Cloning and Functional Expression of UGT Genes Encoding Sterol Glucosyltransferases from Saccharomyces cerevisiae, Candida albicans, Pichia pastoris, and Dictyostelium discoideum*

Dirk WarneckeDagger §, Ralf Erdmann, Annette FahlDagger , Bernhard HubeDagger , Frank MüllerDagger , Thorsten ZankDagger , Ulrich Zähringerparallel , and Ernst HeinzDagger

From the Dagger  Universität Hamburg, Institut für Allgemeine Botanik, 22609 Hamburg,  Freie Universität Berlin, Institut für Biochemie, Limonenstrasse 7, 12203 Berlin, and parallel  Forschungszentrum Borstel, 23845 Borstel, Germany

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Sterol glucosides, typical membrane-bound lipids of many eukaryotes, are biosynthesized by a UDP-glucose:sterol glucosyltransferase (EC 2.4.1.173). We cloned genes from three different yeasts and from Dictyostelium discoideum, the deduced amino acid sequences of which all showed similarities with plant sterol glucosyltransferases (Ugt80A1, Ugt80A2). These genes from Saccharomyces cerevisiae (UGT51 = YLR189C), Pichia pastoris (UGT51B1), Candida albicans (UGT51C1), and Dictyostelium discoideum (ugt52) were expressed in Escherichia coli. In vitro enzyme assays with cell-free extracts of the transgenic E. coli strains showed that the genes encode UDP-glucose:sterol glucosyltransferases which can use different sterols such as cholesterol, sitosterol, and ergosterol as sugar acceptors. An S. cerevisiae null mutant of UGT51 had lost its ability to synthesize sterol glucoside but exhibited normal growth under various culture conditions. Expression of either UGT51 or UGT51B1 in this null mutant under the control of a galactose-induced promoter restored sterol glucoside synthesis in vitro. Lipid extracts of these cells contained a novel glycolipid. This lipid was purified and identified as ergosterol-beta -D-glucopyranoside by nuclear magnetic resonance spectroscopy. These data prove that the cloned genes encode sterol-beta -D-glucosyltransferases and that sterol glucoside synthesis is an inherent feature of eukaryotic microorganisms.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Sterol glycosides are widespread membrane lipids, occurring in all plants, several algae (1-3), some fungi (4-9), slime molds (10-12), Dictyostelium (13), a few bacteria (14-19), and even animals (20-23). The knowledge base for sterol glycosides is rather limited compared with free sterols and sterol esters, where the synthesis, transport, and functions have been studied extensively in animals (24-29), plants, (30-35), and yeast (36-40). The basis for studies on the functions of sterol glycosides is the assumption that free sterols and sterol glycosides differ physiologically. It is obvious that the attachment of a glycosyl moiety to the sterol backbone alters the physical properties of this lipid. As a result, there are changes in the properties of membranes containing different proportions of free sterols and sterol glycosides. Such changes have been studied with artificial membranes in terms of membrane fluidity, permeability, hydration, and phase behavior (41-44). However, we still do not know how free sterols and sterol glycosides differ physiologically in biological membranes and why many eukaryotic organisms synthesize sterol glycosides. One of the main reasons for our limited knowledge in this field is the lack of a genetic approach. The objective of the present work was the isolation and characterization of sterol glycosyltransferase genes from eukaryotic organisms. We expect that genetic manipulation of these genes will facilitate the elucidation of sterol glycoside functions in these organisms.

The predominating sugar moiety in sterol glycosides is glucose. Besides plants, UDP-glucose:sterol glucosyltransferase activity was determined by in vitro enzyme assays in Saccharomyces cerevisiae (45-48), Candida bogoriensis (49), other fungi (50), and Physarum polycephalum (51, 11). Although the lipid composition of S. cerevisiae was studied in detail during the last decades, there are only rare reports on the actual isolation of sterol glucoside (SG)1 from this yeast (8, 9). The expression level of sterol glucosyltransferase gene(s) seems to be so low that very sensitive radioactive assays are required to detect enzyme activity in vitro. However, expression is too low for accumulation of a significant amount of SG in vivo suitable for lipid analysis. Another possibility is the existence of a sterol glucoside hydrolase that counteracts the sterol glucosyltransferase keeping the amount of SG very low. Such a hydrolase has been detected in plants (52, 53) but not in S. cerevisiae.

In contrast to S. cerevisiae, significant amounts of SG were occasionally found in other fungi such as C. bogoriensis (4) and Phytium sylvaticum (5), whereas sterol mannosides were detected in the human pathogen Candida albicans (6, 7).

Here we describe the identification and characterization of sterol glucosyltransferases from three different yeasts as well as from Dictyostelium discoideum. Since the genes cloned in this work all belong to a superfamily of UDP-glycosyltransferases (UGTs) (54, 55), we use the gene symbol UGT for each new member. The UGT51 gene of S. cerevisiae was identified by a homology probing approach with the cDNA sequences of UDP-glucose:sterol glucosyltransferases from plants that were recently cloned from oat (ugt80A1,2 GenBankTM accession number Z83832) and Arabidopsis (ugt80A2, GenBankTM accession number Z83833) (56, 57). The UGT51 genes from C. albicans, Pichia pastoris, and UGT52 from D. discoideum were cloned by either homology probing or PCR-based strategies. Moreover, we identified ergosterol as an in vivo substrate for the sterol glucosyltransferases from S. cerevisiae and P. pastoris. Furthermore, a new glycolipid was identified in S. cerevisiae as the product of the in vivo action of the yeast sterol glucosyltransferase from P. pastoris.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Plasmids used in this study


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Bacterial and Saccharomyces Strains and Plasmids

Escherichia coli strains XL1-Blue (MRF') (Stratagene), BL21(DE3) (Novagen), TOP10 (Invitrogen), and the vectors pGEM-T (Promega), pBluescript (Stratagene, La Jolla, CA), pET19b (Novagen), and pBAD-TOPO (Invitrogen) were used for cloning and expression of different fragments of the sterol glucosyltransferase genes. Yeast strains used in this study were UTL-7A (MATa, ura3-52, trp1, leu2-3,112) and Delta ugt51 (MATa, ura3-52, trp1, leu2-3, 112, ugt51::kanMX4; this study). Expression of different UGT genes in S. cerevisiae was performed with pYES2 (Invitrogen) and pGAL4 (this study). For the construction of pGAL4, the XbaI/BamHI fragment of pTerm1 (58) which contains the CYC1 terminator (59) was subcloned into plasmid pRS316 (60), resulting in pRS316t. The 0.5-kb SpeI/HindIII fragment of pYES2.0 which contains the GAL1 promoter (61) was subcloned into pBluescript KS+, resulting in pGAL1. The GAL1 promoter was excised with XbaI/PvuII and subcloned into XbaI/HincII-restricted pBluescript KS+. The resulting plasmid was designated pGAL2. Subcloning of the XhoI/SacI fragment of pGAL2 into pYES2.0 resulted in pGAL3. The KpnI/XhoI fragment of pGAL3 comprised the GAL1 promoter, and it was subcloned in front of the CYC1 terminator of pRS316t resulting in pGAL4.

Media and Growth of Bacteria and Saccharomyces

E. coli was grown in Luria-Bertani broth (LB, Duchefa, Haarlem, The Netherlands). Ampicillin (100 mg/liter) and 1.5% agar (Difco) was included for solid LB media. Yeast synthetic dextrose medium (SD) was prepared according to Ausubel et al. (62).

