Eur. J. Biochem. 249, 61-69 (1997) 0 FEBS 1997
Cloning and characterization of the Arabidopsis thaliana S Q S l gene encoding squalene synthase Involvement of the C-terminal region of the enzyme in the channeling of squalene through the sterol pathway Rachida KRIBTI', Montserrat ARR02, Ana DEL ARCO', Victor GONZALEZ', Lluis BALCELLS ', Didier DELOURME', Albert FERRER*, Francis KARST' and Albert BORONAT'
' '
Laboratoire de Gtnttique Physiologique et MolCculaire, Institut de Biologie MolCculaire et d'IngCnierie GCnktique, UniversitC de Poitiers, France Unitat de Bioquimica, Facultat de Farmacia, Universitat de Barcelona, Spain Departament de Bioquimica i Biologia Molecular, Facultat de Quimica, Universitat de Barcelona, Spain
(Received 17 June 1997)
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EJB 97 08684
Squalene synthase (SQS) catalyzes the first committed step of the sterol biosynthetic pathway. A fulllength Arabidopsis thaliana SQS cDNA has been isolated by combining library screening and PCR-based approaches. Arabidopsis SQS is encoded by a small gene family of two genes (SQSl and SQS2) which are organized in a tandem array. SQSl and SQS2 have an identical organization with regard to intron positions and exon sizes and encode SQS isoforms showing a high level of sequence conservation (79% identity and 88% similarity). The isolated cDNA has been assigned to the SQSl gene product, SQSl. RNA blot analysis has shown that the 1.6-kb SQSl mRNA is detected in all plant tissues analyzed (inflorescenses, leaves, stems and roots) although the transcript is especially abundant in roots. Arabidopsis SQSl isoform is unable to complement the SQS-defective Saccharomyces cerevisiae strain 5302, although SQS activity was detected in the microsomal fraction of the transformed yeast strain. However, a chimeric SQS resulting from the replacement of the 66 C-terminal residues of the Arabidopsis enzyme by the 111 C-terminal residues of the Schizosaccharomyces pombe enzyme was able to confer ergosterol prototrophy to strain 5302. Labeling studies using ['Hlfarnesyl-P, and microsomal fractions obtained from yeast strains expressing either Arabidopsis SQSl or chimeric Arubidopsis/S. pombe SQS derivatives indicated that the C-terminal region of the enzyme is involved in the channeling of squalene through the yeast sterol pathway.
Keywords: isoprenoid biosynthesis; Arabidopsis thaliana; yeast; squalene synthase; metabolic channeling.
Squalene synthase (SQS) catalyzes the condensation of two molecules of farnesyl diphosphate (farnesyl-P,) to produce squalene, the first committed precursor for sterol biosynthesis [I]. In plants, as in other eukaryotes, farnesyl-P, lies at a multiple branch point in the isoprenoid biosynthetic pathway [2]. Farnesyl-P, serves as a substrate for the first committed reactions of several branched pathways leading to the synthesis of isoprenoid compounds that are required for plant growth and development, such as phytosterols (membrane structure and function), dolichols (glycoprotein synthesis), ubiquinones (electron transport) or sesquiterpenoid phytoalexins (defense against pathogen attack). Farnesyl-P, also acts as a prenyl donor Correspondence to A. Boronat, Departament de Bioquimica i Biologia Molecular, Facultat de Quimica, Universitat de Barcelona, Marti i FranquCs 1, E-08028 Barcelona, Spain E-mail:
[email protected] Abbreviations. SQS, squalene synthase ; farnesyl-P,, farnesyl diphosphate; RACE, rapid amplification of cDNA ends. Enzymes. Squalene synthase, famesyl-diphosphate farnesyl transferase (EC 2.5.1.21); squalene epoxidase (EC 1.14.99.7); lanosterol synthase (EC 5.4.99.7). Note. The Arabidopsis SQS cDNA sequence reported here has been deposited in the GenBank and DDBJ databases under accession number X86692.
in protein prenylation, a process that promotes membrane interactions and biological activities of a variety of cell proteins involved in signal transduction, membrane biogenesis and cell growth control [3, 41. In addition to the proposed role of SQS in the control of phytosterol biosynthesis, changes in SQS activity could also affect the flux of isoprenoid compounds down the various branches of the pathway in competition for the available farnesyl-P,. SQS has been shown to be highly regulated in mammals [5, 61. There are reports indicating that SQS activity is also modulated in plants. In tobacco cell-suspension cultures, the addition of fungal elicitors induces the synthesis of sesquiterpenoid phytoalexins, with a concomitant decline in sterol biosynthesis which correlates with a suppression of SQS activity [7, 81. Similar results have been reported in pathogen- or elicitorchallenged potato tuber disks [9]. SQS is a membrane-bound enzyme that has been purified to homogeneity from microsomal membranes of Saccharomyces cerevisiae [lo] and in a truncated soluble form from rat liver [5]. In plants, the enzyme has been solubilized and partially purified from daffodil microsomal membranes [11] and from tobacco cell-suspension cultures [121. Comparison of the amino acid sequence of SQS from S. cerevisiae [13, 141, rat [6], Schizosaccharomyces pombe [15], human [15, 161 and Arabidopsis
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thuliunu [I71 has revealed that the overall similarity is relatively poor, although some specific regions presumably involved in substrate binding and enzyme catalysis are highly conserved among the different enzymes [6, 151. Conservation is especially poor at the N- and the C-terminal regions of the enzyme. However, in spite of its divergence, the C-terminal region of all known SQS contains a highly hydrophobic sequence that is believed to anchor the enzyme in the endoplasmic reticulum membrane Ll5, 181. A model for the secondary structure of rat SQS has been proposed by McKenzie et al. (61. To gain new insights into the role of SQS in the control of plant sterol biosynthesis, we have isolated and characterized a cDNA clone encoding the Arubidopsis SQSl isoform and its corresponding gene. We also report that Arabidopsis SQS is encoded by two differentially expressed genes ( S Q S l and SQS2) which are organized in a tandem array. Our results also show that the divergent C-terminal region of SQS is involved in the channeling of squalene through the sterol pathway.
