Báo cáo khoa học: Fission yeast decaprenyl diphosphate synthase consists of Dps1 and the newly characterized Dlp1 protein in a novel heterotetrameric structure doc

Fission yeast decaprenyl diphosphate synthase consistsof Dps1 and the newly characterized Dlp1 protein in a novel heterotetrameric structure Ryoichi Saiki, Ai Nagata, Naonori Uchida, Tom

Fission yeast decaprenyl diphosphate synthase consists

of Dps1 and the newly characterized Dlp1 protein in a novel

heterotetrameric structure

Ryoichi Saiki, Ai Nagata, Naonori Uchida, Tomohiro Kainou, Hideyuki Matsuda and Makoto Kawamukai Department of Applied Bioscience and Biotechnology, Faculty of Life and Environmental Science, Shimane University, Matsue, Japan

The analysis of the structure and function of long

chain-producing polyprenyl diphosphate synthase, which

syn-thesizes the side chain of ubiquinone, has largely focused

on the prokaryotic enzymes, and little is known about the

eukaryotic counterparts Here we show that decaprenyl

diphosphate synthase from Schizosaccharomyces pombe is

comprised of a novel protein named Dlp1 acting in

part-nership with Dps1 Dps1 is highly homologous to other

prenyl diphosphate synthases but Dlp1 shares only weak

homology with Dps1 We showed that the two proteins must

be present simultaneously in Escherichia coli transformants

before ubiquinone-10, which is produced by S pombe but

not by E coli, is generated Furthermore, the two proteins

were shown to form a heterotetrameric complex This is

unlike the prokaryotic counterparts, which are homodimers

The deletion mutant of dlp1 lacked the enzymatic activity of

decaprenyl diphosphate synthase, did not produce ubiqui-none-10 and had the typical ubiquinone-deficient S pombe phenotypes, namely hypersensitivity to hydrogen peroxide, the need for antioxidants for growth on minimal medium and an elevated production of H2S Both the dps1 (formerly dps) and dlp1 mutants could generate ubiquinone when they were transformed with a bacterial decaprenyl diphosphate synthase, which functions in its host as a homodimer This indicates that both dps1 and dlp1 are required for the

S pombeenzymatic activity Thus, decaprenyl diphosphate from a eukaryotic origin has a heterotetrameric structure that is not found in prokaryotes

Keywords: Schizosaccharomyces pombe; decaprenyl diphos-phate synthase; ubiquinone; CoenzymeQ

Ubiquinone was identified initially as an essential factor

in aerobic growth and oxidative phosphorylation in the

electron transport system Recently, however, multiple

additional functions of ubiquinone have been proposed

One such function is its apparent role as a lipid-soluble

antioxidant that prevents the oxidative damage of lipids due

to peroxidation [1–4] Studies using ubiquinone-deficient

yeast mutants also suggested that one of the in vivo functions

of ubiquinone is to protect against oxidants [5,6] There is

also a proposed function that links between sulfide

meta-bolism and ubiquinone Sulfide-ubiquinone oxidoreductase,

which was previously thought to occur mainly in

photo-biosynthetic bacteria, and which acts as a component in

an energy metabolic pathway, has now been shown to be

present in S pombe and other eukaryotic organisms [7] In

addition, the clk-1 mutant of Caenorhabditis elegans, which

has perturbed ubiquinone biosynthesis, shows a prolonged

life-span, suggesting a novel role of ubiquinone [8–11]

Furthermore, an elegant study showed that ubiquinone (or menaquinone) accepts electrons that are generated by the formation of protein disulfide in E coli [12] Thus, ubiqui-none appears to play multiple roles

The ubiquinone biosynthetic pathway is composed of

10 steps, including methylation, decarboxylation, hydroxy-lation and isoprenoid transfer The elucidation of this pathway has mostly involved studying respiratory-defici-ent mutants of E coli and S cerevisiae [13,14] The length

of the isoprenoid side chain of ubiquinone varies between organisms For example, S cerevisiae has ubiquinone-6,

E coli has ubiquinone-8, rats and Arabidopsis thaliana have ubiquinone-9, and humans and S pombe have ubiquinone-10 The length of the side chain appears to

be determined by polyprenyl diphosphate synthase, but not

by the 4-hydroxybenzoate-polyprenyl-diphosphate trans-ferases, which catalyze the condensation of 4-hydroxy-benzoate and polyprenyl diphosphate [15–17] The basis

of this notion is that the heterologous expression in E coli and S cerevisiae of polyprenyl diphosphate synthase genes from other sources generated the same type of ubiquinone as that expressed in the donor organisms By this method, Okada et al successfully produced different ubiquinone species (ubiquinone-5 to ubiquinone-10) in the