Cloning of Sterol Glucosyltransferases from S. cerevisiae, C. albicans, P. pastoris, and D. discoideum

S. cerevisiae-- UGT51 (YLR189C, GenBankTM accession number U17246) is a gene located on chromosome XII of S. cerevisiae. We isolated a genomic 6359-bp NdeI/SpeI DNA fragment containing UGT51 by restriction digestion of cosmid 9470 DNA that we received from the American Type Culture Collection. This fragment was cloned into pBluescript II KS giving the plasmid pUGT51g. This plasmid was used to clone the open reading frame (ORF) UGT51 into expression vectors for E. coli (pET19b right-arrow pUGT51x) and S. cerevisiae (pGAL4 right-arrow pUGT51xy). In addition, PCR fragments of UGT51 corresponding to polypeptides with N-terminal or C-terminal deletions of amino acids were cloned into the following plasmids: pN690x, pN722x, pN799x, pC1043x, and pN722xy.

C. albicans-- PCR with genomic DNA of C. albicans was performed with the primer DW3 5'-GSI WCI VGI GGI GAY GTH CAR CC-3' and WA3 5'-GTI GTI CCI SHI CCI SCR TGR TG-3'. The resulting PCR fragment was used to synthesize a digoxigenin-labeled probe for the screening of a fosmid library of genomic DNA of C. albicans strain 1161.3 Three positive fosmid clones were isolated: 2H6, 3E6, and 8C12. An 8.2-kb HindIII fragment of 2H6 was identified by agarose gel separation of the digestion fragments and hybridization with the probe. This fragment contained the ORF UGT51C1 and was ligated with pUC18/HindIII (right-arrow pUGT51C1g). A PCR fragment corresponding to the ORF UGT51C1 was cloned into an expression vector for E. coli (pBAD-TOPO right-arrow pUGT51C1x).

P. pastoris-- PCR with genomic DNA of P. pastoris was performed with the primer DW5 5'-TTY ACI ATG CCI TGG ACI MSI AC-3' and DW30 5'-YKI GRI SHI GCI SCI GTI GTN CC-3'. The resulting PCR fragment was used to synthesize a digoxigenin-labeled probe for the screening of a genomic DNA library of P. pastoris strain GS115 (63, 64). An SphI/EcoRV fragment of a positive clone was isolated according to the method described above for Candida. This fragment contained the ORF UGT51B1 and was ligated with pUC19/SphI/SmaI (right-arrow pUGT51B1g). A PCR fragment corresponding to the ORF UGT51B1 was cloned into expression vectors for E. coli (pBAD-TOPO right-arrow pUGT51B1x) and S. cerevisiae (pYES2 right-arrow pUGT51B1xy).

D. discoideum-- We used the cDNA clone FC-AZ07 (GenBankTM Accession numbers C25752 and C25753 from H. Urushihara, University of Tsukuba)4 from the cDNA library of D. discoideum strain CAX3 for the PCR synthesis of a digoxigenin-labeled probe. With this probe, we screened a lambda -ZAP cDNA library of D. discoideum strain AX4 (from W. F. Loomis, University of California). In vivo excision of a positive clone resulted in the plasmid pUGT52c that had a 3331-bp cDNA insert within the vector pBluescript I SK. A PCR fragment corresponding to the ORF ugt52 was cloned into an expression vector for E. coli (pBAD-TOPO right-arrow pUGT52x). All plasmids used in this study are listed in Table I.

Disruption of the Genomic S. cerevisiae UGT51 Gene

To construct a ugt51 null mutant, the entire UGT51 open reading frame was replaced by the kanMX4 marker gene (65). The PCR-derived construct for disruption comprised the kanMX4 gene flanked by short (45 bp) homology regions to the UGT51 3'- and 5'-noncoding region. Plasmid pFA6a-kanMX4 (65) served as template, and PCR primers were as follows: 5'-TTGCACTTTATGCTTTGGTGAAAATCCGTATAACTTAAAAGAATGCGTACGCTGCAGGTCGAC-3' (S1/ham1) and 5'-TTACGCTTTTTTATAAAAGTGAGAGTGATACTCGGTTTAAATCATATCGATGAATTCGAGCTCG-3' (S2/ham1). The resulting amplification construct was introduced into S. cerevisiae wild-type UTL-7A. Geneticin-resistant clones were selected by growth on YPD plates containing 200 mg/liter G418 (65). The correct replacement of UGT51 by the kanMX4 gene was confirmed by PCR.

Expression of Sterol Glucosyltransferases

E. coli-- Cells of BL21(DE3) and TOP 10 were transformed with plasmids derived from pET19b and pBAD TOPO, respectively. Cells were grown at 30 °C in 25 ml of LB medium containing 100 mg/liter ampicillin to an absorbance between 0.5 and 0.8 at 600 nm. Induction was performed by adding 0.4 mM isopropyl-1-thio-beta -D-galactopyranoside (for BL21(DE3) with pET19b plasmids) or 0.1% arabinose (for TOP10 with pBAD TOPO plasmids) and further incubation for 2-4 h at 30 °C. Cells were harvested by centrifugation (10 min, 2000 × g, 20 °C), resuspended in 1-2 ml of buffer (100 mM Tris/HCl, pH 7.5, 15% glycerol, 5 mM 1,4-dithiothreitol, 200 µM Pefabloc (Serva), 0.5 mg/ml lysozyme), and incubated for 5 min at 20 °C. Cells were cooled in an ice bath, and cell disruption was performed by ultrasonication (probe tip, 6 × 10 s). If necessary, a crude membrane fraction was prepared by centrifugation of the cell homogenate at 4 °C for 20 min at 25,000 × g and resuspension of the sediment with buffer. This procedure was repeated once.

S. cerevisiae-- Yeast cells were grown at 30 °C in 100 ml of the defined medium described above to an absorbance (A600) between 2 and 6. The medium was supplemented with 2% raffinose and 1% galactose (instead of glucose) when the vectors pGAL4 or pYES2 were used. Cells were harvested by centrifugation (10 min, 2000 × g, 20 °C), resuspended in 2 ml of buffer (100 mM Tris/HCl, pH 7.5, 15% glycerol, 5 mM 1,4-dithiothreitol, 200 µM Pefabloc (Serva), 0.5 mg/ml lyticase from Sigma), and incubated for 30 min at 25 °C. 2-3 g of glass beads (inner diameter, 0.4 mm) were added, and cells were disrupted by vortexing for 16 × 30 s.

Sterol Glucosyltransferase Assay

The assay mixture contained in a total volume of 100 µl the following: 80 µl of E. coli or S. cerevisiae cell homogenate or membrane fraction, respectively, either 10 µl of a solution of 4 mM cholesterol in ethanol (400 µM final concentration) or 8 µl of 500,000 dpm [4-14C]cholesterol in ethanol (final concentration 45 µM, specific activity 1.85 GBq/mmol), and either 100,000 dpm UDP-[U-14C]glucose (final concentration 1.5 µM, specific activity 10.8 GBq/mmol) or 360 µM unlabeled UDP-glucose. After incubating for 2 h at 30 °C, the reaction was terminated by the addition of 0.9 ml of 0.45% NaCl solution and 4 ml of chloroform/methanol, 2:1. The extracted lipids were separated by thin layer chromatography with chloroform/methanol (85:15, v/v). The radioactivity on the silica gel plate was detected by radioscanning with a BAS-1000 BioImaging Analyzer (Raytest, Straubenhardt, Germany).