MATERIALS AND METHODS Enzymes and biochemicals. Restriction endonucleases and DNA-modifying enzymes were purchased from Boehringer Mannheim and Promega. [n-”P]dCTP (3000 Ci/mmol) was from Amersham. 11-’H]Farnesyl-P, (1 5 Ci/mmol) was from Isotopchim. Amino acids, uracil, adenine, ergosterol, farnesol, lanosterol, squalene, and Tergitol NP-40 were from Sigma. Squalene 2,3-epoxide was synthesized as described by Van Tamelen and Curphey 1191. Yeast extract, bactopeptone, bactotryptone and yeast nitrogen base without amino acids and (NH,)$O, were from Difco. All the other chemicals were of the highest commercial grade available. Plant material. Arubidopsis thulium plants (ecotype Columbia) were grown as described [20]. Roots were obtained from 3-week-old plants grown on filter papers i n mineral medium supplemented with 1 % (mass/vol.) sucrose and 2% (mass/ vol.) agar. Strains and media. Succhuroinyces cerevisiae strain 5302 (Matn erg9: :HIS3, uru3, his3, ade2, leu2, uux32), devoid of SQS activity [21], was derived from mutant strain erg9-I (ATCC64031). Escherichia coli strain DH5a was used for cloning, maintenance, and propagation of plasmids. Yeast strains were grown in YPG medium, containing 2% (masdvol.) yeast extract, 1 % (mass/vol.) bactopeptone and 2% (masdvol.) glucose, or minimal medium, containing 0.16 c/o (mass/vol.) yeast nitrogen base without amino acids and (NH,),SO,, 0.5 % (masshol.) (NH,),SO, and 1 % (masdvol.) glucose. Yeast cells were grown at 28OC either in liquid culture or on agar plates (media supplemented with 15 g agar/l). When required to supplement auxotrophies, uracil (SO pg/ml), adenine (SO pglml), amino acids (SO pg/ml each), or ergosterol (4 pg/ml in liquid culture or 80 pg/ml i n agar plates) were added to the growth media. Ergosterol was supplied by dilution of a stock solution (4 mg/ml) in a mixture of Tergitol NP-40/ethanol (1 : 1 ) . Yeast strain 5302 was transformed by the modified lithium acetate procedure described by Gietz et al. 1221. Ura+ transformants were selected on medium without uracil and, after 5 days of growth, subcultured on medium without ergosterol to test for ergosterol prototrophy. E. coli cells were grown in LuriaBertani medium containing 1 % (masshol.) bactotryptone, 0.5 % (madvol.) yeast extract and 1% (mass/vol.) NaCl, supplemented with ampicillin (100 mg/ml) when required. Cloning of Arabidopsis SQS cDNA. Amino acid sequences conserved among S. cerevisiue, S. pombe and human SQS were used to design two degenerate oligonucleotide primers for PCR
amplification of DNA obtained from an Arubidopsis cDNA expression library constructed in plasmid pFL61 [23]. The sequence of the primers was: 5‘-CGGAATTCTAYTGYCAYTAYGTTGCTGGIYTIGTNGG-3’ (forward primer) and 5‘-
CGGG ATCCATIGCCATIACYTGNGGRATNGCRCARAA-3' (reverse primer:), where R is G or A, Y is C or T, N is G, C, A or T, and I is inosine. EcoRI and BanzHI restriction sites (underlined) were included in the sequence of forward and reverse primers, respectively. PCR reactions were performed in a 100-p1 reaction mixture containing 10 pM of each primer, 1 pg template DNA, 0.2mM each of dATP, dCTP, dGTP and dTTP, PCR buffer and 2 U of Ampli Tuq DNA polymerase (Perkin-Elmer). The amplification reactions were performed for 35 cycles of 60 s at 94”C, 3 0 s at 58°C and 9 0 s at 72”C, with a 10-min final extension at 72°C. The resulting PCR products were digested with EcoRI and BanzHI and cloned into the corresponding sites of plasmid pUCl8 prior to sequencing. Sequencing of the amplification product revealed a fragment of 386 bp encoding an amino acid sequence corresponding to SQS. The amplified 386-bp cDNA fragment was used as a probe for the screening of 2.5 X 10’ clones of the Arubidopsis cDNA expression library. The probe was ”P-labeled by random priming [24] with [u-’*P]dCTPusing the random primers DNA labeling kit (Boehringer Mannheim). Hybridization of replica filters was for 18 h at 65°C in 6XNaCKit (1XNaCKit = 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0), 2XDenhardt’s, and 100 pg/ml denatured salmon sperm DNA. Nitrocellulose filters (Millipore) were washed at 45°C twice i n 2xNaCl/Cit, 0.1 % SDS and twice in 0.2xNaCl/Cit, 0.1% SDS. Two positive clones (pAB14 and pAB23) were identified and purified. The inserts of plasmids pAB14 and pAB23 were excised as Not1 fragments and cloned into the corresponding site of plasmid pBluescript (Stratagene) prior to sequencing. Sequencing of the inserts revealed that they were chimeric cDNAs in which the 3’region corresponded to SQS but the 5’-region contained unrelated sequences. The two SQS cDNA fragments encoded N-terminal truncated forms of the enzyme. The missing 5‘-coding region of the cloned Arubidopsis SQS cDNA was isolated using the Marathon cDNA amplification kit (Clontech). 2 pg poly(A)rich RNA from Amhidopsis seedlings was reverse transcribed according to the manufacturer’s recommendations, using an antisense gene-specific primer (5’-AACAAGCCCAGCAACATAGTGGC-3’) complementary to the isolated Ambidopsis SQS cDNA. After PCR amplification, the product obtained was cloned into plasmid pGEM-T (Promega) to create plasmid pRK3. A cDNA containing the entire Arubidopsis SQS coding sequence was generated by using appropriate restriction sites that allowed the fusion of the 5’- and the 3’-regions (plasmid pRKl8). Isolation of an Arabidopsis SQS genomic clone. An Arubidopis genomic library constructed in AFIX (2X 104recombinant phages) was screened using a probe derived froin the Arubidopsis SQS cDNA. The probe was ”P-labeled by random priming as described above. Hybridization of replica filters was for 18 h at 65°C in 6XNaCl/Cit, 2XDenhardt’s, and 100pgIml denatured salmon sperm DNA. Nitrocellulose filters (Millipore) were washed at 45°C twice in 2XNaCl/Cit, 0.1 % SDS and twice in 0.2xNaCl/Cit, 0.1 % SDS. A single positive recombinant clone was identified and plaque-purified. DNA sequencing. Appropriate restriction fragments were subcloned into plasmids pBluescript or pUC18. When required, exonuclease 111 deletions were generated using the Erase-a-Base system kit (Promega). Both strands of DNA were sequenced by the dideoxynucleotide chain-termination method [ 251 using an ALF DNA sequencer (Pharmacia).
