at 800 x g for 5 min. The pellet was washed with TES three times and resuspended in sample buffer. Samples were electrophoresed in SDS-polyacrylamide gels, and the target polypeptides were detected by Western blot analysis using rat anti-GFP (a generous gift from S. Fujita) or mouse anti-glutathione S-transferase (GST) (Sigma-Aldrich, St. Louis, MO) antibody.
Subcellular Fractionation
pTN381(GFP-ypt7) was digested with SnaBI and the linear plasmid was integrated into the leu1 locus of wild type (TN8) and the spo9 mutant (YF16) to yield YF51 and YF54, respectively. The YF51 and YF54 strains were grown overnight at 28°C in MM. The cells were harvested in log phase, ruptured with glass beads as described above, and centrifuged at 800 x g for 5 min. The supernatant was further centrifuged at 100,000 x g for 1 h to separate the soluble fraction, the pellet being resuspended in an equal volume of resuspension buffer. The samples from each fraction were subjected to Western blot analysis by using either mouse anti-GFP (Roche Diagnostics), rabbit anti-Rhb1 (Nakase et al., 2006), or rabbit anti-Spo14 (Nakamura-Kubo et al., 2003) antibodies.
Fluorescence Microscopy
Cells were fixed as described previously (Hagan and Hyams, 1988) by use of glutaraldehyde and paraformaldehyde. The Psy1 protein was visualized by fusing it to GFP. The nuclear chromatin region was stained with 4,6-diamidino-2-phenylindole (DAPI). Stained cells were observed under a fluorescence microscope (model BX51; Olympus, Tokyo, Japan), and images were obtained using a CoolSNAP charge-coupled device camera (Roper Scientific, San Diego, CA).
Electron Microscopy
Cells were mounted on copper grids to form a thin layer, and then they were submerged in liquid propane cooled with liquid N2. Frozen cells were transferred to 2% OsO4 in anhydrous acetone, held at –80°C for 48 h in a solid CO2-acetone bath, and then transferred to –35°C for 2 h, 4°C for 2 h, and room temperature for 2 h. After washing with anhydrous acetone three times, samples were infiltrated with increasing concentrations of Spurr's resin in anhydrous acetone, and finally with 100% Spurr's resin. Samples were then polymerized at 50°C for 5 h and 60°C for 50 h. Thin sections were cut on a Reichert Ultracut S ultramicrotome, and then stained with uranyl acetate and lead citrate. Sections were viewed on an electron microscope H-7600 (Hitachi, Tokyo, Japan) at 100 kV.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) (Figure 1A). In addition, the spo9-B261 mutant also exhibited temperature-sensitive vegetative growth (Figure 1B). These results indicate that the spo9+ gene is required not only for sporulation but also for vegetative growth. To identify the spo9+ gene product and its biological function, we isolated the spo9+ gene by functional complementation (see Materials and Methods). Two plasmids were isolated that were able to rescue both the temperature sensitivity and sporulation deficiency of spo9-B261. Subcloning and partial sequencing revealed that the spo9-complementing ability was due to one ORF composed of 1.2 kb (SPBC36.06c) (Figure 2A). The spo9+ gene encodes a 40.0-kDa protein consisting of 351 amino acid residues (Figure 2B). The predicted Spo9 protein shares 42 and 38% identity and 64 and 61% similarity with the S. cerevisiae FPS, Erg20, and human FPS, respectively (Figure 2B). FPS catalyzes chain elongation of the C5 substrate DMAPP to the C15 product FPP by addition of two molecules of IPP. Spo9 contains two aspartate-rich domains, first aspartate-rich motif: consensus DDXX(XX)D (FARM), and second aspartate-rich motif: consensus DDXXD (SARM), conserved among many prenyltransferases, including FPSs and GGPSs (Koyama et al., 1993; Chen et al., 1994; Song and Poulter, 1994; Szkopinska and Plochocka, 2005) (Figure 3A).