Lipid Extraction and Analysis

S. cerevisiae cells were grown at 30 °C on defined, lipid-free media described above. Harvesting of the cells by centrifugation was followed by the extraction of lipids with chloroform/methanol, 1:2, and chloroform/methanol, 2:1. The extracted lipids were separated by thin layer chromatography on silica gel 60 (Merck, Darmstadt, Germany) with chloroform/methanol, 85:15. Glycolipids were visualized with a spray of alpha -naphthol/sulfuric acid mixture. For nuclear magnetic resonance spectroscopy and mass spectrometry, the novel glycolipid was purified by column chromatography on silica gel by elution with acetone. The lipid was acetylated with acetic anhydride in pyridine and subjected to preparative thin layer chromatography in diethyl ether.

Mass Spectrometry

Mass spectrometric analysis of the peracetylated sterol glycoside was carried out with a Hewlett-Packard model 5989 spectrometer using the direct insert probe mode. Temperature was raised from 80 to 325 °C at a rate of 30 °C/min. EI-mass spectra were recorded at 70 eV with an ion source temperature of 200 °C.

Nuclear Magnetic Resonance (NMR) Spectroscopy

NMR spectra of the peracetylated glycoside (200 µg) were recorded at 300 K in CDCl3 (99.96%, Cambridge Isotope Laboratories, Andover, MA) for 1H NMR at 600 MHz (Avance DRX-600 spectrometer) and for 13C NMR at 90.6 MHz (DPX 360) (Bruker, Germany). Signals were referenced to internal tetramethylsilane (delta H = delta C = 0.000). One- and two-dimensional homonuclear (1H, 1H COSY, relayed COSY) and 1H-detected heteronuclear 1H, 13C multiple quantum coherence (HMQC) experiments were performed using standard Bruker software (XWINNMR, version 1.3).

The nucleotide sequences of the sterol glucosyltransferases have been deposited in the GenBankTM data base under accession numbers AF091398 (C. albicans), AF098916 (D. discoideum), and AF091397 (P. pastoris).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Isolation and Cloning of Putative Sterol Glycosyltransferases-- A BLAST (66) data base search with the amino acid sequences of plant sterol glucosyltransferases revealed similarities to Ugt51 (Ylr189c, PIR: locus S51434) which was described as a hypothetical protein from S. cerevisiae with unknown function. The first aim of the present work was to elucidate the function of this protein. UGT51 contains an open reading frame (ORF) of 3594 bp encoding a polypeptide of 1198 amino acids with a calculated molecular mass of 136 kDa. We isolated a 6359-bp NdeI/SpeI fragment of the cosmid 9470 (accession number U17246) from the chromosome XII of S. cerevisiae that contained UGT51. This fragment was cloned into pBluescript (right-arrow pUGT51c) and used to subclone the ORF UGT51 into expression vectors for E. coli and S. cerevisiae.

Furthermore, we wanted to isolate and clone homologous genes from other organisms. The identification of novel sterol glycosyltransferase genes will provide the basis for genetic manipulation of these genes and contribute to the elucidation of sterol glycoside functions that may differ in these organisms. We cloned the homologous genes by a PCR-based strategy with degenerated oligonucleotide primers that were derived from conserved regions of Ugt51 and the plant sterol glucosyltransferases. PCR products obtained from C. albicans and P. pastoris were sequenced, and the deduced amino acid sequences of these fragments showed similarities with Ugt51 and the plant sterol glucosyltransferases. These PCR products were labeled and used to isolate the complete genes from genomic libraries of these organisms. An 8.2-kb fragment of C. albicans that contained the incomplete MDL1 gene and an unknown ORF was cloned into pUC18 (right-arrow pUGT51C1c). Both strands of the unknown ORF were sequenced, and these data were published in nucleotide data bases under the accession number AF091398. The ORF of 4548 bp, called UGT51C1, encoded a polypeptide of 1516 amino acids with a calculated molecular mass of 171 kDa.

From P. pastoris we cloned and sequenced a genomic fragment of 6777 bp (accession no. AF091397). It contained an ORF (UGT51B1) of 3633 bp encoding a polypeptide of 1211 amino acids with a calculated molecular mass of 136 kDa.

A BLAST search (66) with Ugt51 revealed sequence similarity with a cDNA clone from D. discoideum CAX3 (clone FC-AZ07, accession numbers C25752 and C25753). This clone was used to label a PCR fragment that was suitable for the isolation of longer fragments from a D. discoideum AX4 cDNA library. The longest clone (pUGT52c) had an insert of 3331 bp that was sequenced on both strands (accession number AF098916). It contained an ORF from bp 244-3312 (ugt52) that encoded a polypeptide of 1023 amino acids with a calculated molecular mass of 114 kDa.

Fig. 1 shows a comparison of the deduced amino acid sequences of the plant sterol glucosyltransferases with those of Ugt51p and the gene products of the homologous genes from Pichia, Candida, and Dictyostelium. The polypeptides showed only a few similarities in their N-terminal parts, but within a C-terminal region of about 360 amino acids (residues 1030-1425) similarities were significant as follows: 69% identity between the three yeasts, 36-39% between the yeasts and Dictyostelium, 34-37% identity between the yeasts and the plants, 86% between the plants, and 33% between the plants and Dictyostelium. Near their C-terminal ends all sequences showed a 29-amino acid region of striking homology (box 4 in Fig. 1). This region was very similar to a "signature sequence" that is characteristic for a superfamily of nucleoside diphospho-sugar glycosyltransferases and that was suggested to represent a UDP-sugar binding domain (54). For this reason, all open reading frames mentioned in this work were classified as UGTs (UDP-glycosyltransferases) in agreement with the UGT Nomenclature Committee (54) and the Saccharomyces Genome Data base.5 Most of the members of this superfamily, but not all, use UDP-sugars as substrates. The conserved boxes 1 and 3 of the sequences shown in Fig. 1 are also present in some prokaryotic members of the UGT superfamily, e.g. a rhamnosyltransferase from Pseudomonas aeruginosa (GenBankTM accession number L28170), but are absent in the eukaryotic members of the superfamily. The region named psbd in Fig. 1 corresponds to a putative steroid-binding domain found in a solanidine glucosyltransferase from potato (see Ref. 67, GenBankTM accession number U82367) and mammalian steroid UDP-glucuronosyltransferases (e.g. UGT2B7, GenBankTM accession number J05428). The UGTs cloned in the present work showed only a few similarities with this putative steroid-binding domain. These data suggest that the novel UGTs cloned in the present work were more related to bacterial glycosyltransferases than to other eukaryotic glycosyltransferases, although some of them use structurally similar substrates. The conserved box 2 in Fig. 1 is not present in other members of the UGT superfamily and seemed to be characteristic for sterol glucosyltransferases.



View larger version (149K):
[in this window]
[in a new window]
 
Fig. 1.   Sequence alignment of sterol glucosyltransferases from Arabidopsis thaliana (A.t.), A. sativa (A.s.), C. albicans (C.a.), S. cerevisiae (S.c.), P. pastoris (P.p.), and D. discoideum (D.d.). Black boxes indicate identical amino acids in all six sequences. Gray boxes indicate identical amino acids in four or five sequences. The polypeptides of the three yeasts and D. discoideum contain long N-terminal extensions that do not exist in the plant polypeptides (residues 1-820). Within this N-terminal extension there are only a few sequence similarities between the four species. In contrast to this N-terminal region, all sequences show significant similarity in their C-terminal parts (residues 1030-1425). Within this region four highly conserved boxes are underlined and numbered. The conserved box 4 corresponds to the signature sequence of a protein superfamily of UDP-glycosyltransferases (54). The region named psbd corresponds to a putative steroid-binding domain found in solanidine glucosyltransferase (67) and steroid UDP-glucuronosyltransferases. Clusters of asparagine, glutamine, or threonine in the sequence from D. discoideum are indicated by dotted lines.