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Isolation and analysis of nucleic acids. Genomic DNA from 6-day-old dark-grown Arubidopsis seedlings was prepared as described [26]. Genomic DNA (10 mg) was digested with the indicated restriction enzymes, size-fractionated by electrophoresis in 0.8% (masslvol.) agarose gels, and blotted onto HybondC nitrocellulose membranes (Amersham). Hybridization with a ”P-labeled 574-bp HindIII fragment from the Arubidopsis SQS cDNA was for 18 h either at 65 “C or at 60°C in 0.7 M sodium chloride, 40 mM sodium phosphate pH 7.6, 4 mM EDTA, 0.1 % (masslvol.) SDS, 0.2 % (masdvol.) polyvinylpyrrolidone, 0.2% (mass/vol.) Ficoll, 9% (mass/vol.) dextran sulfate and 200 pg/ ml denatured salmon sperm DNA. High stringency washes of membranes hybridized at 65°C were performed twice at room temperature in 2XNaCI/Cit, 0.5% SDS, twice at 68°C in 1X NaCl/Cit, 0.5% SDS and then in 0.2XNaCl/Cit, 0.5% SDS. Low stringency washes of membranes hybridized at 60°C were performed twice in 2XNaCl/Cit, 0.570 SDS at room temperature, and twice at 60°C in 1XNaCVCit, 0.5 % SDS. Total RNA from different tissues of Arubidopsis was isolated as described [27]. When required, poly(A)-rich RNA was obtained using the Rapid mRNA purification kit (Amresco). For northern blot analysis, 30 pg total RNA from each sample was fractionated in a 1% (mass/vol.) agarose gel containing 2.2 M formaldehyde and blotted onto Hybond-N nylon membranes (Amersham). Hybridization with the same probe used for Southem blot analysis was for 18 h at 42°C in 50% (by vol.) f o m amide, 1 M NaCl, 50 mM sodium phosphate pH 6.5, 7.5XDenhardt’s, 1 % SDS, 10% (mass/vol.) dextran sulfate, and 500 mg/ ml denatured salmon sperm DNA. Filters were washed twice at room temperature in 2XNaCI/Cit, 0.5% SDS, twice at 65°C in 2XNaCI/Cit, 0.5% SDS and twice at 65°C in O.lXNaCl/Cit. Mapping of the 5‘-end of Arabidopsis SQSl mRNA. The 5’-end of the Arubidopsis SQSl mRNA was determined by the 5’ RACE technique 1281 using the 5’-Amplifinder RACE kit (Clontech). 5 pg poly(A)-rich RNA from Ambidopsis seedlings was reverse transcribed according to the manufacturer’s recommendations, using an antisense primer (5’-AACAAGCCCAGCAACATAGTGGC-3’) complementary to Arubidopsis SQS 1 cDNA. An anchor oligonucleotide (provided in the kit) was then ligated to the 3’ end of the single-stranded cDNA using T4 RNA ligase. The 5’ end of the SQSl cDNA was amplified by PCR using a forward primer complementary to the anchor oligonucleotide (provided in the kit) and a reverse nested primer (5’-CACACACGGCGTTACGGAGCTCGG-3’) complementary to the nucleotide sequence in the SQS cDNA. Expression of SQS cDNA sequences in S. cerevisiue. For the expression of the Arubidopsis SQS in yeast, we constructed plasmid pRK18, a derivative of plasmid pNEV-N [29], which contains the entire coding region of the Arubidopsis SQS cDNA under the control of the PMAl gene promoter. First, a BuEI fragment excised from plasmid pAB14 was cloned into plasmid pRK3 linearized with BulI, to create plasmid pRK4. Plasmid pRK18 was obtained by a three-piece ligation of a NotI-SphI fragment obtained from plasmid pRK4, a SphI-Not1 fragment from plasmid pAB14 and pNEV-N linearized with NotI. For the expression of chimeric ArubidopsislS. pombe SQS proteins we constructed plasmids pRK15, pRK17 and pRK19. Plasmid pRK15 encodes a chimeric SQS in which the N-terminal region of Arubidopsis SQS was replaced by the equivalent region of the S. ponzbe enzyme. A cDNA fragment encoding the N-terminal region of S. pombe SQS (residues 1- 121) was excised from plasmid pRK7 (Kribii and Karst, unpublished results) as a NotI -EcoRI fragment. A cDNA fragment encoding residues 122-410 of Arubidopsis SQS was amplified by PCR using plasmid pAB14 as a template. The sequence of the primers used was : 5’-ACGAATTCCACCATGTTTCTGCAGCT-3’ (forward
- 21.2 -
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5.2 5.0 4.3 3.5
2.0 1.9 1.6
1.3 1.0 0.8 0.6
Fig. 1. Southern blot analysis of Arubidopsis genomic DNA. Genomic DNA from Arubidopsis (10 pg) was digested with the restriction enzymes indicated at the top, electrophoresed and transferred onto nitrocellulose membranes. Filters were hybridized with a 574-bp HindIII fragment excised from the Avubidopsis SQSl cDNA under conditions of high (A) and low (B) stringency. Numbers on the right indiute the mobility of DNA size markers.
primer) and 5’-CGCTTACAGCGGCCGCTCAGTTTGCTCTGAGATATGC-3’ (reverse primer). EcoRI and NotI restriction sites (underlined) were included in the sequence of forward and reverse primers, respectively. PCR was performed in a 50-pl reaction mixture containing 10 pM of each primer, 5 ng template DNA, 0.2mM each of dATP, dCTP, dGTP and dTTP, PCR buffer and 2 U of Ampli Tuq DNA polymerase (Perkin-Elmer). The amplification reactions were performed for 30 cycles of 30 s at 94”C, 30 s at 56°C and 60 s at 72”C, with a 10-min final extension at 72°C. Plasmid pRK1.5 was obtained by a threepiece ligation of the S. pombe NotI-EcoRI SQS cDNA fragment, the Arubidopsis PCR product digested with EcoRI and NotI and pNEV-N linearized with NotI. Plasmid pRK19 encodes a chimeric SQS in which the C-terminal region of Arabidopsis SQS was replaced by the C-terminal region of the S. pombe enzyme. A cDNA fragment encoding the N-terminal region of Arubidopsis SQS (residues 1-344) was excised from plasmid pRK18 as a Notl-SphI fragment. A cDNA fragment encoding residues 350 to 460 of S. pombe SQS was amplified by PCR using plasmid pRK7 as a template. The sequence of the primers used was : 5’-ACCCGCATGCTTCATTATAAGAACACTC-3’ (forward primer) and 5’-ATAGTTTAGCGGCCGCACTAAAACAAATTAAGCTCTT-3’ (reverse primer). SphI and NotI restriction sites (underlined) were included in the sequence of forward and reverse primers, respectively. PCR amplification was performed as described above. Plasmid pRK19 was obtained by a three-piece ligation of the Arubidopsis NotI-SphI SQS cDNA fragment, the S. pombe PCR product digested with Sphl and NotI, and pNEV-N linearized with NotI. Plasmid pRK17 encodes a chimeric SQS in which both the N- and C-terminal regions of the Arabidopsis SQS were replaced by the correspond-
Kribii et al. ( E m J, Biochem. 24Y)
64
lkb
A
H
H
H
X E
E
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HHH
EX
SQS2
SQSl
0.2 kb
B
H
SQS 1 TGA
ATG
m 2
1
SQS2
3
6
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8
7
9
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13
: 1
2
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Fig. 2. Restriction and structural maps of Arubidopsis SQS genoinic clones. (A) Restriction map of the genomic region containing the SQSI and SQS2 genes. SQSl and SQS2 transcription unit are represented by solid boxes. The cloned regions contained in recombinant plasmids are indicated below the restriction map. Restriction sites are as follows: E, EcoRI; H, HindTTT, X, XbaI. (R) Structural organization of the SQSl and SQS2 genes. Exons are represented by boxes and are numbered from the 5’ end of the genes. Lines between boxes correspond to introns. Coding regions are represented by solid boxes.