S cerevisiae COQ1 mutant, which lacks its own poly-prenyl diphosphate synthase [18] Moreover, 4-hydroxy-benzoate (PHB) polyprenyl diphosphate transferase was confirmed to have a broad substrate specificity by the heterologous expression of COQ2 in the ppt1D S pombe strain [19] and the ubiA-delta E coli strain [20]

Correspondence toM Kawamukai, Department of Applied Bioscience

and Biotechnology, Faculty of Life and Environmental Science,

Shimane University, 1060 Nishikawatsu, Matsue 690–8504, Japan.

Fax: + 81 852 32 6092, Tel.: + 81 852 32 6587,

E-mail: kawamuka@life.shimane-u.ac.jp

Abbreviations: PHB, 4-hydroxybenzoate; GPP, geranyl diphosphate;

FPP, farnesyl diphosphate; GGPP, geranylgeranyl diphosphate;

IPP, isopentenyl diphosphate; GST, glutathione-S-transferase.

(Received 26 May 2003, revised 17 July 2003,

accepted 22 August 2003)

The genes encoding the short-chain polyprenyl

diphos-phate synthases, including geranyl diphosdiphos-phate (GPP, C10)

synthase, farnesyl diphosphate (FPP, C15) synthase and

geranylgeranyl diphosphate (GGPP, C20) synthase, have

been cloned from various organisms ranging from bacteria

to mammals [21–24] The mechanisms that determine the

chain length synthesized by FPP synthase and GGPP

synthase have been extensively studied [25,26] All of these

short-chain polyprenyl diphosphate synthases function as

homodimers, except for the GPP synthase from spearmint,

which functions as a heterotetramer [27,28] In this latter

GPP synthase, one subunit is similar to known prenyl

diphosphate synthases but the other component does not

contain the typical aspartate-rich motifs that are considered

to be the substrate-binding sites Unlike most of the

short-chain polyprenyl diphosphate synthases, the medium-short-chain

polyprenyl diphosphate synthases (C30 and C35) that are

responsible for synthesizing the side chain of the

menaqui-nones in Micrococcus luteus BP26, Bacillus

stearothermo-philus, and Bacillus subtilis are heterodimers [29,30] In

contrast, all the long-chain polyprenyl diphosphate

synth-ases that synthesize the ubiquinone side-chains are thought

to be homodimeric enzymes [14,21] However, it has not

been known what kind of enzyme component(s) the

eukaryotic polyprenyl diphosphate synthase contains even

though its essential gene (e.g COQ1 or dps1) has been

identified [6,31]