Functional Expression of the UGTs in E. coli and in Vitro Determination of Sterol Glycosyltransferase Activity-- The ORFs of the DNA fragments cloned in this work were expressed in E. coli to examine the function of the expression products. This was done by in vitro determination of sterol glycosyltransferase activity in cell-free extracts using radiolabeled substrates. E. coli is suitable for such experiments since untransformed cells do not contain a sterol glycosyltransferase activity. The ORF UGT51 from S. cerevisiae was cloned into the expression vector pET19b (pUGT51x). The other UGTs from C. albicans, P. pastoris, and D. discoideum were amplified by PCR and subcloned into the expression vector pBAD-TOPO (pUGT51C1x, pUGT51B1x, and pUGT52x). Transformed and induced E. coli cells were disrupted, and the homogenates or crude membrane fractions were used for in vitro enzyme assays. Membrane fractions had the advantage that most of the intracellular UDP-glucose was eliminated. It turned out that more than 50% of the expressed protein was not in the soluble fraction but was found in the sediment of membranes and inclusion bodies (data not shown).

To measure the sterol glycosyltransferase activity, we performed in vitro assays with various radiolabeled sugar donors (NDP-sugars) and acceptors (sterols). After thin layer chromatography (TLC), the radioactivity in the lipophilic reaction products was determined. Table II shows a comparison of the substrate specificities of the recombinant enzymes from S. cerevisiae, C. albicans, P. pastoris, D. discoideum, and Avena sativa. These data should not be considered as a quantitative determination of substrate affinities, since the assays were not performed under linearized conditions. These results rather suggest which substrates were accepted or discriminated against by each of the heterologously expressed proteins. With UDP-[U-14C]glucose as the donor, all expressed UGT proteins used various sterols as sugar acceptors, whereas there was no background SG synthesis of a control E. coli homogenate. The UGT proteins glucosylated sterols with a planar backbone such as cholesterol, ergosterol, beta -sitosterol, stigmasterol, and even the steroidal alkaloid tomatidine. However, other lipids, e.g. ceramide and dioleoyl glycerol, were not accepted. Regarding the sugar moiety of the donor, all enzymes exhibited a distinct specificity for glucose (UDP-glucose), whereas UDP-galactose, UDP-glucuronic acid, UDP-mannose, and GDP-mannose were not accepted. This was of particular relevance in the case of the Ugt51C1 from C. albicans in view of the identification of sterol mannoside in this organism (see "Discussion"). UDP-xylose, as a pyranose, mimics UDP-glucose except for the lacking CH2OH moiety. It was incorporated into cholesterol xyloside by the Avena, Saccharomyces, and Pichia enzymes at a low rate. UDP served as the best nucleotide moiety of the glucose donors, but CDP-glucose and GDP-glucose were also used, although at significantly lower rates. TDP-glucose was not accepted. These in vitro data gave strong evidence that all UGT genes cloned in this work encode specific sterol glucosyltransferases using UDP-glucose as sugar donor and different planar sterols as sugar acceptors.

                              
View this table:
[in this window]
[in a new window]
 
Table II
Substrate specificities of different recombinant sterol glucosyltransferases expressed in E. coli
Reaction mixtures contained in a total volume of 100 µl: 80 µl of E. coli cell-free homogenate or membrane preparation, NDP-U-14C-sugar or unlabeled NDP-sugar and unlabeled acceptor or [4-14C]cholesterol. E. coli expressed the sterol glucosyltransferases from A. sativa (A.s.), C. albicans (C.a.), D. discoideum (D.d.), P. pastoris (P.p.) or S. cerevisiae (S.c.). Incorporation with UDP-glucose and cholesterol was taken as 100%. Results are averages of two determinations. ND, not determined.

Only a C-terminal Conserved Region of UGT51 Was Essential for Enzyme Activity-- An alignment of the different sterol glucosyltransferases (Fig. 1 and Fig. 2) showed that the sequences shared significant similarities only at their C-terminal parts and that the polypeptides from the three yeasts and from Dictyostelium had large N-terminal extensions that did not exist in the plant polypeptides. This phenomenon raised the question which parts of the polypeptides were necessary for their function. We selected one of the proteins, Ugt51p from S. cerevisiae, to investigate this phenomenon. Therefore, various fragments of Ugt51p, obtained by deleting different N- or C-terminal sequences, were expressed in E. coli (Fig. 2). The cell-free homogenates of the transgenic E. coli were used for in vitro enzyme assays performed with cholesterol and UDP-glucose. Ugt51p fragments with N-terminal deletions of either 690 or 722 amino acids synthesized SG (Fig. 3). These fragments contained the conserved region indicated in Fig. 2. In contrast, elimination of parts of the conserved region by deletion of either 799 N-terminal or 155 C-terminal amino acids resulted in complete loss of enzyme activity. These data indicate that at least 722 N-terminal amino acids of Ugt51p are not required for in vitro enzyme function. Ugt51p fragments containing the complete conserved region of the sterol glucosyltransferases are sufficient for in vitro SG synthesis.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 2.   Schematic alignment of amino acid sequences of the sterol glucosyltransferases Ugt80A1p from A. sativa and Ugt51p from S. cerevisiae. The gray boxes indicate a region of significant amino acid similarity between these glucosyltransferases (region 1030-1425 in Fig. 1) The black boxes indicate the signature sequence of UGTs (54) that contains highly conserved amino acids. In the lower part of the figure four fragments of Ugt51p are shown resulting from the deletion of sequences with a length of either 690, 722, and 799 N-terminal amino acids (N690, N722, and N799) or 1043 C-terminal amino acids (C1043). These fragments were expressed in E. coli to determine which parts of the polypeptide were essential for enzyme function. A oplus  indicates in vitro sterol glucosyltransferase activity and a odash  indicates a lack of activity. The detailed results of these experiments are given in Fig. 3.


View larger version (41K):
[in this window]
[in a new window]
 
Fig. 3.   In vitro activity of recombinant S. cerevisiae sterol glucosyltransferase (Ugt51p) expressed in E. coli as full-length or as N- or C-terminally shortened fragments. Cell-free homogenates from transgenic E. coli were used for in vitro enzyme assays. The assay mixtures contained UDP-glucose and cholesterol with either radiolabeled glucose (A) or radiolabeled cholesterol (B). The labeled lipophilic reaction products were separated by thin layer chromatography with subsequent analysis by a BAS 1000 BioImaging Analyzer. SG was identified by co-chromatography with an authentic standard. The transgenic E. coli harbored the control plasmid pET19b (lanes 1, 1'), pUGT51x (encoding the complete polypeptide Ugt51p, lanes 2, 2'), pN690x (encoding Ugt51p that lacked 690 N-terminal amino acids, lanes 3, 3'), pN722x (encoding Ugt51p that lacked 722 N-terminal amino acids, lanes 4, 4'), pN799x (encoding Ugt51p that lacked 799 N-terminal amino acids, lanes 5, 5'), and pC1043x (encoding Ugt51p that lacked 155 C-terminal amino acids, lanes 6, 6').