ing regions of the S. pornhe enzyme. This plasmid was obtained by replacing the 3’ SphI-NoA fragment of plasmid pRK15 by the 3’ SphI-Not1 fragment from plasmid pRK19. All constructs were confirmed by DNA sequencing. The yeast strain 5302 was transformed with plasmids pRKIS, pRK17, pRK1S and pRK19 as described above. Assay for SQS activity. Yeast strains were grown in minimal medium containing ergosterol and/or the amino acids required to supplement auxotrophies. All subsequent rnanipulations were performed at 4°C. Yeast cells were harvested by centrifugation at 5OOOXg for 10 min, washed in SO mM potassium phosphate pH 7.5, and resuspended in breakage buffer (SO mM potassium phosphate pH 7.5, 1 mM dithiothreitol and 1 m M phenylmethylsulfonyl fluoride). Cells were disrupted by vigorous vortexing i n the presence of 0.45-mm glass beads. After centrifugation at 12000Xg for 30 min, the pellet was discarded and the supernatant was centrifuged at 105000Xg for 40 min. The supernatant (cytosolic fraction) was collected and the pellet (microsomal fraction) was resuspended in breakage buffer and centrifuged again at 105000Xg for 40 min. The pelleted microsomes were resuspended in SO mM potassium phosphate pH 7.5 at a final protein concentration of 1 mg/ml. The SQS activity was assayed for 30 min at 30°C in a reaction mixture (0.5 ml) containing 50 m M potassium phosphate pH 7.5, 5 mM MgCI,, 10 mM KF, 1 mM NADPH, 4 mM glucose 6-phosphate, 2 U glucose-6-phosphate dehydrogenase, 25 pM [l-’H]farnesyl-PL (48 nCi) and either microsomal or cytosolic fractions (100 pg protein). The reaction was stopped by adding 0.25 ml 4 0 % KOH and 0.25 ml ethanol. Saponification was allowed to proceed for 30 min at 60°C and the non-saponifiable lipids were extracted three times with 1 ml hexane. Farnesol resulting from the dephosphorylation of farnesyl-P, by endoge-
nous phosphatases was removed by adding silica powder to the hexane phase. The radioactivity remaining in the hexane extract was quantified by liquid scintillation counting. Assay for squalene epoxidase and lanosterol synthase activities. The enzymes were assayed in the same reaction mixture (100 pl) described above for the measurement of SQS activity supplemented with 40 pM FAD (required for squalene epoxidase activity) [30]. [I-’HIFarnesyl-P, was increased to 240 nCi. After incubation at 30°C for 1 h, SO ng each of squalene, squalene 2,3-epoxide, lanosterol, farnesol and ergosterol was added to the mixture as carriers. Aliquots (30 pl) of the reaction mixture were separated on TLC silica plates (Sigma) using cyclohexane/ethyl acetate (9 : 1) as a solvent. Radioactivity incorporated into squalene, squalene 2,3-epoxide and lanosterol was detected using an automatic TLC linear analyzer (Berthold 2832).
RESULTS Cloning of a cDNA encoding Arabidopsis SQS. The cloning of Arabidopsis SQS cDNA was first attempted by functional complementation of S. cerevisiue strain 5302, carrying a disrupted SQS gene (ergY::HIS3),using an Arubidopsis cDNA expression library constructed in plasmid pFL6I [23]. About 3 X 1Oi Ura’ transformants were screened for ergosterol prototrophy without success. Alternatively, a full-length SQS cDNA was isolated by combining a library-screening approach, using a homologous SQS probe generated by PCR, and RACE-based approaches to clone the missing 5‘-region of the cDNA sequence (for details, see Materials and Methods). While this study was in progress, a cDNA encoding Arabidopsis SQS was reported [171.
65
Kribii et al. ( E M J. Riochenz. 249) TATA box
-104
+l
GMTC~PTGTGAGTTGGTTCA~~~~C~G~G~TTCAGAAGCCTTTG~TTCATCGATGMT~~MCPIP~PIG~~~TA~ATATTCG~GGCCGAATCGC~TCG exon 1
136
1 ,176 17
TGAGGGTTCffGCMmATCCCTCGTGGT~CTGMT~CAGATCGTCGTCMCGMTCCTCCA~~ffGMTC~T~TffffiAAAC~~~AGffT~ACGATGCTGAGATATCC~ATGACATATAT H C S L D T M L R Y P D D I Y P
G~~CCTGAAGATGAAACGAGCGA~GA~GC~AGMG~GATCCCTC~AGCCACA~~~T~~~GCTA~CGATG~CCA~GGTTTCCC~GC~T~CTCG~A~~GCMCTC L L K M X R A I E K A E K Q I P P E P E W G F C Y S M L E K V S R S F S L V I Q Q L N T E L
intron 1 +316
63 1456 +596 66
GTAACGCCgtaagttcttctegctctcqgtct=tqtgttqgtcaacgt=tt=aatt~ttggagga~tccaqatatctg~~act~atttattgttgacgtcqgcgatgattggtttcttctgtagaatccgatggctttga
R
N
A
qagtttgtqttccacttagtteaatatgagttatgcagattcttattaattgattctg~agttt~ttctgagctqcagtaaatgcagattqtattttaqtqa~ct~tqattcgatatgtt~~acataa~aaaatggtgtg exon 2 lntron 2 tqtqttcqatcacatqctatcaqtgtagctqtgttcagtcttgcagcattttcttattcaatttttttcatttttttcattttgtagGTGTGTGTG~~A~TGG~~CCGAGff~GATA~G~~ttagtqtct
V
C
V
P
Y
L
V
L
R
A
L
D
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V
+736 80
cttaactttgctttcaaqattacettgatacqatqaggcttctccaaaatctgctatatttctqttttqaaatgcttttatgttaqaqccatctttcttcttcatqcttctgctqctaatattattttttgtacga~agA exon 3 Lntron 3 E
+876
GGATGATACTAGCATACCAGATG~~CC~TC~GATAG~CACC~~CATATACGATACTGA~CA~A~~Tqcaaqtctqaagagtctcttaacatcttcatctttcacattccattaatc D D T S I P T D E K V P I L I A F H R H I Y D T D W E Y S
01
exon 4 t1016 110 +1156 13 6 +1296 137 11436 +1576 15 9 +1716 186 +I856
cacttctgttctactttatagattttttctgctcatgttgtctcacgtttattg~gttactcaatttqttqatactcttatctgctttctgacaatactaatta~ct~atttttaacatatqcttttqtatatgaagGTA exon 5 intron 5 Y T~GA~CTATCGA~~A~AG~GMT~~~~AT~C~G~ATff~~GA~tc~qtqatacagaq~agtttcgtagttqatttqtttttgaqttatqqtgacaaaatgtcttgagct94a