In this study, we characterized the decaprenyl

diphos-phate synthase of S pombe We found that the prenyl

diphosphate synthase homologue of S pombe, Dps1, forms

a heterotetramer with another component that we identified

and denoted Dlp1 Dlp1 is needed to make a functional

enzyme as the dlp1 disruptant produced no ubiquinone, is

sensitive to H2O2 and requires an antioxidant to grow on

glucose-containing medium, which is typical of the

ubiqui-none-deficient S pombe strains [6,19] This is the first

molecular characterization of a long chain-producing

poly-prenyl diphosphate synthase that synthesizes the side-chain

of ubiquinone in a eukaryote

Materials and methods

Materials

Restriction enzymes and other DNA-modifying enzymes

were purchased from Takara Shuzo Co Ltd and New

England Biolabs, Inc [1–14C]IPP (1.96 TBqÆmol)1) was

purchased from Amersham Pharmacia Biotech Ltd IPP,

all-E-farnesyl diphosphate, geranylgeraniol, solanesol

(all-E-nonaprenol), and polyprenols (C40–C60) from Ailanthus

altissimawere purchased from Sigma Chemical Co Kiesel

gel 60 F254thin-layer plates were purchased from Merck

Reverse-phase LKC-18 thin-layer plates were purchased

from Whatman Chemical Separation

Strains and plasmids

E colistrains DH10B and DH5a were used for the general

construction of plasmids [32] Plasmids pBluescript SK+/–,

pT7Blue-T (Novagen), pSTV28 (Takara Shuzo), pQE31

(Qiagen), pGEX-KG (Amersham Pharmacia), pREP1 [33],

pREP2 (the LEU2 marker of pREP1 was exchanged with

the ura4 marker), pDS473 (ura4 marker), pDS472 (ura4 marker) and pSLF173 (LEU2 marker) were used as vectors [34,35] The S pombe homothallic haploid wild-type strain SP870 (h90leu1–32 ade6-M210 ura4-D18) and the diploid strain SP826 (h+leu1–32 ade6-M210 ura4-D18/h+ leu1–32 ade6-M216 ura4-D18) [36] were used to pro-duce Ddlp1::ura4 strains by homologous recombination KS10 (h+leu1–32 ade6-M216 ura4-D18Ddps1::ura4) and NU609 (h90leu1–32 ade6-M210 ura4-D18 Dppt1::ura4) have been described previously [6,19] Yeast cells were grown in YE (0.5% yeast extract, 3% glucose) or PM minimal medium with appropriate supplements as described

by Moreno et al [37] YEA and PMA contain 75 lg of adenine per ml in YE and PM, respectively The concen-tration of the supplemented amino acids was 100 lgÆmL)1

DNA manipulations Cloning, restriction enzyme analysis, and preparation of plasmid DNAs were performed essentially as described previously [32] The dlp1 gene was cloned and expressed in

E coliand S pombe as follows Two oligonucleotides were used to amplify the 1.8 kb fragment containing the dlp1 gene and the surrounding region, namely, 19G12X, 5¢-TCGAATTCGATGAGCTTTCCGTTC-3¢ (creates an EcoRI site) and 19G12Y, 5¢-CATGGATATCGCATTC-3¢ (contains an EcoRV site) The resulting 1.8 kb fragment was digested with EcoRI and EcoRV, cloned into the SmaI sites of pSTV28 to yield pSTVDLP1 and the EcoRI-EcoRV sites of pBluescript SK+ to yield pBSDLP1 To make pDISDLP1, it was necessary to remove the BamHI site of pBluescript SK+ After pBSDLP1 was digested with NotI and SmaI, it was blunt-ended using the Klenow Fragment (Takara Shuzo) and self-ligated The resulting plasmid was digested with BamHI and ligated with the ura4 cassette derived from pHSG398-ura4 Two other oligonu-cleotides were also used to amplify the dlp1 gene, namely, 5-dlp1, 5¢-TCGTCGACGAGCTTTCCGTTC-3¢ (creates

a SalI site) and 3-dlp1, 5¢-TCCCCGGGATTACTTCG AAAC-3¢ (creates a SmaI site) The amplified fragment was cloned into the SalI-SmaI sites of pREP1 to yield pRDLP1

To construct pGSTDLP1, the 5-dlp1Fu oligonucleotide (5¢-TCGCGGCCGCATGAGCTTTCCG-3¢, which creates

a NotI site) and 3-dlp1 were used to amplify the dlp1 gene The amplified fragment was cloned into the NotI and SmaI sites of pDS473 to yield pGSTDLP1 To construct pGEXDLP1, pBSDLP1 was digested with SmaI and SalI and then cloned into the SmaI and SalI sites of pGEX-KG

to yield pGEXDLP1

To construct pBSDPS1, the oligonucleotides 5-dps1 (5¢-TCCTGCAGCATGATTCAGTATGTA-3¢, which cre-ates a PstI site) and 3-dps1 (5¢-TCGTCGACTCACTTC TTTCTCGTTAT-3¢, which creates a SalI site) were used to amplify the dps1 gene The plasmid pKS18 containing the dps1region from the S pombe cDNA library was used as a template for PCR The amplified fragment was cloned into the PstI and SalI sites of pBluescript SK+ to yield pBSDPS1 To construct pHADPS1, the oligonucleotides 5-dps1Fu (5¢-TCGCGGCCGCATGATTCAGTAT-3¢, cre-ates a NotI site) and 3-dps1 were used to amplify the dps1 region from the S pombe cDNA library The amplified fragment was cloned into the NotI and SalI sites of