Deletion and Overexpression of UGT51 in S. cerevisiae-- To address the question whether the in vitro synthesis of SG reflects the in vivo function of Ugt51p, we deleted UGT51 in S. cerevisiae strain UTL7A. The null mutant was still viable and grew like the parental strain on complex and minimal media, at low and elevated temperatures, under different conditions of osmotic stress and in the presence of nystatin. Fig. 4 shows that cell-free homogenates of the parental strain UTL7A synthesized low amounts of SG in vitro (1700 dpm of radiolabeled product), whereas the enzyme activity was completely lost in the null mutant. The labeled compound near the front probably was sterol ester. Then we transformed the null mutant with a plasmid that led to the expression of complete UGT51 under the control of a galactose-induced promoter. The in vitro SG synthesis was restored in these cells and was more than 10-fold higher (22,000 dpm of labeled product) than with UTL7A cells (Fig. 4). These data prove that the expression of UGT51 in S. cerevisiae resulted in the biosynthesis of an enzymatically active sterol glucosyltransferase. The expression of the N722 fragment in the null mutant also resulted in a detectable enzyme activity (1500 dpm of labeled product). However, the activity resulting from the expression of the truncated enzyme was much lower than that observed for the expression of the wild-type enzyme.


View larger version (38K):
[in this window]
[in a new window]
 
Fig. 4.   In vitro determination of the sterol glucosyltransferase Ugt51p from S. cerevisiae. Cell-free homogenates of wild-type and transgenic S. cerevisiae cells were used for in vitro enzyme assays. The assays contained UDP-glucose and radiolabeled cholesterol. The labeled lipophilic reaction products were separated by thin layer chromatography with subsequent analysis by a BAS 1000 BioImaging Analyzer. SG was identified by co-chromatography with an authentic standard. Lane 0, radiolabeled cholesterol (the spot with slightly lower Rf value represents a degradation/oxidation product of cholesterol, which is only absent in freshly purified cholesterol); lane 1, S. cerevisiae strain UTL7A; lane 2, UTL7A Delta ugt51 (sterol glucosyltransferase deletion mutant); lane 3, UTL7A Delta ugt51 harboring the control plasmid pGAL4; lane 4, UTL7A Delta ugt51 harboring pN722xy (encoding Ugt51p that lacked 722 N-terminal amino acids); lane 5, UTL7A Delta ugt51 harboring pUGT51xy (encoding the complete polypeptide Ugt51p).

Isolation of a Novel Glycolipid from S. cerevisiae and Its Identification as Ergosterol-beta -D-Glucopyranoside-- The lipids of S. cerevisiae were extracted and separated by thin layer chromatography to address the question whether SG was accumulated in vivo (Fig. 5A). The total lipid extracts of the S. cerevisiae wild-type cells and the null mutant did not contain detectable amounts of SG. Also, expression of the N722 fragment in null mutant cells did not result in the biosynthesis of significant amounts of SG, whereas cells expressing the complete UGT51 contained a new glycolipid that co-chromatographed with an authentic SG standard.


View larger version (82K):
[in this window]
[in a new window]
 
Fig. 5.   Search for in vivo accumulation of SG in wild-type and transgenic strains of S. cerevisiae. Lipid extracts were separated by thin layer chromatography. A, lane 1, S. cerevisiae strain UTL7A; lane 2, UTL7A Delta ugt51 (sterol glucosyltransferase deletion mutant); lane 3, UTL7A Delta ugt51 harboring the control plasmid pGAL4; lane 4, UTL7A Delta ugt51 harboring pN722xy (encoding Ugt51p that lacked 722 N-terminal amino acids); lane 5, UTL7A Delta ugt51 harboring pUGT51xy (encoding the complete polypeptide Ugt51p). Authentic SG was chromatographed as control (at right). B, expression of the P. pastoris Ugt51B1p in S. cerevisiae. Lane 1, UTL7A Delta ugt51 harboring pUGT51B1xy (encoding the complete polypeptide Ugt51B1p from P. pastoris). A lipid extract of this strain was used to isolate SG for subsequent analysis by spectroscopy (see Figs. 6 and 7).

We found that a heterologous expression of UGT51B1 from P. pastoris in the S. cerevisiae knock-out mutant led to the accumulation of larger amounts of the novel glycolipid (Fig. 5B) as compared with the homologous expression of UGT51. We made use of this large accumulation of the glycolipid for its purification from lipid extracts of S. cerevisiae expressing UGT51B1 from P. pastoris. This was done by column chromatography on silica gel, acetylation of the isolated glycolipid, and subsequent thin layer chromatography. The peracetylated glycolipid was subjected to structural analysis by mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy.

Electron impact (EI) mass spectrometry analysis of the peracetylated glycolipid was performed using the direct insert probe mode. Two characteristic fragments were obtained, one (m/z = 331) from a terminal single tetra-O-acetylated hexosyl residue and the other (m/z = 378) from the steroid (Fig. 6). The molecular ion [M - H]- (m/z = 726) was found to be in agreement with the calculated mass for tetra-O-acetylhexosyl-ergosterol (C42H62O10; Mr = 726.94).


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 6.   Structure and electron-impact (EI) mass spectrum of per-O-acetylated beta -glucosyl-ergosterol isolated from transgenic S. cerevisiae expressing the sterol glucosyltransferase from P. pastoris.

The structure of the purified tetra-O-acetylhexosyl-ergosterol was further investigated by 1H NMR spectroscopy at 600 MHz. The 1H NMR spectrum revealed signals for four O-acetyl groups (delta  2.011, 1.984, 1.954, and 1.937), indicating one terminal hexose and no additional OAc group derived from a putative OH group of the steroid. The anomeric signal of the hexose (H-1, delta  4.551) expressed a coupling constant J1, 2 of 8 Hz (Table III), thus showing beta -configuration. All other hexose ring protons showed high coupling constant values (J2, 3 9.5, J3, 4 9.6, and J4, 5 9.8) characteristic for the gluco configuration in a pyranoside that was further supported by the chemical shift data (Table III). These data are in full agreement with a terminal tetra-O-acetyl beta -glucopyranose (68).

                              
View this table:
[in this window]
[in a new window]
 
Table III
NMR spectroscopic identification of ergosterol-beta -D-glucoside
600-MHz 1H and 90.6-MHz 13C NMR data of 3beta -O-(2,3,4,6-tetra-O-acetyl-beta -D-glucopyranosyloxy)-ergosta-5,7,22E-trien that was isolated in its nonacetylated form from S. cerevisiae expressing UGT51B1 from P. pastoris (see "Experimental Procedures"; CDCl, 300 K; internal tetramethylsilane, delta Hdelta C = 0.000). Published data for 3-OAc-ergosterol are given in parentheses; 1H (69); 13C (70). Signals for four OAc are as follows: delta H 2.011, 1.984, 1.954, 1.937; delta C 169.3, 169.2, 168.4, 168.3; 19.6 (2×), 19.7, and 19.8 ppm.

Characteristic signals for the steroid were found at delta  5.501 and 5.312 and were diagnostic for the conjugated diene system in ring B (H-6 and H-7) of ergosterol (69). Signals for H-22 and H-23 at the isolated double bond between C-22 and C-23 (delta  5.156 and 5.105, respectively) expressed a high coupling constant value of J22, 23 15.2 Hz, thus indicating E configuration (Fig. 7). Signals for H-3, H-4alpha , and H-4beta were at delta  3.531, 2.367, and 2.173, respectively. All other signals could be assigned and part of the coupling constant values determined based on relay COSY and 1H,13C HMQC experiments (Fig. 7 and Table III).