Q
E
A
I
E
E
I
T
R
R
H
G
A
+2136 255 +2276
G
M
A
K
F
I
C
Q
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~gqtctctgttt~aqttaqaattetqtcatgcttaatcttttcataagtcataaatattgqaatgcctcatgtqatgcttctat~atatqacaagtaqtaatt~tattgaaagtaccaaaacttattt~taaqttttgta exon 6 aaqaaatqaaatcatttqtttae=g=tccagttqatccaqttttcactattatacaca~TAG~ffG~GATGAffACGATGMTA~GCCAffA~~CTTG~GG~AGG~GTCG~ff~CCT
V
E
T
V
D
D
Y
D
E
Y
C
H
Y
V
A
G
L
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G
L
G
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S
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L
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L
intron 6 GffGCA~ATCA~C~GA~~CGA~C~TTC~T~A~ffACA~ttacaacaatgtattttgaggtgtacaatgtttcttcttcctcaaatcttttqcaatttcqqacaa
A A G S E V L T P D W E A I S N S M G L F L Q exon 7 tatcattgaatgatttataacactggtcttatttgcaq~~~~ATCAGAGA~AT~GA~A~~MTGAGATACC~TCCCG~TG~C~CGCGAGA~~CAAATA
K
209 t1996 243
mtron 4
ttgactgtctaactqacaaattttacttgttcaactttaqGT~ACGRAGG1\GTIICRAGTA~Ga~~~TOGACCM~~CCATG~~G~G~~~~~ttagttctaatattt C Q T K E Y K I L M D Q F H B V S A A P L E L E K G
T
N
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I
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S
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W
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intron 7 exon 8 GaGgtgcatacataccactctcaagctctcatcttaggtctqatgttacccatattgactattttctgaacagtgcctttttqgtgatttgtaaattqtaatqtagGa~~TACGaGGAGAACA~~TCCG E
D
L
K
Y E E lntron 8
N
T
N
K
S
T A C A G m C T T A R P I T G ~ ~ G ~ A C ~ ~ ~ G A T G ~ T A ~ M ~ ~ G C f f G A A A T A C A T ~ ~ C ~ ~ T G A T C ~ C ~ T A ~ C G G T T ~ ~ C C A T C C ~ ~ ~ t t a q a q a a c q a
V
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L
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Y
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P
Q
ttaagtctttaaacttcacattqagaa~tgtttatattgttttcc~cttcatatggaattaqgtattctcctctttaactacqtctgcactaatatagaatqccttagaaqttaatattgcttt~c~tggqtqtattttc
+2416 293 +2556 318 +2696 330 +2836 340
exon 10 aaaqqaPaPcccgttatcttttattatcaacgagcttqagaacgtgtagtaqtaqct~q~~ttatt~tctgaaq~ctaatatcgctgaa~aa~tatctattgtttagGT~A~G~~~~~GATCG
G L T A K V I D R T K T intron 10 TGGCTGATGTCTATOGTG~ATGA~C~TGff~GA~~taqtcatcaqttccatgtttgtgattaqtttttcactcaqtttctgtttaaaqaaaag~atcatatgtg~attttctatcgacata M A D V Y G A F Y D P S C M L T T K exon 1 1 intron 11 t a t g a c a t a q G T T G A C C G A T C C ~ T ~ ~ G T ~ ~ ~ ~ ~ C C G A ~ ~ ~ ~ C A G ~ ~ ~ ~ G A G A f f i ~ G G A G ~ ~ ~ ~ ~ t a c a g t c t t t t a t t t a a t c c c a
V
D
X
N
D
P
N
A
S
K
T
L
N
R
L
R
A
V
Q
K
L
C
R
D
A
G
V
L
Q
N
R
exon 12 +2976
379 +3116 383
cctctcactaagaaactggtcacaaacacctataactaatcttccaaqtattctagaataqqatgatattatcataatctttctttctgqttgqgtttctgctqaatcattqtqtctcttcctttcagAAAATCTTATGT K S Y V intron 12 exon 13 TAATGACPIPrPIGGACC~~GTGT~~qtaaqtta~atagtttccctttt~catataagtat~tcatttttaaaaaa~ta~ataaaa~tt~tattct~tatattctacccgaaa~tgc~qA~ATA N D K G Q P N S V P I I M V V
13256
398 r3396 13536
Fig.3. Nucleotide sequence of the Arabidopsis SQSI gene and its derived amino acid sequence. Introns are in lower-case letters whereas all other sec1uences are in upper-case letters. Nucleotides are numbered (left) by assigning position +1 to the transcription start site (in bold). The putative TATA box and the start and stop codons are underlined. The polyadenylation site is indicated by an asterisk. The deduced amino acid sequence of the encoded protein is shown below the nucleotide sequence and is numbered on the left.
Southern blot analysis. DNA blot analysis was performed using Arubidopsis genomic DNA digested with restriction endonucleases CluI, EcoRI, EcoRV, HindIII, PstI and Xbal. A 574bp HindIII fragment derived from the Arubidopsis SQS cDNA clone was used as a probe. The results are shown in Fig. 1. Under high stringency conditions, a single major band was observed in the lanes corresponding to CluI, EcoRV, HindlII, Pstl and Xbul digestions, whereas two bands were detected in the lane corresponding to EcoRI (Fig. 1A). This simple pattern of bands indicated that the fragments detected derived from the gene corresponding to the cloned SQS cDNA. However, under low stringency conditions, additional bands were observed in the lanes corresponding to HindIII, PstI and XbaI digestions (Fig. 1B), thus suggesting that Arubidopsis contains at least two SQS genes. Interestingly. no additional bands were observed in the lanes corresponding to ClaI, EcoRI and EcoRV digestions under low stringency conditions. A plausible explanation for these results was that the Arubidopsis SQS genes were close to one another in the genome, as was later verified. Isolation and characterization of the Arubidopsis SQSl gene. To isolate the gene corresponding to the cloned Arubidopsis SQS
1.6 kb
+ SQS
Total RNA Fig. 4. Northern blot analysis of‘ Arnbidopsis RNA. Total RNA samples from different tissues of Arubidupsis (30 @lane) were electrophoresed in a 1 % agarose/formaldehyde gel and transfeii-ed onto a nylon membrane. The filter was hybridized with the same probe described in the legend of Fig. 1. Exposure time was 24 h. Ethidium bromide staining of the gel before transfer is shown below the autoradiography.