pSLF173 to yield pHADPS1 To construct

pSTVHI-SDPS1, the oligonucleotides 5-Sph I-dps1 (5¢-GCGCATG

CGATGATTCAGTATGTA-3¢, creates a SphI site) and

3-dps1 were used to amplify the dps1 gene The plasmid

pBSDPS1 was used as a template for PCR The amplified

fragment was cloned into the SphI and SalI sites of pQE31

The pQE31 plasmid harboring the dps1 gene was digested

with XhoI and SalI and cloned into the Sal I site of pSTV28

to yield pSTVHISDPS1

The ddsA gene that encodes the decaprenyl diphosphate

synthase of Gluconobacter suboxydans was endowed with

the putative mitochondrial transit sequences of the gene

encoding PHB polyprenyl diphosphate transferase (ppt1) as

follows The mitochondrial transit sequences were amplified

by PCR using the oligonucleotides 5¢-AGGTCGACAGA

TTAGCATGTAAATAG-3¢ (creates a SalI site) and

(cre-ates a HindIII site) The PCR products were cloned into

the SalI and HindIII sites of pBluescript SK+ to yield

pBSTP Two oligonucleotides, namely, 5-ddsA (5¢-GCA

AGCTTAAAGCTGTGGTTCAGGGTGCAG-3¢, creates

a HindIII site) and 3-ddsA (5¢-TAGCATGCTTAGCGGG

CCCGATTC-3¢, creates a SphI site), were used to amplify

the 1.0 kb fragment containing the ddsA gene and an

additional 23 amino acid segment beyond the first

methio-nine The amplified fragment was then cloned into

pT7Blue-T to yield ppT7Blue-T7DDSA ppT7Blue-T7DDSA was then digested with

HindIII and BamHI (this site was in pT7Blue-T) and cloned

into the HindIII and BamHI sites of pBSTP to yield

pBSTPDDSA To construct pRDDSA, pBSTPDDSA was

digested with SalI and BamHI, and the fragment was cloned

into the SalI and BamHI sites of pREP1

Gene disruption

The one-step gene disruption technique was performed

according to the procedure of Rothstein [38] The

pDISDLP1 plasmid was linearized using the appropriate

restriction enzymes, and the linear plasmid was used to

transform SP826 for uracil prototrophy Southern

hybridi-zation was performed as described previously [32]

Ubiquinone extraction and measurement

Ubiquinone was extracted as described previously [6,19]

The crude extract of ubiquinone was analyzed by

normal-phase TLC with authentic ubiquinone-10 as the standard

Normal-phase TLC was carried out on a Kieselgel 60 F254

plate with benzene/acetone (97 : 3, v/v) The band

contain-ing ubiquinone was collected from the TLC plate followcontain-ing

UV visualization and extracted with chloroform/methanol

(1 : 1, v/v) Samples were dried and redissolved in ethanol

The purified ubiquinone was further analyzed by HPLC

with ethanol as the solvent

Prenyl-diphosphate synthase assay and product analysis

Prenyl diphosphate synthase activity was measured by the

method described previously [15] S pombe cells were

grown on the mid-to-late log phase in PMA-based medium

All subsequent steps were performed at 4C Cells were

harvested by centrifugation, suspended in buffer A (100 m

potassium phosphate, pH 7.4, 5 mM EDTA, and 1 mM

2-mercaptoethanol) The washed cells were ruptured by vigorous shaking with glass beads 14 times for 30 s at 60 s intervals in an ice bath After centrifugation of the homogenate, the supernatant was used as a crude enzyme extract The incubation mixture contained 2 mM MgCl2, 0.2% (w/v) Triton X-100, 50 mM potassium phosphate buffer (pH 7.4), 5 mMKF, 10 mM iodoacetamide, 20 lM

[14C]IPP (specific activity 0.92 MBqÆmol)1), 100 lM FPP, and 1.5 mgÆmL)1protein of the enzyme in a final volume of 0.5 mL The sample mixtures were incubated for 60 min at

30C Reaction products, such as prenyl diphosphates, were extracted with 1-butanol-saturated water and hydro-lyzed with acid phosphatase The hydrolysis products were extracted with hexane and analyzed by reverse-phase TLC with acetone/water (19 : 1, v/v) Radioactivity on the plate was detected with a BAS1500-Mac imaging analyzer (Fuji Film Co.) The plate was exposed to iodine vapor to detect the spots of the marker prenols

Immunoprecipitation of Dps1 protein with Dlp1 protein

To test whether Dlp1 and Dps1 form a heterologous complex, pGSTDLP1, which produces the glutathione-S-transferase (GST) and Dlp1 fusion protein, GST-Dlp1, and pHADPS1, which produces HA-fused Dps1, were trans-formed into S pombe strain SP826 Transformants were grown to the stationary phase in PMA medium and 0.5 mL

of culture was then inoculated into 50 mL of the same medium The cultures were grown to the mid-to-late log phase After the cells were collected by centrifugation at

3000 g for 5 min, the pellets were suspended in 0.1 mL of buffer A The suspended cells were ruptured by vigorous shaking with glass beads 14 times for 10 s at 60 s intervals in

an ice bath After the addition of 0.3 mL of buffer A kept at

4C, the homogenate was centrifuged at 2000 g for 5 min The supernatant solution was then mixed with glutathione Sepharose 4B (Amersham Pharmacia Biotech) at 30C for