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 7.   Two-dimensional 1H, 13C HMQC spectrum of 3beta -(2,3,4,6-tetra-O-acetyl-beta -D-glucopyranosyloxy)ergosta-5,7,22E-trien which was isolated in its nonacetylated form from S. cerevisiae expressing UGT51B1 from P. pastoris. The spectrum was measured in a solution of CDCl3 at 300 K. Assignments of protons to the glycosyl and ergosterol part (in italics) are indicated. The corresponding 1H NMR spectrum (600 MHz) is displayed along the horizontal (F2) axis and the 13C NMR spectrum (90.6 MHz) along the vertical (F1) axis.

In the HMQC spectrum (Fig. 7) the positions of the signals for sugar carbons, including the anomeric carbon (C-1, delta  98.6), were in agreement with a 2,3,4,6-tetra-O-acetyl-beta -D-glucopyranoside (Table III). The steroid moiety expressed diagnostic resonances for the isolated double bond (C-23, delta  134.4; C-22, delta  131.1) and for the two conjugated double bonds (C-6, delta  119.3; C-7, delta  115.2), further confirming ergosterol to be the aglycon in the glycolipid. Other carbon resonances were assigned as well and found to be in agreement with published data (70).

Based on the EI-mass spectrometry and 1H and 13C NMR spectroscopy data, the per-O-acetylated product of the UDP-glucose:sterol glucosyltransferase Ugt51B1 from P. pastoris, as expressed in transgenic S. cerevisiae, could be unambiguously identified as 3beta -(2, 3, 4, 6-tetra-O-acetyl-beta -D-glucopyranosyloxy)ergosta-5,7,22E-trien (Fig. 6) thus demonstrating the enzyme to be a beta -glucosyltransferase with ergosterol as acceptor.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

This work reports the cloning, identification, and characterization of novel UDP-glucose:sterol glucosyltransferases from S. cerevisiae, C. albicans, P. pastoris, and D. discoideum. We also isolated a sterol glucoside from transgenic S. cerevisiae expressing UGT51B1 from P. pastoris. This is the first report on the structural analysis of a glycosylated sterol from S. cerevisiae which we identified as ergosterol-beta -D-glucoside. These data prove that UGT51B1 encodes a UDP-glucose:sterol beta -D-glucosyltransferase. Combined with in vitro data on the substrate specificities of the identified glucosyltransferases from different organisms, we assume that all the recombinant enzymes that we characterized in this work are sterol beta -D-glucosyltransferases. The enzymes show a narrow specificity for the sugar donor using predominantly UDP-glucose. In contrast, the in vitro specificity for the sterol acceptor is broad, pointing to the biosynthesis of glucosyl sterols with different sterol moieties depending on the individual sterol composition of each organism.

The isolation of novel sterol glucosyltransferases represents one step in our approach to elucidate the functions of sterol glucosides in these organisms. Amino acid sequence comparisons showed that these genes represent a gene family with members in plants, fungi, and other eukaryotic organisms (Fig. 1). This gene family is part of a superfamily of UDP-glycosyltransferases (UGTs) (54), which is characterized by a C-terminal signature amino acid sequence (Fig. 1).

Glucose is the main sugar moiety in the sterol glycosides of many organisms, but other sugars have also been found (1). This raises the question whether there are physiological differences between sterol glycosides having different hydrophilic head groups. Since C. albicans was reported to contain mainly cholesterol mannoside (6, 7), it was our intention to isolate the gene encoding the corresponding mannosyltransferase. Our data on the substrate specificity of Ugt51C1p from C. albicans showed that it is not a mannosyltransferase as expected but a glucosyltransferase. The discrepancy between the glucose preference of Ugt51C1p and the presence of mainly sterol mannoside in C. albicans points to the possibility that this organism may contain two sterol glycosyltransferases with different sugar specificities. The ongoing C. albicans genome sequencing project should answer the question whether there is a gene homologous to UGT51C1 which may encode a mannosyltransferase.

The expression of different fragments of UGT51 in E. coli and S. cerevisiae showed that at least 722 N-terminal amino acids of the polypeptide are not required for the in vitro glucosyltransferase activity of the protein (Fig. 3 and Fig. 4). No significant difference in enzyme activity was observed between the wild-type and the truncated protein upon expression in E. coli. Interestingly, upon expression in S. cerevisiae, a significantly lower sterol glycosyltransferase activity was observed for the truncated form (Fig. 4). In line with this observation, expression in S. cerevisiae of the wild-type form but not of the truncated form led to the biosynthesis of detectable amounts of SG (Fig. 5). These observations might suggest that the N-terminal region contains yeast-specific regulatory elements for the activity, expression, or stability of the protein. However, it remains to be investigated what function this large N-terminal extension of about 700 amino acids may have. There are only low sequence similarities between these N-terminal extensions of the three yeasts and D. discoideum and they do not show significant similarity to any sequence from the data bases. They do not contain known membrane targeting motifs nor membrane spanning domains. Interestingly, this N-terminal extension is not present in the plant enzymes (Fig. 1).

By deletion of UGT51 in S. cerevisiae we showed for the first time that SG is non-essential for growth of S. cerevisiae. In addition, no differences in growth were observed under various stress conditions. This is consistent with three genome-wide expression studies of S. cerevisiae which showed no alterations in UGT51 expression during the sporulation process (71), shift from anaerobic to aerobic conditions (72), and when grown in rich or minimal media (73). The expression level of UGT51 seems to be very low, and we do not know whether a low amount of SG is sufficient to fulfill its physiological function or whether larger amounts need to be biosynthesized, e.g. under special growing conditions, that are sufficient for altering membrane properties. Lipid analyses of other yeasts and fungi showed that some of them contain significantly larger amounts of sterol glycosides than Saccharomyces and that an increase in its synthesis was induced under stress conditions.6

In this context, it should be mentioned that insertional mutagenesis experiments with the rice blast fungus Magnaporthe grisea and a subsequent screening procedure revealed mutants with reduced pathogenicity (74). One of these mutants identifies the gene PTH8 (GenBankTM accession number AF027983), the partial sequence of which shows striking similarity to the sterol glucosyltransferases from yeasts. These data suggest that SG may be involved in the pathogenicity of phytopathogenic fungi.

Another interesting observation concerns some regions of the cDNA of the D. discoideum sterol glucosyltransferase which are rich in AAC codons pointing to clusters of asparagine, glutamine, or threonine (see Fig. 1). Such clusters were reported to be characteristic for developmentally regulated genes (75-77). A hypothetical change of SG synthesis during development would be consistent with the observations that SG synthesis alters during development of D. discoideum (13) and that SG accumulation was induced by the process of differentiation from amoeboid stage to plasmodium in P. polycephalum (78, 51).

In summary, we have shown that SG synthesis is not restricted to plants but that it is also a common feature of yeasts, fungi, and other eukaryotes. We have identified and characterized four novel sterol glucosyltransferases from yeasts and D. discoideum. Our work provides the basis for new approaches to elucidate the functions of SG in these organisms.

    ACKNOWLEDGEMENTS

We thank J. M. Cregg for providing us with a DNA library from P. pastoris; D. Fuller and W. F. Loomis for a Dictyostelium cDNA library; H. Urushihara for the Dictyostelium cDNA clone FC-AZ07; M. Strathman and S. Scherer for the C. albicans fosmid library; R. Swoboda for replicating a copy of it; D. Hess and M. Bossenz for preparing dot-blots of this library; and P. Mackenzie for help regarding gene nomenclature.

    FOOTNOTES

* This work was supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 470, and Fonds der Chemischen Industrie.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF091398 (C. albicans), AF098916 (D. discoideum), and AF091397 (P. pastoris).

§ To whom correspondence should be addressed: University of Hamburg, Institut fuer Allgemeine Botanik, Ohnhorststrasse 18, 22609 Hamburg, Germany. Tel.: 49 40 42816 364; Fax: 49 40 42816 254; E-mail: warnecke{at}botanik.uni-hamburg.de.