66 SQSl
SQS2
Kribii et al. ( E M J. Biochem. 249) MGSLGTMLRYPDDIYPLLKMKRAIEKAEKQIPPEPHWGFCYSMLHKVSRS 5 0 . . . .S.1 ..H . .EL . . . . .L.L . .T..Q....L...LA....I.....K. 5 0 V V FSLVIQQLNTELRNAVCVFYLVLRALDTVEDDTSIPTDEKVPILIAFHRH
. . . . . . . . G . . . . . . . . . . . . J............V.FEI........... V
cDNA probe were subcloned. Sequence analysis revealed that clones pgABl and pgAB2 (Fig. 2A) contain overlapping inserts including the entire coding region of an SQS gene, hereafter referred to as S Q S I , as well as its 5'- and 3-flanking regions. The nucleotide sequence of the transcription unit of the Arahidopsis SQSl gene is shown in Fig. 3. The alignment of the nucleotide sequence of the SQSI gene with that of the SQSl cDNA showed that the gene consists of 13 exons and 12 introns (Figs 2B and 3). The transcription start site of the SQSl gene, mapped by the 5' RACE technique, is located 129 bp upstream of the translation start site and is preceded by a consensus TATA box located 31 bp upstream (Fig. 3).
100 100
V
IYDTDWHYSCGTKEYKILMDQFHHVSAAFLELEKGYQEAIEEITRRMGAG 150 . . .G . . . F........L.............K..........D..K..... 150 n
MAKFICQEVETVDDYDEYCHYVAGLVGLGLSKLFLAAGSEVLTPDWEAIS 200 . . . . . . x . . . . I.........A..........I.I.SEL.I.....KQ.. 200 V V NSMGLFLQKTNIIRDYLEDINEIPKSRMFWPREIWGKYADKLEDLKYEEN 250 ..T . . . . . . . . . . K........R...............V.....F.N.. K 250
V TNKSVQCLNEMVTNALMHIEUCLKYMVSLRDPSIFRFCAIPQIMAIGTLA 3 0 0 A T . A . . . . . . . . . . . . N.V . . . . .SLA .....A . .QS ...... V..... T 3 0 0 V V LCYNNEQVFRGVVKLRRGLTAKVIDRTKTMADVYGAFYDFSCMLKTKVDK 350 . . . . .V . . . . . . . R M . . . . .............................. N 3 5 0
"
Fig.5. Amino acid sequence alignment of Arubidopsis SQSl and SQSZ. Identical residues are represented by dots, intron positions by open triangles. The predicted hydrophobic sequences involved in the anchoring of the enzyme to the endoplasmic reticulum membrane are shaded.
cDNA, the 574-bp HindIII cDNA fragment was used to screen a genomic library. A single positive clone was isolated (see Materials and Methods). DNA from this genomic clone was digested with EcoRI or XbaI and the fragments hybridizing to the
Expression pattern of the S Q S i gene. The expression pattern of Arabidopsis SQSl mRNA was studied by northern blot analysis using total KNA isolated from inflorescences, leaves, stems, roots and seedlings. Hybridization was performed under high stringency conditions using as a probe the 574-bp HindIII fragment derived from the Arubidopsis SQSl cDNA. A transcript of 1.6 kb was identified in all samples, although the highest level of SQSl mRNA was detected in roots (Fig. 4). Similar amounts of RNA were loaded in each lane, as shown by ethidium bromide staining of the RNA samples before transfer (Fig. 4). Arabidopsis contains two SQS genes organized in a tandem array. Sequencing of the overlapping plasmids pgAB2 and pgAB3 (Fig. 2A) revealed the presence of a second SQS gene (SQS2) located downstream from the SQSI gene. The organization of exons and introns of the SQS2 gene was deduced by comparing its nucleotide sequence with that of the SQSl gene. The assignement of introns for the SQS2 gene (Fig. 2B) is reinforced by the fact that all follow the GT/AG rule. In both genes,
A
pRKl8
COMPLEMENTATION OF STRAIN 5302
ENCODED PROTEIN
PLASMlD
1 NH2
A. thaliana
410
-
m
pRK15
pRKl9
, pRK17
NHZ
I
S.pombe1211,22
A. thaliana
344,350
S.pombe
460
a m +
6
Fig. 6. Complementation analysis of S. cerevisiae strain 5302 with Arubidopsis SQSl and chimeric derivatives. (A) Schematic representation of the SQS proteins expressed by plasmids pRK15, pRK17, pRK18 and pRK19. Hatched and open boxes represent Arabidopsis and S. pornbe sequences, respectively. Numbers above the boxes indicate the position of amino acid residues. (B) Complementation analysis o f yeast strain 5302 transforined with plasmids pRK18 and pRK19. Strains 5302, 5302 [pRK18] and 5302[pRK19] were streaked onto YPG plates or YPG plates supplernented with ergosterol (80 pg/ml) and incubated at 28°C for 4 days.
Kribii et al. (Eur: J. Biochem. 249)
Table 1. Squalene synthase activity in cytosolic and microsomal fractions of plasmid-hearing yeast strains. Microsomal and cytosolic fractions prepared from yeast strains were assayed for SQS activity i n triplicate assays as described in Materials and Methods. The specific activity found in the wild-type strain FLIOO was assigned a relative acitvity of 100; n.d.. activity not detectable. Strains
SQS specific activity cytosol
microsomes
0.03 t 0.01 n. d. n. d. n.d. n.d.
B
h
Ai
FL100
Microsoma1 relative activity
U/mg
FL100 5302 5302 [pRK17] 5302 [pRK18] 5302 [pRK19]
E
67
5302
0.38 t 0.04 n. d. 0.04 -+ 0.01 0.03 t 0.01 0.04 i0.01
100 -
11 8 11
introns are located at equivalent positions relative to the coding sequences (Fig. 5). The transcript corresponding to the SQS2 gene has been identified by reverse transcriptase PCR followed by sequencing of the amplified cDNA product. The predicted protein encoded by the SQS2 gene contains 413 amino acid residues and shows 79% identity (88% similarity) with the SQSl protein (Fig. 5).
