Sterols are major components of eukaryotic cell membranes. End
products of the sterol biosynthetic pathway differ depending on
species; cholesterol is encountered in animals, ergosterol is the most
common sterol in fungi, while sitosterol, campesterol, and stigmasterol
are typical plant sterols(1) . Most of terminal sterols feature
a similar four-ring structure including a C-5-C-6 unsaturation on
ring B and a more or less branched side chain. Additional features
depend on the origin; cholesterol has a C
side chain,
whereas plant sterols bear an additional alkyl (methyl or ethyl) group
on C-24. Ergosterol, in addition to a C-24 methyl group, contains two
additional unsaturations on the C-7-C-8 bond of the B ring and on
the C-22-C-23 bond of the side chain. These unsaturations are
specific to fungal sterols and make them the target for antifungal
polyene drugs like nystatin(2) .
The enzymatic steps that
allow conversion of lanosterol resulting from the oxidative cyclization
of squalene to cholesterol have been
documented(3, 4, 5, 6, 7) .
Most of steps are also found in Saccharomyces cerevisiae ergosterol biosynthesis(8, 9) . Few of the
involved mammalian genes like the P450 lanosterol demethylase have been
isolated (10, 11) . From zymosterol
(cholesta-8,24-dieneol) to cholesterol (cholesta-5-eneol) or to
ergosterol (ergosta-5,7,22-trieneol), there are multiple alternative
pathways leading to the terminal sterol. In mammals, the main flow
involves the following sequence: isomerization of the double bond from
C-8 to C-7, introduction of a double bond at C-5, and two reduction
steps at the C-24 and C-7 double bonds. These reduction steps are
absent in yeast but are catalyzed by the sterol
24-reductase and
the
7-red, (
)respectively, in higher eukaryotes. Very
little is known about these two reactions excepted that they can be
reproduced in vitro using a reconstituted system involving
partially purified fractions from rat liver (12, 13) or plant (14) microsomes. Indeed,
inhibitors of the
7-red have been tested as therapeutic drugs
against hypercholesterolemia(15) , and it was recently reported
that a recessive autosomal disorder, the Smith-Lemli-Optiz syndrome
responsible for multiple congenital anomalies, corresponds to a
deficient activity of this enzyme(16) . Lipoproteins of
affected patients are enriched in
7-cholesterol
(cholesta-
5,7-dieneol) and are highly depleted in
cholesterol(17) . Despite its interest, identification of the
gene encoding the
7-red appeared to be a puzzling task due to the
total lack of sequence information as well as of purified protein or
related antibodies.
Here we present the cloning of the Arabidopsis thaliana
7-red based on the observation that
nystatin toxicity is highly dependent on the presence of sterol
carrying a
5,7-dienic structure. Selection for functional
expression in yeast of a plant cDNA encoding
7-red activity was
based on the expectation that reduction of the
5,7-endogenous
yeast sterols would decrease nystatin toxicity. Nevertheless, the
possibility to substitute ergosterol by its reduction product was a
major concern since disruption of ergosterol biosynthesis is the
classical mode of action of antifungal drugs like ketoconazole. In
fact, the bulk (structural) function of the sterols can be fulfilled in
yeast by different sterols such as cholestanol, cholesterol,
lanosterol, or intermediates of sterol biosynthesis pathway, provided
that some residual level of ergosterol be present (18, 19) . This is known as the ``sparking
effect,'' which is probably related to a cell cycle control
mechanism in wild type strains(20) . This requirement is
abolished in cells harboring fen1 and/or fen2 gene
mutations (21, 22) suggesting to use strain
FY1679-28C, a naturally occurring fen1 mutant, as a host for
the
7-red screening. We also chose a plant, A. thaliana as cDNA source because of the high
7-red activity present in
plant microsomes (
)and its low relative genome
complexity(23, 24) .
EXPERIMENTAL PROCEDURES
Yeast Strains
FY1679-28C yeast strain (MAT
a, ura3-52, trp1
63, leu2
1, his3
200, GAL2) is
a derivative of S288C constructed by Thierry et
al.(25) . The pol5 strain (gift from Dr. F.