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) was viable but displayed temperature sensitivity and sporulation deficiency like the original spo9-B261 mutant (Figure 1, A and B). Because most of the meiosis-defective mutants isolated to date are unable to sporulate (Bresch et al., 1968; Kishida and Shimoda, 1986), one cannot rule out the possibility that the spo9 mutant has a defect in meiosis. Therefore, we analyzed the meiotic nuclear divisions in spo9. The first and second meiotic divisions were found to proceed with kinetics similar to that observed in an isogenic wild-type strain, with the final yield of tetranucleate cells reaching 90% (data not shown). These results suggest that the spo9 mutant is able to complete meiosis but that it is defective in ascospore formation.
Sequencing of the spo9-B261 allele revealed a direct duplication of 20-base pairs (from the 926th to 945th nucleotide of wild-type spo9+) (Figure 2C). This duplication caused a frameshift, resulting in a truncated protein of 272 amino acid residues. This truncated protein lacked the conserved hydrophilic C terminus, which is important for FPS activity (Song and Poulter, 1994). The spo9-B261 mutant protein seems to have lost all activity, like the spo9 disruptant.
Although the S. pombe genes responsible for mating, meiosis, and sporulation are generally transcribed under conditions of nutritional starvation (Yamamoto et al., 1997; Watanabe et al., 2001; Shimoda, 2004), Northern analysis revealed that spo9+ transcription occurred during vegetative growth and that it was not enhanced, but rather diminished, after the shift to a nitrogen-free medium (Figure 4).
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) indicated that only two spores per ascus were viable and produced Ura– colonies. Microscopic observation of the inviable spores showed that most germinated, but they seemed to have undergone only one or two divisions before growth arrest. Therefore, we conclude that fps1+ is essential for cell viability, but not for germination.
Genetic Relationship among S. pombe and S. cerevisiae FPS- and GGPS-related Genes
To test the possibility of functional redundancy between Fps1 and Spo9, we examined whether overexpression of fps1+ could rescue the phenotypes of spo9. fps1+ was placed under control of the thiamine-repressible nmt1 promoter. The resultant expression plasmid, pREP1(fps1), rescued both the temperature-sensitive growth and sporulation deficiency, when introduced into spo9 cells (Figure 5, C and D). In contrast, pREP1(spo9) could not rescue the lethality of the fps1 null mutant (data not shown).
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; Blanchard and Karst, 1993). To assess the level of functional relatedness between S. pombe and S. cerevisiae FPSs, spo9+ and fps1+ were expressed under control of the GAL1 promoter in an erg20 mutant of S. cerevisiae. Ectopic expression of fps1+ but not spo9+ rescued the lethality of the erg20 null mutation in S. cerevisiae (Figure 5A). In a reciprocal manner, S. cerevisiae ERG20 was placed under the control of the thiamine-repressible nmt1 promoter and introduced into S. pombe fps1 and spo9 mutants. ERG20 rescued both the lethality of the fps1 mutant (Figure 5B) and the temperature sensitivity and sporulation deficiency of a spo9 mutant (Figure 5, C and D). These data establish that fission yeast Fps1 is able to fulfill all essential Erg20 functions.