5302 [pRK18]
Expression of Arabidopsis SQS in S. cerevisiae. The coding sequence of the Aunbidopsis SQSl cDNA was cloned in the expression plasmid pNEV-N under the control of the PMAI promoter to create plasmid pRK18 (see Materials and Methods). The PMAI promoter was chosen since it has been reported to drive efficient expression of plant cDNAs in yeast 129, 311. Unexpectedly, plasmid pRK18 was unable to confer ergosterol prototrophy to strain 5302 (Fig. 6) As a first approach to elucidating the molecular basis underlying this lack of complementation, a series of constructs expressing chimeric Ambidopsis/S. pombe SQS proteins were assayed for their ability to complement strain 5302 (Fig. 6). The simultaneous replacement of the divergent N- and C-terminal regions of the Arabidopsis SQS by the corresponding regions of the yeast enzyme (plasmid pRK17) resulted in a chimeric enzyme able to confer ergosterol prototrophy to strain 5302. The same result was obtained when the C-terminal region of the Arabidopsis enzyme was replaced by the C-terminal region of the S. pombe enzyme (plasmid pRKl9). In contrast, the chimeric enzyme resulting from the substitution of the N-terminal region of Arabidopsis SQS by the equivalent region of the S. pombe enzyme (plasmid pRKl5) was unable to complement strain 5302. To analyze whether the complementation of strain 5302 by the different chimeric enzymes was dependent on the synthesis of an active form of the enzyme, we measured SQS activity in microsomal fractions obtained from strains FL100, 5302, 5302[pRK17], 5302[pRKlX] and 5302[pRK19] (Table 1). The SQS activity level detected in the microsoinal fraction of strains 5302 [pRK17] and 5302 [pRK19] was similar to that found in strain 5302 [pRK18]. No SQS activity was detected in the cytosolic fraction of strains 5302, 5302[pRKl7], 5302[pRKI 81 or 5302[pRK19] (Table 1). These results indicated that the lack of complementation of the erg9 mutation by the Arabidopsis enzyme was not due to the synthesis of an inactive form of the enzyme or to the inability of the plant enzyme to be targeted to the endoplasmic reticulum membrane. A plausible explanation for the lack of complemen-
5302 [pRKl9]
Fig. 7. Analysis of the reaction products of SQS, squalene epoxidase and lanosterol synthase in the microsomal fractions of plasmid-bearing strains. Microsomal fractions obtained from the yeast strains indicated to the right were incubated with ['H]farnesyl-P, and the reaction products were analyzed by TLC. Radioactivity was measured by using an automatic TLC linear analyzer (Berthold 2832). Radioactivity peaks correspond to farnesyl-Pz ( l ) , farnesol (2), squalene (3), squalene 2,3epoxide (4), and lanosterol (5). Peak 6 corresponds to an unidentified product.
tation of strain 5302 was that the squalene synthesized by the plant enzyme in the yeast endoplasmic reticulum membrane could not be used as a substrate for the following enzymes of the pathway. To address this question, labeling studies were performed to measure the synthesis of squalene 2,3-epoxide and lanosterol in rnicrosomes isolated from strains FL100, 5302, 5302[pRKl8] and 5302[pRK'19] after incubation in the presence of ['H]farnesyl-P,. The results, shown in Fig. 7, indicated that microsomes isolated from strain 5302[pRK19] were able to synthesize squalene 2,3-epoxide and lanosterol in addition to squalene, like microsomes isolated from the wild-type strain FLI 00. In contrast, microsornes isolated from strain 5302[pRK18] synthesized squalene but not squalene 2,3-epoxide or lanosterol. Taking all these data together, the following conclusions can be drawn: (a) Arubidopsis SQS is targeted to the yeast endoplasmic reticulum membrane, where it catalyzes the synthesis of squalene; (b) the squalene synthesized by the plant
68
Kribii et al. ( E m J. Biochem. 249)
t A. thaliana 1 A. thaliana 2 Rat Mouse Human S . cerevisiae S . pombe consensus
FCAIPQIMAI GTLALCYNNE QVFRGVVKLR RGLTAKVIDR TKTMADVYGA SCAIPQIVAI GTLTLCYNNV QVFRGVVRMR RGLIAKVIDR TKTMDDVYGA FCAIPQVMAI ATLAACYNNH QVFKGWXIR KGQAVTLMMD ATNMPAVKAI FCAIPQVMAI ATLAACYNNQ QVFKGWKIR KGQAVTLMMD ATNMPAVKAI FCAIPQVMAI ATLAACYNNQ QVFKGAVKIR KGQAVTLMMD ATNMPAVKAI FCAIPQVMAI ATLALVFNWR EVLHGDVKIR KGTTCCLILK SRTLRGCVEI FCAIPQVMAI ATLAAVFRNP DVFQTNVXIR KGQAVQIILH SVNLKNVCDL -cAIpQ--AI -TL-----N- -V----VK-R-G--------_ _ _ _ _ _ _ _ _ _
FYDFSCMLKT 346 FYDFSCMLQT 346 IYQYIEEIYH 347 IYQYIEEIYH 347 IYQYMEEIYH 347 FDYYLRDIKS 354 FLRYTRDIHY 351 -_________
A. thaliana 1 A. thaliana 2 Rat Mouse Human S . cerevisiae S. pombe Consensus
KVDKNDPNAS KTLNRLEAVQ .. KVDNNDPNAM KTLNRLETIK RVPNSDPSAS KAKQLISNIR .............. ......... ............ RIPNSDPSSS KTKQVISKIR RIPDSDPSSS KTRQIISTIR .............................. KLAVQDPNFL KLNIQISKIE QFMEEMYQDK LPPNVKPNET PIFLKVKERS KNTPKDPNFL KISIECGKIE QVSESLFPRR FREMYEKAW SKLSEQKKGN -----DP---K---------_ _ _ _ _ - _ -- __- -_ _ _ _ _ _ _ _ - _ - _ _ _ _ _ _
..KLCRDAGV 374 ..KVCRENGV 374 ..TQSLPNCQ 375 ..TQNLPNCQ 375 ..TQNLPNCQ 375 RYDDELVPTQ 414 GTQKAILNDE 411
A. thaliana 1 A. thaliana 2 Rat Mouse Human S . cerevisiae S . pombe Consensus
LQNRKSYVND KGQ LHXRKSYVND ETQ LISRSHY . . . . . . . LISRSHY... .. LISRSHY... QEEEYK .... QKELYRKDLQ
............ .........
RX. QVT QVT QVT RA. IHW
EDYVQREH. EDYVQREH. EDYVQTGEH
.........
SDFKELNLF
_---_-____
410 413 416 416 417 444 460
Fig. 8. Amino acid sequence alignment of the C-terminal region of SQS from Arubidopsis (SQSl and SQSZ), rat [6], mouse [39], human [IS, 161, S. cerevisiae [13, 141 and S. pombe 1151. Identical residues in all proteins are shown in bold. Dots indicate gaps introduced to optimize the aligment. Amino acid residues are numbered on the right. Arrow indicates the breakpoint in the Ambidopsis sequence used to create the C-terminal chimeric SQS enzymes. Shaded amino acid stretches correspond to the hydrophobic region involved in the anchoring to the endoplasmic reticulum membrane
enzyme in the yeast endoplasmic reticulum membrane cannot be further used as substrate by the subsequent enzymes of the pathway; (c) the replacement of the C-terminal region of the Arubidopsis SQS by the corresponding region of yeast SQS restores the channeling of squalene through the sterol pathway.