60 min This mixture was washed five times with 140 mM

NaCl, 2.7 mMKCl, 10 mMsodium phosphate and 1.8 mM

potassium phosphate, and then once with 50 mMTris/HCl (pH 8.0) and 10 mMglutathione (glutathione elution buffer)

to elute the GST-Dlp1 protein To detect Dps1-Dlp1 heterotetramers, a crude protein extract of E coli harboring pGEXDLP1 and pSTVHISDPS1 was incubated with the water-insoluble cross linker disuccinimidyl suberate (Pierce) (1 mM in final concentration) and subjected to Western blotting

Results

Cloning of thedlp1 gene and construction

of thedlp1D strain

We hypothesized that the decaprenyl diphosphate synthase

of S pombe might be a heteromeric enzyme because we observed that expression of the dps1 gene alone in E coli did not give any enzymatic activity [6] In contrast, when single bacterial genes encoding prenyl diphosphate synthases are expressed in E coli, functional enzymes are produced [16,39,40] We looked for a potential partner of Dps1 by searching the S pombe genomic DNA sequence in the

National Center for Biotechnology Information database

for a gene with homology to dps1 We found meaningful

sequence similarity with an uncharacterized gene

(SPAC19G12.12) The deduced amino acid sequence of

the SPAC19G12.12 gene possessed the conserved domains

I to VII of Dps1, but did not contain DDXXD sequence

motifs that are typically found in all prenyl diphosphate

synthases (Fig 1) We designated this gene dlp1 (for D-less

polyprenyl diphosphate synthase) and characterized it

further

Fig 1 Alignment of the amino acid sequences of long chain-producing

polyprenyl diphosphate synthases (1) Decaprenyl diphosphate synthase

encoded by ddsA from Gluconobacter suboxydans (NCBI accession no.

AB006850); (2) octaprenyl diphosphate synthase encoded by ispB from

E coli (accession no NP417654); (3) component of decaprenyl

diphosphate synthase encoded by dps1 from S pombe (accession no.

D84311); (4) hexaprenyl diphosphate synthase encoded by COQ1 from

S cerevisiae (accession no J05547); (5) a novel component of

deca-prenyl diphosphate synthase encoded by dlp1 from S pombe

(acces-sion no AB118853) Residues conserved in more than two of the five

sequences are boxed Conserved regions (I–VII) are underlined.

Numbers on the right indicate amino acid residue positions.

Fig 2 Plasmid constructs used in this study and Southern hybridization analysis of genomic DNAs from SP826, SP826Ddlp1 and RS312 (A) Plasmid constructs pRDDSA and pRDLP1 contain the entire length

of the ddsA and dlp1 genes, respectively pHADPS1 and pGSTDLP1 express the HA–Dps1 and GST–Dlp1 fusion proteins, respectively pRDDSA, pRDLP1, pHADPS1 and pGSTDLP1 are under the control of the strong nmt1 promoter pGEXDLP1 contains the entire length of the dlp1 gene fused with the GST gene in pGEX-KG vector pSTVHISDPS1 contains the entire length of the dps1 gene fused with His 6 -tag that allows to express the His–Dps1 fusion protein in E coli.

In pDISDLP1 the dlp1 gene was disrupted by the ura4 cassette on the vector pBluescript SK+ (B) Southern hybridization analysis (I) Restriction map of the dlp1 and the dlp1::ura4 regions Genomic DNAs of wild-type and dlp1 disruptants were prepared, separated on agarose gel, and probed with the ura4 gene (II) and the dlp1 gene from pSTVDLP1 (III) Arrows and the size calculated from the sequences in (I) matched with arrows indicated in (III) Lane 1, wild-type SP826 (diploid); lanes 2 and 3, SP826Ddlp1 (diploid); lane 4, RS312 (haploid) TP: Transit peptide from P p +1 [19] Ba, BamHI; EI, EcoRI; EV, EcoRV; H, HindIII; N, NotI; P, PstI; Sa, SalI; Sp, SphI; Sm, SmaI; Xh, XhoI.