GGPS and FPS belong to a family of prenyltransferases that catalyze consecutive condensations of IPP with allylic primer substrates. A number of genes of GGPS (Misawa et al., 1990; Ohnuma et al., 1994; Jiang et al., 1995; Zhu et al., 1997; Kainou et al., 1999; Kuzuguchi et al., 1999; Okada et al., 2000; Engprasert et al., 2004) and FPS (Chambon et al., 1990; Fujisaki et al., 1990; Koyama et al., 1993; Delourme et al., 1994; Cunillera et al., 1996; Koyama, 1999; Szkopinska and Plochocka, 2005) have been isolated from various organisms. Sequence alignment of the encoded enzymes reveals limited partial homology (Figure 3A). Apparently, the enzymes have been grouped based on their primary structures (Chen et al., 1994) (Figure 3B). However both enzymes share two aspartate-rich motifs as described above. A BLAST search of the nonredundant proteins in the database at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/BLAST/) was carried out using human GGPS (accession no. AAH05252) as a template to identify its S. pombe orthologue. We selected proteins whose e-value is <1.0. Two S. pombe prenyl diphosphate syntheses, Dps1 (e-value 4e-06) and Fps1 (e-value 2e-02) were hit. Dps1 has been known to function as a component of decaprenyl diphosphate synthase (Saiki et al., 2003), but Fps1 remained to be elucidated. These facts suggest that S. pombe has no apparent orthologue of GGPS as deduced from the primary structure, and they also imply the possibility that Fps1 and/or Spo9 function(s) as a GGPS. To test this possibility, we examined the relationship between S. pombe FPS-like proteins and S. cerevisiae GGPS. Both fps1+ and spo9+ complemented the cold sensitivity of an S. cerevisiae bts1 mutant (Figure 5E) and reciprocally, BTS1 complemented the temperature sensitivity and sporulation deficiency of an S. pombe spo9 mutant (Figure 5, C and D), but not the lethality of the fps1 mutant (data not shown). Erg20 also complemented the cold sensitivity of the S. cerevisiae bts1 mutant (Figure 5E). However, BTS1 could not rescue the lethality of the S. cerevisiae erg20 mutation (data not shown).
S. pombe fps1+ Complements an E. coli ispA Mutant
We next examined whether Fps1 and Spo9 possessed FPS activity by expressing the genes in an E. coli ispA mutant that lacks FPS (Fujisaki et al., 1990). The ubiquinone level in the ispA mutant is significantly lower than that in wild type, because ubiquinone is one of the end products of FPP (Fujisaki et al., 2005, Figure 6, A and B). If fps1+ and/or spo9+ encoded an FPS, we presumed that the ubiquinone level of the ispA strain expressing these genes would be restored. Ubiquinone was extracted from ispA strains either harboring the empty vector, fps1+-, or spo9+-carrying plasmids, and it was assayed by HPLC. As shown in Figure 6, the ubiquinone level in fps1+-expressing cells (Figure 6C) was significantly higher than in cells harboring the control vector (Figure 6B). In contrast, the level in the spo9+-expressing strain was almost identical to that in the control strain (Figure 6D). These data suggest that Fps1 but not Spo9 has FPS activity.
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-glucosidase), crtY (lycopene cyclase), crtI (phytoene desaturase), crtB (phytoene synthase), and crtZ (-carotene hydroxylase) genes (Misawa et al., 1990). Because E. coli does not possess GGPS, E. coli cells transformed with plasmid pACCAR25crtE containing all the crt genes except for crtE from E. uredovora, are only able to accumulate zeaxanthin, which has a yellow color, if GGPP is produced (Zhu et al., 1997; Kainou et al., 1999; Engprasert et al., 2004). Therefore, we tested whether spo9+ has GGPS activity by use of this carotenoid formation test. fps1+, spo9+, and S. cerevisiae BTS1 were cloned into a bacterial expression plasmid, and introduced into DH10B harboring pACCAR25crtE. BTS1 transformants (positive control) produced the expected yellow pigment (Figure 7). However, neither fps1+ nor spo9+ transformants formed yellow colonies. Because it is known that FPS and GGPS from many organisms form a dimer and that dimer formation is essential for enzyme activity (Tarshis et al., 1994; Szkopinska and Plochocka, 2005), we speculated that if coexpression of Fps1 and Spo9 is needed for GGPS activity, cotransformation of these genes in E. coli might result in formation of a functional GGPP-producing enzyme. As shown in Figure 7, transformants expressing fps1+ and spo9+ simultaneously produced a notable yellow pigment. These data suggest that Fps1 and Spo9 together form a heteromeric GGPS.