2 We use lowercase italics for the gene symbols of plants and Dictyostelium due to the gene nomenclature rules which differ from that practiced with S. cerevisiae. It must be emphasized that in contrast to yeasts, lowercase italics do not indicate recessive alleles but mean the same as uppercase italics for S. cerevisiae.

3 Information available from M. Strathmann and S. Scherer at the following on-line address: http://alces.med.umn.edu.candida/fosmidinfo/html.

4 Information available on-line at the following address: http: //www.csm.biol.tsukuba.ac.jp/Sexual-cDNA/SexualcDNA/html.

5 Saccharomyces Genome Data base available at the following on-line address: http://genome-www.stanford.edu/.

6 T. Sakaki, D. Warnecke, U. Zaehringer, and E. Heinz, unpublished data.

    ABBREVIATIONS

The abbreviations used are: SG, sterol glucoside(s); ORF, open reading frame; PCR, polymerase chain reaction; UGT, UDP-glycosyltransferases; kb, kilobase pair(s); bp, base pair(s); EI, electron impact.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
  1. Wojciechowski, Z. A. (1991) in Physiology and Biochemistry of Sterols (Patterson, G. W., and Nes, W. D., eds), American Oil Chemists Society, Champaign, IL
  2. Ullmann, P., Ury, A., Rimmele, D., Benveniste, P., and Bouvier-Navé, P. (1993) Biochimie (Paris) 75, 713-723[Medline] [Order article via Infotrieve]
  3. Veron, B., Billard, C., Dauguet, J.-C., and Hartmann, M.-A. (1996) Lipids 31, 989-994[Medline] [Order article via Infotrieve]
  4. Kastelic-Suhadolc, T. (1980) Biochim. Biophys. Acta 620, 322-325[Medline] [Order article via Infotrieve]
  5. McMorris, T. C., and White, R. H. (1977) Biochim. Biophys. Acta 486, 308-312[Medline] [Order article via Infotrieve]
  6. Ghannoum, M. A., Janini, G., Khamis, L., and Radwan, S. S. (1986) J. Gen. Microbiol. 132, 2367-2375[Medline] [Order article via Infotrieve]
  7. Ghannoum, M. A., Swairo, I., and Soll, D. R. (1990) J. Med. Vet. Mycol. 28, 103-115[Medline] [Order article via Infotrieve]
  8. Työrinoja, K., Nurminen, T., and Suomalainen, H. (1974) Biochem. J. 141, 133-139[Medline] [Order article via Infotrieve]
  9. Baraud, J., Maurice, A., and Napias, C. (1970) Bull. Soc. Chim. Biol. 52, 421-432[Medline] [Order article via Infotrieve]
  10. Murakami-Murofushi, K., Nishikawa, K., Hirakawa, E., and Murofushi, H. (1997) J. Biol. Chem. 272, 486-489[Abstract/Free Full Text]
  11. Wojciechowski, Z. A., Zimowski, J., Zimowski, J. G., and Lyznik, A. (1979) Biochim. Biophys. Acta 570, 363-370[Medline] [Order article via Infotrieve]
  12. Duperon, R., Doireau, P., Verger, A., and Duperon, P. (1980) in Biogenesis and Function of Plant Lipids (Mazliak, P., Benveniste, P., Costes, C., and Douce, R., eds), pp. 445-447, Elsevier/North-Holland, New York
  13. Hase, A. (1981) Arch. Biochem. Biophys. 210, 280-288[Medline] [Order article via Infotrieve]
  14. Haque, M., Hirai, Y., Yokota, K., Mori, N., Jahan, I., Ito, H., Hotta, H., Yano, I., Kanemasa, Y., and Oguma, K. (1996) J. Bacteriol. 178, 2065-2070[Abstract]
  15. Hirai, Y., Haque, M., Yoshida, T., Yokota, K., Yasuda, T., and Oguma, K. (1995) J. Bacteriol. 177, 5327-5333[Abstract]
  16. Livermore, B. P., Bey, R. F., and Johnson, R. C. (1978) Infect. Immun. 20, 215-220[Medline] [Order article via Infotrieve]
  17. Mayberry, W. R., and Smith, P. F. (1983) Biochim. Biophys. Acta 752, 434-443[Medline] [Order article via Infotrieve]
  18. Patel, K. R., Smith, P. F., and Mayberry, W. R. (1978) J. Bacteriol. 136, 829-831[Medline] [Order article via Infotrieve]
  19. Smith, P. F. (1971) J. Bacteriol. 108, 986-991[Medline] [Order article via Infotrieve]
  20. Abraham, W., Wertz, P. W., Burken, R. R., and Downing, D. T. (1987) J. Lipid. Res. 28, 446-449[Abstract]
  21. Wertz, P. W., Stover, P. M., Abraham, W., and Downing, D. T. (1986) J. Lipid. Res. 27, 427-435[Abstract]
  22. Hungund, B. L., Dayal, B., Dayal, V. K., and Salen, G. (1994) Chem. Phys. Lipids 69, 167-173[Medline] [Order article via Infotrieve]
  23. Muhiudeen, I. A., Koerner, T. A., Samuelsson, B., Hirabayashi, Y., DeGasperi, R., Li, S. C., and Li, Y. T. (1984) J. Lipid. Res. 25, 1117-1123[Abstract]
  24. Duncan, E. A., Dave, U. P., Sakai, J., Goldstein, J. L., and Brown, M. S. (1998) J. Biol. Chem. 273, 17801-17809[Abstract/Free Full Text]
  25. Oelkers, P., Behari, A., Cromley, D., Billheimer, J. T., and Sturley, S. L. (1998) J. Biol. Chem. 273, 26765-26771[Abstract/Free Full Text]
  26. Lange, Y., Ye, J., and Steck, T. L. (1998) J. Biol. Chem. 273, 18915-18922[Abstract/Free Full Text]
  27. Schroeder, F., Frolov, A. A., Murphy, E. J., Atshaves, B. P., Jefferson, J. R., Pu, L., Wood, W. G., Foxworth, W. B., and Kier, A. B. (1996) Proc. Soc. Exp. Biol. Med. 213, 150-177[Abstract]
  28. Cases, S., Novak, S., Zheng, Y. W., Myers, H. M., Lear, S. R., Sande, E., Welch, C. B., Lusis, A. J., Spencer, T. A., Krause, B. R., Erickson, S. K., and Farese, R. V., Jr. (1998) J. Biol. Chem. 273, 26755-26764[Abstract/Free Full Text]
  29. Underwood, K. W., Jacobs, N. L., Howley, A., and Liscum, L. (1998) J. Biol. Chem. 273, 4266-4274[Abstract/Free Full Text]
  30. Bach, T. J., and Benveniste, P. (1997) Prog. Lipid Res. 36, 197-226[CrossRef][Medline] [Order article via Infotrieve]
  31. Bouvier-Nave, P., Husselstein, T., Desprez, T., and Benveniste, P. (1997) Eur. J. Biochem. 246, 518-529[Abstract]
  32. Hartmann, M.-A. (1998) Trends Plant Sci. 3, 170-175[CrossRef]
  33. Klahre, U., Noguchi, T., Fujioka, S., Takatsuto, S., Yokata, T., Nomura, T., Yoshida, S., and Chua, N-H. (1998) Plant Cell 10, 1677-1690[Abstract/Free Full Text]