To assess the relevance of the dlp1 gene in ubiquinone

biosynthesis, we constructed an S pombe strain whose dlp1

gene has been disrupted (dlp1D) To do this, we constructed

the plasmid pDISDLP1, in which the dlp1 gene is disrupted

by the ura4 gene (Fig 2A) This plasmid was then linearized

by the appropriate restriction enzymes and the fragment

was used to transform the S pombe wild-type diploid strain

SP826 About 20 Ura+ transformant colonies could be

picked and they were grown on YEA medium After the

stability of the ura4+ marker was examined by replica plating, two stable Ura+transformants were obtained One

of these strains, designated SP826Ddlp1, was allowed to make spores, and the germinated haploid cells were plated

in replicates on plates containing YEA and PMA+Leu While all cells grew well on YEA medium, some grew only very slowly on the PMA+Leu plate One of these haploid strains, designated RS312, and the parental diploid SP826Ddlp1 strain were subjected to Southern hybridization analysis to confirm the proper disruption of dlp1 by ura4 (Fig 2B) RS312 (dlp1::ura4) was then examined for ubiquinone synthesis as described in the Materials and methods No ubiquinone was detected in RS312, although the RS312 strain that harbored the plasmid expressing dlp1 did show ubiquinone synthesis (Fig 3) This encouraging result indicates that the dlp1 gene is involved in ubiquinone biosynthesis

Phenotypes of thedlp1 disruptant

It was reported previously that KS10 (Ddps1::ura4), a strain

of S pombe whose decaprenyl diphosphate synthase-encoding dps1 gene has been disrupted, and NU609

Fig 3 Detection of ubiquinone-10 in S pombe strains Ubiquinone was

extracted from Wild-type SP870, KS10 (Ddps1::ura4), RS312

(Ddlp1::ura4), KS10 harboring pRDDSA or pRDPS1, and RS312

harboring pRDDSA or pRDLP1 Ubiquinone was first separated by

thin-layer chromatography and then further analyzed by high-pressure

liquid chromatography.

Fig 4 Recovery of RS312 growth on minimal medium by adding cys-teine or glutathione (A) Wild-type SP870 harboring pREP2 (ura4 marker), NU609 (Dppt1::ura4), RS312 (Ddlp1::ura4) and RS312 har-boring pRDLP1 were grown on PM medium supplemented with

75 lgÆmL)1adenine and 100 lgÆmL)1leucine (B) The same strains were grown on PM medium supplemented with adenine, leucine and

200 lgÆmL)1glutathione (C) The same strains were grown as in B except that cysteine was used instead of glutathione NU609 and RS312 could not grow on PM medium (A) unless it was supplemented with glutathione (B) or cysteine (C) RS312 harboring pRDLP1 could grow on PM medium lacking glutathione or cysteine (A).

(Dppt1::ura4), a strain of S pombe whose PHB polyprenyl

diphosphate transferase-encoding ppt1 gene has been

disrupted, are unable to produce ubiquinone and have

some notable additional phenotypes [6,19] These include

H2O2and Cu2+sensitivity and a requirement of cysteine,

glutathione or a-tocopherol for growth on minimal medium

[19] We thus tested RS312 for these phenotypes RS312

(Ddlp1::ura4) was first grown on PM-based medium with

and without supplementation with 200 lgÆmL)1of cysteine

or glutathione RS312 cells did not grow on the minimal

medium but the addition of cysteine or glutathione

effect-ively caused their growth to recover (Fig 4) RS312 cell

growth also recovered when they were grown on minimal

medium containing 1 mMa-tocopherol, a well-known lipid

antioxidant (data not shown) In addition, the good growth

of RS312 on supplemented medium was severely inhibited

when 1 mMH2O2or 1 mMCu2+was added (Fig 5) Thus,

RS312 cells bear the same phenotypes as the

ubiquinone-nonproducers KS10 and NU609

RS312 was similar to KS10 and NU609 in another

phenotype We previously found that the S pombe strains

that were deficient in either dps1 or ppt1 produced H2S but

the wild-type cells did not [6,19] When we tested for the

presence of H2S by assaying for its chemical reaction with

lead acetate (which produces PbS), the RS312 culture was

found to produce H2S (data not shown) In addition, when

we measured the amount of acid-labile sulfide present in the

cells, we found that the ubiquinone-less mutants of RS312

(Ddlp1) and NU609 (Dppt1) produced 12-fold-higher

amounts of S2– (1064.9 and 1110.1 nmol per 109 cells,

respectively) than the wild-type strain SP870 (83.3 nmol per

109 cells) All these phenotypes of RS312 are identical to

those of the two ubiquinone-less mutants that had been

constructed earlier [6,19] Thus, dlp1 is essential for

ubi-quinone synthesis in S pombe

Complementation ofdlp1-disrupted cells by expressing

G suboxydans ddsA

To determine whether dlp1 is directly involved in the

activity of decaprenyl diphosphate synthase, we

expres-sed ddsA in the dlp1 and dps1 disruptants The ddsA gene encodes the decaprenyl diphosphate synthase of