DISCUSSION To gain new insights into the role of SQS in the isoprenoid biosynthetic pathway in higher plants we first isolated a cDNA encoding Arabidopsis SQS. This was first attempted by functional complementation of a SQS-defective S. cerivisiae strain using an Arubidopsis cDNA expression library. Unexpectedly, no positive clones were detected after the screening of 3x10' transformants. Alternatively, a full-length SQS cDNA was isolated by combining library screening and PCR-based approaches. During the course of our studies, a cDNA encoding the same Arabidopsis SQS isoform was reported [17]. However, no information was provided on the structure of the corresponding gene or its expression pattern. By means of DNA blot analysis and gene cloning, we have shown that Ambidopsis contains two SQS genes (SQS! and SQS2) which are organized in a tandem array. The SQS! gene corresponds to the cloned SQS cDNA. The two SQS genes have an identical organization with regard to intron position and exon sizes and encode SQS isoforms that show a high level of sequence similarity. RNA blot analysis performed under high stringency hybridization conditions allowed the detection of the SQSl mRNA in all plant tissues tested. In contrast, attempts to detect the expression of the SQS2 mRNA by northern blot analysis were unsuccessful. However, the occurrence of a transcript corresponding to SQS2 has been detected by using reverse-transcriptase PCR. These results indicate that SQS2 corresponds to an active gene that i s expressed either at very low levels or at specific tissues or developmental stages. The occurrence of multiple SQS genes in Arubidopsis raises the question about the role of each individual SQS
isoform in the plant sterol biosynthetic pathway. The differential expression of the SQSI and SQS2 genes might be indicative of an specialized function of each SQS isoform within the sterol pathway in Arilbidopsts. The occurrence of SQS isoforms, together with the observation that other key enzymes of the plant isoprenoid pathway are also present in multiple isoforms [32351, give support to the recent hypothesis proposing that specific classes of isoprenoids are synthesized by independently regulated metabolic channels within the pathway [36]. An intriguing result concerning the characterization of Aruhidopsis SQSl isoform was its inability to complement S. cerevisiue strain 5302, impaired in SQS activity as a consequence of a disruption in the ERG9 gene. This finding, although initially unexpected, was not unprecedented since it has been reported that expression of the human SQS cDNA cannot confer ergosterol prototrophy to yeast cells bearing an ERG9 gene disruption [IS]. However, it was reported that the expression of a chimeric SQS cDNA containing the human 5' region and the S. cerevisiae 3' region reverted the ergosterol requirement of the ERG9 disrupted strain, thus tracing the lack of complementation to the 3' end of the human SQS cDNA. It was suggested that the underlying problem might involve protein instability, misreading of membrane targeting signal(s), or inefficient translation of the human SQS mRNA as a result of a different codon bias of the yeast strain LlS]. To elucidate the molecular basis behind the lack of complementation of the yeast strain 5302 by the Arubidopsis SQSl isoform, a series of constructs expressing chimeric SQS proteins, in which the N- and/or the C-terminal regions of plant SQS were replaced by the corresponding region(s) of the S. pombe enzyme, were assayed for their ability to complement strain 5302. The rationale for the replacement of these specific regions was that similarity among Arubidopsis, mammalian and fungal SQS is especially poor at the N- and C-terminal regions. In addition, S. poinbe SQS was chosen because of its high level of expression i n S. cerevisiue [15] (and Kribii and Karst, unpublished results).
Kribii et al. (Eur: J. Biochern. 249)
The results obtained demonstrate that the chimeric enzyme resulting from the replacement of the 66 C-terminal residues of the Arubidopsis enzyme by the I1 1 C-terminal residues of the S. pombe enzyme was able to confer ergosterol prototrophy to strain 5302. The region replaced contains the C-terminal divergent region (Fig. s), which includes the hydrophobic sequence involved in the anchoring of the enzyme to the endoplasmic reticulum membrane [15, IS]. Our results have shown that, despite the lack of complementation, the expression of Arubidopsis SQSl resulted in a significant level of SQS activity in the microsomal fraction of the transformed yeast strain. Since such a level of enzyme activity is enough to support the growth of erg9 mutant cells [13], we envisaged that, in the case of Arabidopsis SQSI, the underlying problem was most likely related to an intrinsic feature of the SQSt protein rather than to mRNA stability or translatability. In addition, our results ruled out the possibility that the lack of complementation was due to the synthesis of an inactive form of the enzyme or to the inability of plant SQS to be targeted to the yeast endoplasmic reticulum membrane. The results of the complementation assays and the enzyme activity measurements in the transformed yeast strains suggested that the squalene synthesized by Arabidopsis SQSl in the yeast endoplasmic reticulum membrane could not be used as a substrate for the following enzymes of the pathway. To address this question, we performed labeling studies using [’Hlfarnesyl-P, to identify the presence of metabolites derived from squalene in the microsomal fractions obtained from transformed yeast strains expressing either Arabidopsis SQSl or its chimeric derivatives. The results obtained indicated that squalene 2,3-epoxide and lanosterol were only detected when squalene was synthesized by the chimeric enzyme containing the S. pomhe C-termnal region, thus showing that this region of the yeast enzyme is required for the proper channeling of squalene through the sterol pathway in yeast. One possible explanation is that the C-terminal region of the yeast SQS contains sequence(s) that allow the specific interaction between SQS and squalene epoxidase, the next enzyme in the pathway, which has also been reported to be associated with the endoplasmic reticulum membrane [37]. The interaction between these two enzymes has previously been proposed from the observation that squalene is a poor substrate for yeast squalene epoxidase when it is exogenously added to yeast microsomal fractions, in contrast to farnesyl-P, which is efficiently converted to squalene 2,3-epoxide [30].Alternatively, it cannot be ruled out that specific sequence(s) within the C-terminal region of yeast SQS are required for the proper targeting of the enzyme to specific subdomain(s) in the endoplasmic reticulum to allow its co-localization with other enzymes of the sterol pathway (i.e. squalene epoxidase). In this respect, it has recently been proposed that yeast endoplasmic reticulum might contain specific regions specialized for sterol biosynthesis [38]. Work is currently in progress to identify the specific element(s) within the yeast C-terminal region required for the proposed channeling of squalene through the sterol pathway. We thank Dr F. Lacroute for the Arubidopsis cDNA expression library. This work was supported by grants PB93-0753 from the Direccidn General de lnvestigacicin CienrLficu y Te‘cnicu and GRQ94-1034 and 1995SGR-00457 from the Comissici lnterdepurtamentel de Recercu i Innovacid Tecnolbgicu de la Generalitat de Cutulunyu (to A. B.), GREG/ INRA no. 9:95 (to E K.), and Accicin lntegrudu Hispano-Fruncesu HF95-290B (to A. F. and F. K.). V. G. and A. A. are recipients of a predoctoral fellowship from the Direccid General de Recercu de la Generalitat de Cutulunyu.
69
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