Karst), which is deficient in sterol
22-desaturase, was originally
isolated by Molzahn and Woods (26) as a polyene-resistant
mutant. For construction of PLC1051 (MAT
, erg 5,
ura3-52, his3D200, trp
631, leu2D1, GAL2), pol5 strain was crossed with FY 1679 (MAT a ) and the
resulting diploid cells were sporulated. Spore purification by
hydrophobic binding to polypropylene tubes was performed as
described(27) . The PLC1051 haploid strain was selected after
germination as an ura
, his
,
trp
, leu
, erg5 clone
(accumulation of ergosta-5,7-dieneol detected by gas chromatography on
a SE30 capillary column) able to grow on galactose (GAL2 phenotype) and
to use glycerol as carbon source (respiration competency). The PLC 1451 (MAT
, erg5, erg4, ura3-52, trp
631, leu2
1,
GAL2, rho+), a double sterol mutant carrying deficiencies in
the sterol
22-desaturase and in the sterol
24(28)-reductase
genes, was constructed as follows: a spontaneous nystatin-resistant
mutant (5 µg/ml nystatin) of the pol5 strain which
accumulates ergosta-5,7,24(28)-trieneol instead of ergosta-5,7-dieneol
was isolated by direct screening for alteration in the sterol
composition among nystatin-resistant clones. This latter strain was
crossed with FY1679-28C, and the diploid was sporulated as described
previously. After spore purification and germination, the haploid
strain PLC1451 was isolated. This strain accumulates
ergosta-5,7,24(28)-trieneol, is auxotrophic for uracil, tryptophan and
leucine and can grow in presence of galactose or glycerol as carbon
source. PLC1061 (MAT
, erg6, ura3-52,
his
200, GAL2) was isolated by sporulation of the
diploid formed after crossing pol6 with FY1679-28C.
Media
Synthetic media are SGI (7 g/liter yeast
nitrogen base (Difco), 1 g/liter bactocasamino acid (Difco), 20 g/liter
glucose, and 20 mg/liter DL-tryptophan) or SLI in which
glucose is replaced by galactose (20 g/liter). The complete medium
without carbon source is named YP (10 g/liter yeast extract (Difco), 10
g/liter bactopeptone (Difco)).
Vectors
The plasmid pUC9-N was derived from pUC9
(Pharmacia Biotech Inc.) by insertion of a NotI restriction
site in the filled EcoRI site (gift from F. Lacroute).
pBlueScript (Stratagene) was used to subclone different fragments of
the
7-red gene. The Escherichia coli-S. cerevisiae shuttle vectors are pYeDP1/8-2 (named V8), which carries a yeast
``2µ'' origin of replication, the URA3 selection
marker, an expression cassette based on the galactose inducible GAL10-CYC1 promoter, a multiple cloning site, and the
phosphoglycerate kinase gene (PGK) terminator (28) and
pFL61(29) .
Screening of the A. thaliana cDNA Expression Library in
Yeast
The cDNA library from full seedling A. thaliana at the two-leaf stage was kindly provided by Dr. F.
Lacroute(29) . In this library, cDNAs are cloned as a NotI cassette placed under the transcriptional control of PGK transcription promoter and terminator sequences in a pFL61 E. coli-S. cerevisiae shuttle vector. A ``2µ''
origin of replication and a URA3 selection marker are used for
propagation in yeast, whereas the E. coli propagation part is
derived from pUC19. The FY1679-28C yeast strain was transformed with
the cDNA library using the lithium acetate procedure(30) .
Cells were plated on synthetic medium lacking uracil (SGI with 2% agar
(Difco)). Transformation yielded 10
primary transformants
prototrophic for uracil. The cells were pooled and plated at 5
10
cells per plate containing SGI solid medium and either 2
or 5 µg/ml nystatin. After 3 days of incubation at 28 °C, a
hundred clones were growing in the presence of 2 µg/ml nystatin.