  34. Rahier, A., Smith, M., and Taton, M. (1997) Biochem. Biophys. Res. Commun. 236, 434-437[CrossRef][Medline] [Order article via Infotrieve]
  35. Schaller, H., Bouvier-Nave, P., and Benveniste, P. (1998) Plant Physiol. 118, 461-469[Abstract/Free Full Text]
  36. Leber, R., Landl, K., Zinser, E., Ahorn, H., Spok, A., Kohlwein, S. D., Turnowsky, F., and Daum, G. (1998) Mol. Biol. Cell 9, 375-386[Abstract/Free Full Text]
  37. Ness, F., Achstetter, T., Duport, C., Karst, F., Spagnoli, R., and Degryse, E. (1998) J. Bacteriol. 180, 1913-1919[Abstract/Free Full Text]
  38. Palermo, L. M., Leak, F. W., Tove, S., and Parks, L. W. (1997) Curr. Genet. 32, 93-99[CrossRef][Medline] [Order article via Infotrieve]
  39. Tomeo, M. E., Palermo, L. M., Tove, S., and Parks, L. W. (1997) Yeast 13, 449-462[CrossRef][Medline] [Order article via Infotrieve]
  40. Yang, H., Bard, M., Bruner, D. A., Gleeson, A., Deckelbaum, R. J., Aljinovic, G., Pohl, T., Rothstein, R., and Sturley, S. L. (1996) Science 272, 1353-1356[Abstract]
  41. Grunwald, C. (1971) Plant Physiol. 48, 653-655
  42. McKersie, B. D., and Thompson, J. E. (1979) Plant. Physiol. 63, 802-805
  43. Mudd, J. B., and McManus, T. T. (1980) Plant. Physiol. 65, 78-80
  44. Webb, M. S., Irving, T. C., and Steponkus, P. L. (1995) Biochim. Biophys. Acta 1239, 226-238[Medline] [Order article via Infotrieve]
  45. Lehle, L. (1980) Eur. J. Biochem. 109, 589-601[Abstract]
  46. Parks, L. W., McLean-Bowen, C., Taylor, F. R., and Hough, S. (1978) Lipids 13, 730-735[Medline] [Order article via Infotrieve]
  47. Lenart, U., Haplova, J., Magdolen, P., Farkas, V., and Palamarczyk, G. (1995) Acta Biochim. Pol. 42, 269-274[Medline] [Order article via Infotrieve]
  48. Parodi, A. J. (1976) Acta Physiol. Pharmacol. Latinoam. 26, 430-433
  49. Esders, T. W., and Light, R. J. (1972) J. Biol. Chem. 247, 248-259
  50. Wojciechowski, Z. A., and Zimowski, J. (1979) Phytochemistry 18, 39-42[CrossRef]
  51. Murakami-Murofushi, K., Nakamura, K., Ohta, K., Suzuki, M., Suzuki, A., Murofushi, H., and Yokota, T. (1987) J. Biol. Chem. 262, 16719-16723[Abstract/Free Full Text]
  52. Kalinowska, M., and Wojciechowski, Z. A. (1986) Phytochemistry 25, 45-49[CrossRef]
  53. Bouvier-Nave, P., and Benveniste, P. (1995) Plant Sci. 110, 11-19[CrossRef]
  54. Mackenzie, P. I., Owens, I. S., Burchell, B., Bock, K. W., Bairoch, A., Belanger, A., Fournel-Gigleux, S., Green, M., Hum, D. W., Iyanagi, T., Lancet, D., Louisot, P., Magdalou, J., Chowdhury, J. R., Ritter, J. K., Schachter, H., Tephly, T. R., Tipton, K. F., and Nebert, D. W. (1997) Pharmacogenetics 7, 255-269[Medline] [Order article via Infotrieve]
  55. Campbell, J. A., Davies, G. J., Bulone, V., and Henrissat, B. (1997) Biochem. J. 326, 929-939[Medline] [Order article via Infotrieve]
  56. Warnecke, D. C., and Heinz, E. (1994) Plant. Physiol. 105, 1067-1073[Abstract/Free Full Text]
  57. Warnecke, D. C., Baltrusch, M., Buck, F., Wolter, F. P., and Heinz, E. (1997) Plant Mol. Biol. 35, 597-603[CrossRef][Medline] [Order article via Infotrieve]
  58. Erdmann, R. (1994) Yeast 10, 935-944[Medline] [Order article via Infotrieve]
  59. Zaret, K. S., and Sherman, F. (1982) Cell 28, 563-573[Medline] [Order article via Infotrieve]
  60. Sikorski, R. S., and Hieter, P. (1989) Genetics 122, 19-27[Abstract/Free Full Text]
  61. Lorch, Y., and Kornberg, R. D. (1985) J. Mol. Biol. 186, 821-824[Medline] [Order article via Infotrieve]
  62. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (eds) (1989) Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York
  63. Cregg, J. M., Barringer, K. L., Hessler, A. Y., and Madden, K. R. (1985) Mol. Cell. Biol. 5, 3376-3385[Medline] [Order article via Infotrieve]
  64. Liu, H., Tan, X., Russel, K. A., Veenhuis, M., and Cregg, J. M. (1995) J. Biol. Chem. 270, 10940-10951[Abstract/Free Full Text]
  65. Wach, A., Brachat, A., Poehlmann, R., and Philippsen, P. (1994) Yeast 10, 1793-1808[Medline] [Order article via Infotrieve]
  66. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. (1990) J. Mol. Biol. 215, 403-410[CrossRef][Medline] [Order article via Infotrieve]
  67. Moehs, C. P., Allen, P. V., Friedmann, M., and Belknap, W. R. (1997) Plant J. 11, 227-236[CrossRef][Medline] [Order article via Infotrieve]
  68. Jorasch, P., Wolter, F. P., Zähringer, U., and Heinz, E. (1998) Mol. Microbiol. 29, 419-430[CrossRef][Medline] [Order article via Infotrieve]
  69. Kirk, D. N., Toms, H. C., Douglas, C., and White, K. A. (1990) J. Chem. Soc. Perkin Trans. 2, 1567-1594
  70. Smith, W. B. (1977) Org. Magn. Res. 9, 644-648
  71. Chu, S., DeRisi, J., Eisen, M., Mulholland, J., Botstein, D., Brown, P. O., and Herskowitz, I. (1998) Science 282, 699-705[Abstract/Free Full Text]
  72. DeRisi, J. L., Iyer, V. R., and Brown, P. O. (1997) Science 278, 680-686[Abstract/Free Full Text]
  73. Wodicka, L., Dong, H., Mittmann, M., Ho, M.-H., and Lockhart, D. L. (1997) Nat. Biotechnol. 15, 1359-1367[Medline] [Order article via Infotrieve]
  74. Sweigard, J. A., Carroll, A. M., Farrall, L., Chumley, F. G., and Valent, B. (1998) Mol. Plant-Microbe Interact. 11, 404-412[Medline] [Order article via Infotrieve]
  75. Shaw, D. R., Richter, H., Giorda, R., Ohmachi, T., and Ennis, H. L. (1989) Mol. Gen. Genet. 218, 453-459[Medline] [Order article via Infotrieve]
  76. Kimmel, A. R., and Firtel, R. A. (1979) Cell 16, 787-796[Medline] [Order article via Infotrieve]
  77. Kimmel, A. R., and Firtel, R. A. (1985) Mol. Cell. Biol. 5, 2123-2130[Medline] [Order article via Infotrieve]
  78. Murakami-Murofushi, K., and Ohta, J. (1989) Biochim. Biophys. Acta 992, 412-415[Medline] [Order article via Infotrieve]


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.