G suboxydans This ddsA gene is known to be completely functional when it is expressed in E coli [40], which suggests that DdsA is a homomeric enzyme A 45 amino-acid mitochondrial transfer signal from Ppt1, which is known to locate in the mitochondria [19], was first added

to the ddsA gene product so that it would be located in mitochondria The resulting ppt1-ddsA fusion gene suc-cessfully complemented both the dps1 and dlp1 disruptants

as both cell types were able to produce ubiquinone (Fig 3) Thus, the lack of ubiquinone synthesis in the dlp1 mutant, as well as in the dps1 mutant, is due to the lack

of decaprenyl diphosphate synthase activity

We further confirmed that the Ddlp1 strain, as well as the dps1D strain, is defective in decaprenyl diphosphate synthase activity by measuring the in vitro activity of this enzyme as described in the materials and methods Both the Ddlp1 and Ddps1 strains did not produce decaprenol, while the Dppt1 strain and wild-type retained their activities (Fig 6) However, when the Ddlp1 strain was transfected with the plasmid containing dlp1, the enzy-matic activity was restored Thus, both dlp1 and dps1 are essential for decaprenyl diphosphate synthase activity in

S pombe

Fig 5 Sensitivity of the ubiquinone-less mutant to oxygen radical

pro-ducers Wild-type (circles) and RS312 (Ddlp1::ura4) (triangles) strains

were pregrown and then placed in fresh YEA medium with 1 m M H 2 O 2

(A), 1 m M Cu2+(B) or neither Cell numbers were counted at 4-h

intervals.

Fig 6 Thin-layer chromatogram of the product of decaprenyl diphos-phate synthase The decaprenyl diphosdiphos-phate synthase reactions of KS10 (Ddps1::ura4, lane 1), RS312 (Ddlp1::ura4, lane 2), SP870 (Wild-type, lane 3), NU609 (Dppt1::ura4, lane 4) and RS312 harboring plasmid pRDLP1 (lane 5) were measured using [14C]IPP and FPP as substrates The products were hydrolyzed with phosphatase and the resulting alcohols were analyzed by reverse-phase thin-layer chroma-tography The same amounts of radiolabeled products (5000 d.p.m.) were applied to the TLC plate The arrowhead indicates the position of the synthesized decaprenols The positions of standard alcohols are indicated on the right: GGOH, all-E-geranylgeraniol; SOH, all-E-solanesol; Ori., origin; S.F., solvent front.

Expression ofdps1 and dlp1 in E coli

We speculated that if the dps1 and dlp1 are sufficient for

decaprenyl diphosphate synthase activity, cotransformation

of E coli DH5a with the pSTVHISDPS1 plasmid that

expresses the dps1 gene and the pGEXDLP1 plasmid that

expresses the dlp1 gene may result in the formation of a

functional enzyme that generates the ubiquinone-10 species that is produced by S pombe We found that while the cells that were transformed only with pSTVHISDPS1 or pGEXDLP1 produced only ubiquinone-8, which is syn-thesized by the endogenous E coli octaprenyl diphosphate synthase, the pSTVHISDPS1 and pGEXDLP1 double transformant produced a small amount of ubiquinone-10

Fig 7 Detection of ubiquinone in E coli

transfected with constructs expressing dlp1 and/

or dps1 Ubiquinone was extracted from

wild-type DH5a and DH5a harboring

pGEXDLP1 and/or pSTVHISDPS1.

Fig 8 Tetrameric formation of Dlp1 and

Dps1 (A,B) Crude proteins were extracted

from SP826 harboring pHADPS1 and

pGSTDLP1 (lanes 1 and 3) or pHADPS1 and

pDS472 (lanes 2 and 4) The crude proteins

were incubated in buffer A at 30 C for 1 h

and then purified by a GST column (lanes 1

and 2) Western blot analysis was performed

using an anti-HA (A) or anti-GST Ig (B).

Arrows indicate the positions of HA-Dps1 (A)

and GST-Dlp1 (B) protein The asterisk in B

indicates the GST protein (C,D) Crude

proteins were extracted from E coli BL21

harboring pGEXDLP1 and pSTVHISDPS1

(lane 2) The crude proteins were incubated

with a cross-linker, disuccinimidyl suberate, at

30 C for 30 min and then purified by a GST

column (lane 1) Western blot analysis was

performed using an anti-GST (C) or anti-His

Ig (D).