Individual clones were finally analyzed for their sterol composition by
HPLC. Among them, one named F22 was resistant to 5 µg/ml nystatin
and exhibited a sterol composition with a lowered
5,7 content
based on the 280 nm HPLC traces.E. coli was transformed by
the plasmidic DNA extracted from clone F22 as described
previously(31) . The plasmidic DNA from individual E. coli transformants was digested by NotI. Two different classes
of cDNA inserts were identified which correspond to 600-bp and 1.6-kb NotI inserts respectively. The plasmid carrying the 1.6-kb
cDNA insert was found to be also rearranged at the pFL61 level as
judged by the altered restriction pattern. The FY1679-28C yeast strain
was retransformed with this plasmid and the sterol composition of
transformants analyzed. All transformants exhibit the same anomalous
sterol pattern as compared with the void pFL61 transformed strain.
Nucleotide Sequence Determination
The NotI cDNA insert in pFL61 was extracted and subcloned into the
unique NotI site of pUC9-N. The nucleotide sequence was
determined using the Sequenase kit (U. S. Biochemical Corp.), the
direct and reverse primers of pUC9 and of pBlueScript (T3 and T7
primers) and specific oligonucleotide sequences belonging to the
sequenced gene. After completing the full sequence of one strand, the
complementary strand was fully sequenced using a series of specific
oligonucleotides as primers.
Reformatting and Cloning
7-red cDNA into Expression
Vector pYeDP1/8-2
Deletion of the 5`- and 3`-non-coding regions
of the
7-Red cDNA was performed by PCR amplification using
specific primers designed to introduce a BamHI restriction
site immediately upstream of the initiation codon and a KpnI
site immediately downstream of the stop codon.

Sequences identical or complementary to the cDNA are shown as
uppercase, and restriction sites are underlined. The
7-red cDNA
was amplified using 33 thermal cycles with 2 units of Pfu DNA
polymerase (Stratagene) in the presence of 10 pmol of each primer and
0.2 mM of each dNTP, in the recommended buffer. The
temperature cycles were 10 s at 94 °C, 50 s at 52 °C, followed
by 1 min 30 s at 74 °C. The 1300-bp PCR product was BamHI/KpnI digested and inserted between the BamHI and KpnI sites of pYeDP1/8-2, resulting in
plasmid
7red/V8. The integrity of the PCR-amplified fragment was
confirmed by sequencing.
Integration of Reformatted
7-Red cDNA into the ADE2
Locus of FY1679-28C Strain
The BglII DNA fragment of
the yeast ADE2 gene included in the plasmid pASZ11 (32) was first subcloned into the BamHI site of
pBlue-Script, resulting in pBS-ADE2. Primers:
5`-agatctTGAGAAGATGCGGCCAGCAAAAC-3` (hybridizing the 3`-ends of URA3) 5`-GATTACGCCAAGCTTTTCGAAAC-3` (hybridizing the 3`-end of
the PGK terminator) were designed to amplify the full
7-Red/V8 expression cassette including the GAL10-CYC1 promoter, the
7-red cDNA coding sequence and the PGK
transcription terminator: 80 ng of
7red/V8 template, 0.5
µM phosphorylated primers, 0.2 mM of each dNTP
diluted in the commercial buffer were first denatured 1 min at 95
°C, after which 1 unit of native Pfu DNA polymerase was
added and the reaction mixture cycled for 35-fold using 5 s at 95
°C, 30 s at 56 °C, and 4 min 30 at 70 °C. The amplified
2440-bp fragment was purified and subcloned blunt-end into the unique StuI site of pBS-ADE2, giving pAD
7. The 4720-bp NotI- PstI fragment of pAD
7 containing the
disrupted ADE2 sequence was isolated and used to transform
strain FY1679-28C according to previously described
methods(30) . The resulting yeast strain was called ELR01.