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and spo9, respectively. An E. coli strain that harbored plasmid pACCAR25crtE coexpressing either Fps1 plus Spo9 R109Q or Fps1R104Q plus Spo9R109Q failed to produce the yellow pigment. Surprisingly, the same E. coli strain expressing Fps1R104Q plus Spo9 formed yellow colonies (Figure 7). These data suggest that GGPS is dependent on the enzymatic activity of Spo9 but not on that of Fps1.
If spo9+ encodes a component of GGPS, GGPS activity should be detected in S. pombe wild type, but not in the spo9 mutant. In vitro GGPS activity was then measured in cell-free extracts from these strains. Cells were homogenized, and [14C]IPP and FPP were used as substrates to detect prenyl diphosphate synthase activity. The extracts were subsequently hydrolyzed with acid phosphatase, and the products were separated by reversed phase TLC. Although wild type was able to produce geranylgeraniol (GGOH), the spo9 strain barely showed any GGPS activity (Figure 9). The GGOH spot in the spo9 strain was intensified by the overexpression of the spo9+ gene. These data indicate that Spo9 indeed has GGPS activity.
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, 1994; Schafer and Rine, 1992; Omer et al., 1994). A number of proteins are known to be prenylated, and in the most cases, the prenylation is essential for their membrane localization. Indeed, in the S. cerevisiae bts1 mutant, the membrane association of the small GTPases, Ypt1 and Sec4, is defective due to lack of geranylgeranylation (Jiang et al., 1995). Ypt7, a fission yeast orthologue of Rab7 GTPase, mediates fusion of endosomes to vacuoles and homotypic vacuole fusion (Iwaki et al., 2004). Ypt7 has a Cys-X-Cys at its C terminus, which is recognized by geranylgeranyl transferase. If spo9+ encodes a sole GGPS, disruption of the gene should result in depletion of GGPP and loss of geranylgeranylation of Ypt7. To test this hypothesis, we examined the membrane association of Ypt7 in the spo9 mutant. Cell extracts were fractionated into a membrane fraction and a cytosolic fraction, and the distribution of Ypt7 was examined in each by Western blot analysis. Spo14, a membrane-bound protein was used as a probe for the membrane fraction (Nakamura-Kubo et al., 2003). Most Ypt7 was detected in the membrane fraction prepared from wild-type cells. However, in spo9 cells, Ypt7 was detected in both cytosolic and membrane fractions (Figure 10). Thus, the membrane association of Ypt7 is partially defective in spo9 cells. In the unfractionated extract from the spo9 cells, Ypt7 was detected as a doublet. In contrast, in both cytosolic and membrane fractions from spo9 cells, Ypt7 was detected as a single band, although migration of Ypt7 detected in the membrane fraction was slightly faster than that in the cytosolic fraction. Generally, the prenylated protein migrates faster than its unmodified form (Figure 10). These data support the notion that the extent of geranylgeranylation was lower in spo9 cells. Rhb1, a fission yeast orthologue of the human small GTPase Rheb, is known to be farnesylated and the farnesylation is important for membrane association (Yang et al., 2001; Nakase et al., 2006). If Spo9 mainly functions as a GGPS and not an FPS, the distribution of Rhb1 should not be affected by the spo9 mutation. As shown in Figure 10, the distribution of Rhb1 in the spo9 cells in cytosolic and membrane fractions was almost identical to that in wild-type cells. Together, these data are consistent with the possibility that Spo9 is an essential component of GGPS in fission yeast.