(Fig 7) This result indicates Dps1 and Dlp1 together form

a heteromeric decaprenyl diphosphate synthase

Heterotetrameric formation of Dps1 and Dlp1

That dps1 and dlp1 are both required for the decaprenyl

diphosphate synthase activity suggests that Dps1 and Dlp1

might form a complex Consequently, we tested whether

Dps1 and Dlp1 interact with each other to form a

heteromer HA-linked Dps1 and GST-linked Dlp1 were

expressed together in S pombe and subjected to pull-down

assays When the GST–Dlp1 fusion was pulled down by the

GST-column, HA–Dps1 was coprecipitated, although when

HA–Dps1 was expressed with GST alone, HA–Dps1 was

not pulled down by the GST-column (Fig 8A,B) Thus,

Dlp1 and Dps1 bind to each other in S pombe

To analyze the size of the Dps1–Dlp1 heteromer, we

prepared the crude proteins of E coli expressing the GST–

Dlp1 and His–Dps1 fusion proteins and incubated them

with the protein crosslinker disuccinimidyl suberate The

crosslinked proteins were then purified by a GST column

and subjected to SDS electrophoresis Western blotting with

anti-GST and anti-His Igs detected a band with a molecular

mass near the marker of 175 kDa, which can be considered

to be a heterotetrameric molecule because the calculated

molecular mass of heterotetromeric GST–Dlp1 and His–

Dps1 is 188 kDa (Fig 8C,D) Thus, wild-type Dlp1 and

Dps1 form a heterotetramer in E coli

Discussion

We identified a novel gene named dlp1 that encodes a

partner of Dps1, which together constitute the active

decaprenyl diphosphate synthase in S pombe The Dlp1

protein is weakly similar in sequence to Dps1 but lacks the

conserved regions of domains II and VI that are likely to be

the prenyl diphosphate synthase substrate-binding sites

Dlp1 and Dps1 form a heterotetrameric complex in

S pombe and together reconstitute

ubiquinone-10-gener-ating enzymatic activity in E coli The heterotetrameric

structure of Dps1 and Dlp1 is novel since other long

chain-producing polyprenyl diphosphate synthases that

synthesize the side chain of ubiquinone appear to exist as

homodimers [14,39] That the S pombe prenyl diphosphate

synthase exists as a heteromer, unlike its known

homodi-meric counterparts, may relate to the fact that the latter are

prokaryotic enzymes In other words, eukaryotes may have

evolved heteromeric prenyl diphosphate synthases from the

homodimeric prokaryotic synthases Supporting this notion

is our study with the homodimeric long chain-producing

polyprenyl diphosphate synthase from E coli, namely, the

IspB octaprenyl diphosphate synthase [39] Our group

previously showed that when E coli is transfected with a

construct encoding a functionally inactive IspB molecule

due to a mutation, an active enzyme is nonetheless formed

when the mutant is paired with the wild-type enzyme [39]

This observation suggests that the components of the

homodimeric enzyme could be subjected to evolutionary

alteration wherein they act in a heteromeric form with

another molecule At present, it is not clear whether this

heteromeric enzyme form occurs commonly in eukaryotes,

but our preliminary data do suggest that the human

decaprenyl diphosphate synthase is not a homomeric enzyme (data not shown) Heteromeric prenyl diphosphate synthases may be widely spread than it was thought as the medium chain-producing heptaprenyl diphosphate synth-ases from B subtilis and M luteus, which synthesize the side-chain of menaquinone [29,30], do occur as hetero-dimers and it was recently observed that the short chain-producing geranyl diphosphate synthase from spearmint forms a heterotetramer [27,28]

To date, we have obtained three ubiquinone-less mutants of S pombe due to the disruption of dps1, ppt1 and dlp1 All three mutants displayed essentially the same phenotypes, namely sensitivity to H2O2 and Cu2+, the need for an antioxidant such as glutathione for growth on minimal medium, and the production of H2S The former two phenotypes reflect the role ubiquinone plays as an antioxidant, while the latter phenotype supports the notion that ubiquinone acts as a sulfide oxidant Unlike

S pombe, ubiquinone-less S cerevisiae do not produce

H2S The lack of H2S production may be due to the fact that, unlike S pombe and other eukaryotes, S cerevisiae does not possess the genes encoding sulfide ubiquinone reductase, which oxidizes sulfide by ubiquinone Thus, ubiquinone may play an common and important role in eukaryotes by participating in a sulfide detoxication pathway

Acknowledgements

This work is supported by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan We thank Asuka Honma for technical assistance.

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