Southern Blot Analysis
Genomic DNAs from A.
thaliana, yeast, and different species were restricted overnight
in the appropriate buffers. DNA fragments were separated by
electrophoresis on a 0.8% agarose gel. DNA was transferred onto
nitrocellulose filters (BA85; Schleicher & Schuell). The open
reading frame of the
7-red cDNA was isolated from
7-red/V8 on
agarose gel and purified with Jetsorb extraction kit (Bioprobe). The
hybridization of the
P labeled probe was performed at 42
°C for 3 days using standard procedures excepted that formamide
concentration was reduced from 50 to 30% (by volume) to reduce
stringency. Filters were washed using as final conditions 0.1
SSC, 0.1% SDS, and 37 °C before exposition in a PhosphorImager.
Cell Culture and Subcellular Fractionation
Yeast
cells transformed with pFL61 and p
7red/V8 were grown,
respectively, into SGI and SGRI synthetic medium until stationary
phase. SGRI is similar to SGI except that the glucose concentration was
5 g/liter. The saturated culture was diluted with an equal volume of YP
medium complemented with ethanol (final concentration 1.5% by volume)
as carbon source and grown until cell density reached a minimal value
of 7
10
cells/ml (A
=
10).
7-Red expression was induced by addition of D-galactose at a final concentration of 20 g/liter. The
culture was stopped when density reached 1.5 to 2
10
cells/ml (A
= 20-30).
Alternatively, the gene expression was also induced by culture up to 2
10
cells/ml (A
= 3)
in synthetic medium SLI. Harvested yeast cells were broken after
spheroplasts preparation by enzymatic digestion of cell wall and
subcellular fractionation as described previously(28) .
Alternatively, the subcellular fractionation was performed by
ultracentrifugation. The cells were collected, washed twice in buffer
TE-KCl (50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 0.1 M KCl) and suspended in TE, 0.6 M sorbitol. Glass beads
(0.45-0.5 mm diameter, Braun) were added until skimming the top
of the cell suspension and cell walls were disrupted mechanically by
handshaking for 5 min in cold room. The membrane debris, nuclei and
mitochondria were pelleted by centrifugation at 20,000
g, 13 min at 4 °C. The ultracentrifugation of the
supernatant at 100,000
g, 1 h at 4 °C allows the
separation of microsomes, which are pelleted, and cytosol.
Sterol Analysis
Total sterols were prepared by
alkaline saponification as described previously(33) . The
sterol analysis and purification was performed at 55 °C on reverse
phase HPLC operated on a 100
2.1 mm (or 100
4.6 mm for
purification) reverse phase C
column (Applied Biosystems).
The column was eluted with a linear gradient of aqueous methanol from
50 to 100% (by volume) at 1.0 ml/min (or 3.0 ml/min for purification).
The sterol composition was also analyzed by gas chromatography with a
SE30 capillary column (30 m
0.32 mm, Altech) and helium as
carrier gas. The structure of purified sterols from transformed yeast
strains was determined based on comparison of their relative retention
times to standards in GC and HPLC and further confirmed by GC-mass
spectrometry fragmentation and NMR analysis when appropriate.
Enzymatic Assays
For
7-reductase, assays were
performed according to a previously described method(14) .
Subcellular fractions (700 µg of protein in 200 µl) were
incubated for 90 min at 37 °C in 100 mM Tris/HCl buffer,
pH 7.3, containing 150 µM cholesta-
5,7-dieneol
(7-dehydrocholesterol), emulsified with Tween 80 (final concentration
1.5 g/liter) and 2 mM NADPH. The reaction was stopped by
addition of an equal volume of methanol-dichloromethane (50:50, by
volume). The lower phase was collected and air-dried, and products were
dissolved in methanol prior to GC analysis. The authentic products
cholesta-5,7-dieneol and cholesterol were well separated on the column,
and their retention times are quite different from the endogenous yeast
sterols.