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, 2005; Nakase et al., 2001; Nakamura-Kubo et al., 2003; Onishi et al., 2003; Koga et al., 2004; Kashiwazaki et al., 2005). To examine in detail how the spo9 mutation impairs sporulation, the assembly of forespore membrane in the spo9-B261 mutant was observed using GFP-tagged Psy1, a syntaxin-like protein (Nakamura et al., 2001). Progression of meiosis was monitored by observing the formation and elongation of spindle microtubules. In wild-type cells, most haploid nuclei produced by meiotic second divisions were encapsulated by the forespore membrane. In the spo9-B261 mutant, forespore membrane formation initiated normally during meiosis II (Figure 11A), but subsequent development was blocked (Figure 11B). Only 40% of the zygotes contained four complete sets of nucleated prespores (membrane-bounded spore precursors), although they were remarkably small (type I in Figure 11B). Anucleated prespores were observed in 50% of the zygotes (type II in Figure 11B). In type III, assembly of the forespore membrane seemed to be arrested halfway (Figure 11B). These results indicate that forespore membrane formation initiates normally, but that subsequent development and integrity of the forespore membrane are impaired in spo9. Essentially identical data were obtained when spo9 cells were used.
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cells by thin-section electron microscopy. As shown in Figure 12, membrane vesicle accumulation was notable in the cytoplasm of immature spo9 asci, but such vesicle accumulation was not observed in wild-type asci. The vesicles in the spo9 mutant had an average diameter of 79 ± 42 nm, consistent with previous measurements of post-Golgi secretory vesicles in S. cerevisiae. Furthermore, Golgi-like structures were often observed in the spo9 mutant (Figure 12B). In addition, apparently abnormal forespore membranes were also observed. Anucleated spore-like bodies were often observed as well (Figure 12, B–D). These data are essentially identical to those obtained by fluorescence microscopy described above. Thus, spo9+ is important for the assembly of the forespore membrane, especially the expansion of the membrane.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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20%) with typical GGPSs. Thus, both Fps1 and Spo9 more closely resemble FPS rather than GGPS in amino acid sequence. Based on database search and phylogenetic analysis, S. pombe has no other GGPS-encoding gene. In S. cerevisiae, Erg20 and Bts1 are specifically recognized as an FPS and a GGPS, respectively. Overexpression of fps1+ in an E. coli ispA mutant that lacks FPS restored the production of ubiquinone, which is one of the end products of FPP, suggesting that Fps1 functions as a typical FPS in S. pombe. Because GGPP can be a substrate for octaprenyl diphosphate synthase, a key enzyme of ubiquinone synthesis, it is also possible that the same complementation of the ispA mutation is due to the GGPS activity of Fps1. However, this possibility seems unlikely by the following reasons. First, the overexpression of Fps1 but not S. cerevisiae GGPS Bts1 could complement the lethality of an S. cerevisiae erg20 mutant that lacks FPS. Second, ERG20 but not BTS1 could complement the lethality of an S. pombe fps1 mutant. Third, overexpression of fps1+ alone in E. coli cells cannot produce the substantial amount of GGPP by judging from the result of carotenoid producing assay (Figure 7). In similar experiments, no evidence was obtained to support the possibility that Spo9 functions as an FPS, although it resembled other FPSs in primary structure. The failure of spo9+ to complement the phenotype of either fps1 or erg20 makes it unlikely that Spo9 functions as an FPS.
S. pombe Geranylgeranyl Diphosphate Synthase Is Composed of a Typical FPS, Fps1, and an FPS-like Protein, Spo9
Although S. pombe has two FPS-like proteins, a protein that shares significant similarity with GGPS in the S. pombe genome database was not found. Several lines of evidence obtained in this study suggest that both Spo9 and Fps1 form a heteromer and that this complex functions as GGPS. First, spo9+ was able to complement the phenotype of an S. cerevisiae bts1 mutant, which lacks GGPS, and BTS1 was able to rescue spo9. Second, GGPS activity was significantly lower in S. pombe spo9 cells than in wild type. Third, the spo9 mutation perturbed the membrane association of a geranylgeranylated protein but not that of a farnesylated protein. Fourth, Spo9 directly interacts with Fps1. Fifth, E. coli cells that express a set of genes required for carotenoid biosynthesis but without a GGPS gene, only produced carotenoid when spo9+ and fps1+ were expressed simultaneously.
Most short-chain prenyltransferases are known to function as homodimers (Koyama, 1999). However, in other cases, as in spearmint (Burke et al., 1999), geranyl diphosphate synthase forms a heterodimer and decaprenyl diphosphate synthase in S. pombe and humans forms a heterotetramer (Saiki et al., 2003, 2005). Each of these heteromers includes a subunit that has significant homology with typical prenyltransferases that contain two aspartate-rich motifs. For example, Dps1, a component of decaprenyl diphosphate synthase from S. pombe has a high similarity (40%) with typical long-chain prenyl diphosphate synthases, whereas the other component, Dlp1, has only weak similarity (20%) with prenyl diphosphate synthases (Suzuki et al., 1997; Saiki et al., 2003, 2005). In marked contrast to these proteins, both subunits of S. pombe GGPS share high similarity with FPSs, but only weak similarity with typical GGPSs. We attempted to determine whether the S. pombe GGPS is composed of a heterodimer or a heterotetramer by a gel filtration analysis. However, neither protein could be detected in any fractions corresponding to heterodimers or heterooligomers (data not shown). This fact suggests that the interaction between Fps1 and Spo9 is so weak that the complex readily dissociates during gel filtration. In the experiment showing the coimmunoprecipitation of Spo9 and Fps1 in S. pombe cells, a density of Spo9 relative to Fps1 in lane 2 (IP) is lower than that in lane 2 (lysates) (Figure 8B). This result might be attributed to their dissociation during the immunoprecipitation. Alternatively, the result could be interpreted by the formation of homodimers of Fps1, as revealed in Figure 8A.
We obtained clear evidence that S. pombe GGPS is composed of Fps1 and Spo9, but at the same time, we cannot rule out the possibility that Fps1 might have weak GGPS activity for the following reasons. First, although GGPS activity was significantly reduced in spo9 cells, it was not absent (Figure 9). In addition, geranylgeranylation-dependent membrane association of Ypt7 was observed to some extent in spo9 cells (Figure 10). These results suggest that GGPP is still synthesized in spo9 cells. Second, overexpression of fps1+ was able to suppress the temperature sensitivity and sporulation deficiency of spo9, and GGPS activity was detected in those cells (data not shown). Several FPSs have been known to retain GGPS activity as well (Chen and Poulter, 1993; Fujiwara et al., 2004). It is reasonable that GGPS activity was detected under nonphysiological conditions, i.e., overexpression of the Fps1 protein. At present, we cannot examine the GGPS activity and membrane association of Ypt7 and Rhb1 in the fps1 disruptant, because fps1+ is essential for growth. Analysis of conditional fps1 mutants will allow us to elucidate the role of Fps1 as a GGPS.
Previous studies have revealed that the amino acid residue located on the fifth position before the FARM of FPS is important for the determination of chain length of a final product. In all of known FPSs, including S. pombe Fps1, the corresponding amino acid is either phenylalanine or tyrosine, but the corresponding amino acid is cysteine in Spo9 (Figure 3A). Ohnuma et al. (1996) constructed 20 FPS mutant proteins from Bacillus stearothermophilus, each of which has a different amino acid at position 81 located on the fifth position before the FARM, and revealed that the average chain length of products is inversely proportional to the accessible surface area of substituted amino acids. Interestingly, B. stearothermophilus FPS Y81C mutant, in which tyrosine 81 was replaced with cysteine, effectively produced GGPP (Ohnuma et al., 1996). These data suggest the possibility that cysteine 95 is important for the Spo9 function as GGPS. To test this possibility, we constructed the mutant protein Spo9C95F, in which cysteine 95 was replaced with phenylalanine, and introduced it into fps1. However, the mutant protein could not complement the lethality of fps1, and it did complement the phenotypes of spo9. Conversely, the fps1-F90C mutant gene could complement the lethality of fps1. Thus, it is unlikely that cysteine 95 is the only important determinant for the function of S. pombe Spo9 as a GGPS.