RESULTS
Isolation of the cDNA Encoding
7-Red
Activity
Effects of polyene antifungal like nystatin,
amphotericin B, or filipin are critically dependent on the presence of
a
7-bond unsaturation (2) as demonstrated by the highly
nystatin-resistant phenotype of erg2 mutants, which are
deficient in the sterol
8-7 isomerase gene product. The
selection is based on the assumption that such situation could be
mimicked in a wild type strain expressing heterologous
7-red
activity. Yeast FY1679-28C was transformed with an A. thaliana cDNA library placed under the transcriptional control of the host
phosphoglycerate kinase transcription promoter and terminator
sequences. Selection of transformants was first performed on the basis
of the ura3 mutation complementation by the plasmid-borne
marker. A second selection of primary transformants was performed for
an increased nystatin resistance. Resistant clones were finally
screened individually by HPLC for an alteration of the sterol profile
leading to a significant decrease in the proportion of 280 nm-absorbing
hexane-extractable material (
5,7-dienic system signature) compared
with 205 nm-absorbing compounds (any unsaturation) as detailed under
``Experimental Procedures.'' Out of more than 10
primary transformants, only one clone F22, which is resistant up
to 80 µg/ml nystatin, was finally selected. The sterol profile of
this strain was examined by HPLC and GC techniques; ergosterol was no
longer the major component as observed in the parent strain transformed
by a void plasmid (data not shown). Two new major sterols lacking any
280 nm absorption accumulate in similar amounts in F22, representing
globally more than 90% (by weight) of the total sterol content based on
the GC profile. The sterol profile alteration was found to be
plasmid-dependent by transfer of the F22 plasmid into E. coli and back-transformation of the FY1679-28C strain.Individual
sterols from yeast strain back-transformed with pF22 were purified by
preparative HPLC (about 8 mg each recovered) and their structure
determined by GC-mass spectrometry and Fourier-transform NMR analysis.
The more lipophilic sterol (second main peak in HPLC and GC) exhibits a
molecular peak at m/e = 400 in GC-MS and was identified
as ergosta-5-eneol or dihydrobrassicasterol on the basis of NMR. The
compound co-migrates in HPLC and GC with its commercial epimer:
campesterol. Detailed comparison of NMR data with the spectra of
authentic compounds in the literature is indicative of an
``S'' absolute configuration at the C-24 position of
ergosta-5-eneol (data not shown). The more polar additional sterol
(first to elute from HPLC and GC) has a GC-MS molecular peak at 398,
and its fragmentation and
H NMR spectra are fully
consistent with an ergosta-5,22-dieneol structure. This sterol is thus
the direct reduction product of ergosterol (ergosta-5,7,22-trieneol) at
the C-7 double bond level. Accumulation of these two compounds highly
suggests that the plasmid borne cDNA is encoding a sterol
7-red
activity. Concomitant accumulation of ergosta-5-eneol with the
ergosterol reduction product suggests that reduction of the
-7
bond can also occur in vivo at the level of the biosynthesis
intermediate ergosta-5,7-dieneol and/or of its precursor
ergosta-5,7,24(28)-trieneol.
Nucleotide Sequence of the A. thaliana
7-Red
The cDNA in pF22 was extracted and subcloned before
sequencing on both strands. The cloned cDNA (Fig. 1) without its
poly(A) tail is 1488 bp long and contains an open reading frame of 1290
bp, which is coding for a protein of 430 amino acids with a calculated
molecular mass of 49,458 Da. The 5`- and the 3`-untranslated regions
are 76 and 121 bp long, respectively. A possible consensus
polyadenylation signal is located 34 bp upstream of the poly(A) tail. A
computer search on a sequence data base reveals that the deduced amino
acid sequence of
7-red exhibits a significant similarity with the
ones of
14- and
24(28)-sterol reductases (Fig. 2),
suggesting that all known sterol reductases belong to a single sequence
family. These reductases are the S. cerevisiae sterol
C-14-reductase(34) , the Neurospora crassa sterol
C-14-reductase (accession no. X77955 in EMBL data base), the YGL022
open reading frame later identified as the S. cerevisiae sterol
24(28)-reductase (35) , and the Schizosaccharomyces pombe SST1 gene product (27) . The
latter protein product is likely to be a sterol
24(28)-reductase
too(36) . In addition, the
7-red shows a striking
similarity with the 400 C-terminal amino acids of the lamin B receptors
from chicken and human as already evidenced for other sterol
reductases(37, 38, 39) . The N-terminal end
of these two proteins contains a typical DNA binding
domain(40) , absent in all identified sterol reductases
including the
7-red one.