Role of Spo9 in Sporulation
Our study also demonstrates that GGPS plays a crucial role in sporulation. Fluorescence microscopy by using the forespore membrane marker protein GFP-Psy1 revealed that the forespore membrane formation initiates normally, but that the subsequent process, expansion of the membrane was severely impaired in spo9. GGPP is the precursor for several important metabolites, including dolichols, ubiquinones, and heme a in S. pombe. One possibility is that the decrease in these metabolites leads to the defect in sporulation. If so, various steps in sporulation should be affected. However, the spo9 mutation perturbed only expansion of forespore membrane. The other events of sporulation, the meiotic nuclear division and the initiation of forespore membrane formation, seemed to proceed normally in spo9. Therefore, it seems unlikely that the decrease of end products such as heme a and/or ubiquinone directly affects the sporulation-defective phenotype of spo9. At least there is no defect in sporulation in ubiquinone-deficient strains (unpublished observations). Alternatively, it is possible that the spo9 mutation perturbs the membrane association of a geranylgeranylated protein necessary for sporulation. Indeed, the membrane association of Ypt7, a putative geranylgeranylated protein, is significantly diminished in spo9. Our previous study revealed that Ypt7 is essential for proper spore formation (Kashiwazaki et al., 2005). Thus, the defective membrane association of Ypt7 could plausibly result in the spo9 mutant phenotype. Nonetheless, the phenotype of spo9 is much severe than that of ypt7, suggesting that other geranylgeranylated proteins are involved in sporulation as well (Giannakouros et al., 1992). In an S. cerevisiae bts1 mutant, the failure to geranylgeranylate Ypt1 and Sec4 leads to a defect in the membrane association of these proteins. The bts1 mutation may impair intracellular membrane trafficking, because these proteins play essential roles in this process (Rossi et al., 1991; Jiang et al., 1995). A number of genes that play roles in the general secretion machinery have been reported to be necessary for sporulation in both S. cerevisiae and S. pombe (Neiman, 1998; Nakase et al., 2001; Nakamura et al., 2001, 2005; Jantti et al., 2002; Nakamura-Kubo et al., 2003; Nakanishi et al., 2006). Among them, sec4 mutant also exhibits a sporulation deficiency in S. cerevisiae (Neiman, 1998). S. pombe possesses a Sec4 homologue, Ypt2, which also has a consensus sequence for geranylgeranylation (Haubruck et al., 1990). Defective geranylgeranylation of Ypt2 in the spo9 mutant might cause a sporulation deficiency. This possibility is further supported by our observation that many membrane vesicles accumulate in the cytoplasm during sporulation in spo9 cells.
In summary, we report here that S. pombe FPS is composed of Fps1 and that GGPS is composed of a typical FPS, Fps1, and an FPS-like protein, Spo9. To our knowledge, a heteromer of two FPS-like proteins is a novel type of GGPS that has not been previously reported. We speculate that S. pombe developed this GGPS by duplicating an FPS gene followed by limited mutational changes in the duplicated copy resulting in the present heteromeric structure.
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ACKNOWLEDGMENTS |
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Footnotes |
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Address correspondence to: Taro Nakamura (taronaka{at}sci.osaka-cu.ac.jp).
Abbreviations used: DMAPP, dimethylallyl diphosphate; FARM, first aspartate-rich motif; FPP, farnesyl diphosphate; FPS, farnesyl diphosphate synthase; GGPP, geranylgeranyl diphosphate; GGPS, geranylgeranyl diphosphate synthase; GPP, geranyl diphosphate; IPP, isopentenyl diphosphate; SARM, second aspartate-rich motif; TLC, thin-layer chromatography.
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