Figure 1:
Nucleotide and deduced amino acid
sequences of
7-red cDNA. Open reading frame flanking sequences are
indicated by lowercase letters. The cDNA poly(A) tail starts
at nucleotide 1487.
Figure 2:
Sequence alignment of the sterol reductase
family. Alignment was performed using the PILEUP program of the UGCG
package run with the default parameters. Positions with a consensus
residues present in five or more sequences are indicated in bold
uppercase letters. 2428sc and sst1 stand,
respectively, for the
24(28)-sterol reductases from yeast S.
cerevisiae and S. pombe; hlr440 and clr440 stand, respectively, for the human and chicken lamin receptor
sequences starting at residues 440; 14str, 14strpb,
and nc-14red stand, respectively, for the S.
cerevisiae, the S. pombe, and the N. crassa sterol
14-reductases. This last sequence is probably
incomplete on the N-terminal side based on alignments. The
7-red
sequence is labeled d7red.
Consensus sequences involved in the
binding sites of NADPH or NADH and/or flavin have been described in
different reductases family like P450 reductases or nitrate
reductases(41) . However, it was not possible to identify
similar motif in the sterol reductase protein family, even with the
newly included sequence. In addition detailed sequence comparison
between
7-,
14-, and
24(28)/sterol reductases (eight
sequences) did not allow identification of a clear sequence signature
corresponding to the different regio-specificities for sterol reduction (Fig. 2). Globally, sequence conservation within the family is
high in the C-terminal half of the enzymes, with a clear
LLXSGWWGXXRH signature almost perfect in all members.
In contrast, a more limited sequence similarity is present on the
N-terminal half. Particularly the EFGGXXG signature common to
24(28),
14, and lamin receptor is not present in
7-red.
Interestingly enough, the hydrophobic profiles remain very similar
among all family members even within the N-terminal half (starting
residue 440 for lamin B receptors). The lamin B receptor sequence
cannot be distinguished from that of other family members either on
sequence or on hydrophobic profile criteria.
Southern Blot Analysis of
7-Red Locus
A.
thaliana genomic DNA was probed with the open reading frame of
7-red cDNA (Fig. 3). Based on PstI digestion
(absent site from the cDNA) yielding a single hybridizing band in low
stringency conditions, the presence of a single
7-red gene can be
deduced. Absence of overlapping bands was confirmed by double digestion
with BamHI. Cleavage by BamHI alone (two bands: c and
c1) or in combination with PstI (three fragments: a, a1, and
a2) are indicative of the presence of at least one intron in the gene.
PCR applied to genomic DNA using a primer situated at both extremities
of the open reading frame led to amplification of a single 3.6-kb
fragment confirming the presence of a total of 2.5 kb of intronic
sequences within the open reading frame of a unique gene. Digestion
with PvuII, which cuts once within the cDNA, gave rise as
expected to two hybridizing bands (e1 and e). In a second experiment,
genomic DNAs of different origins (human, quail, Drosophila
melanogaster, Xenopus laevis, maize, and yeast) were
tested (Fig. 3B). EcoRI-restricted DNA from
parental yeast FY1679-28C exhibits three weak bands at 4.2, 2.5, and
2.3 kb upon low stringency hybridization, which could correspond to
endogenous
14 and
24(28)-reductase genes. As a control,
strain ELR01 (see later), which contains an expression cassette for A. thaliana
7-red integrated within the yeast genome, was
tested. The strong hybridization signals corresponding to the two
expected EcoRI fragments were observed in addition to two weak
signals also found with the parental strain. Interestingly enough, a
well defined hybridization signal was found with quail DNA.This
hybridization signal was absent under high stringency conditions,
suggesting detectable but limited interspecies sequence conservation.
Weak but defined signals were also found with maize, but not with
human, X. laevis, and D. melanogaster DNAs, thus
illustrating the limits of interspecies cross-hybridization approaches.
Figure 3:
Southern blot analysis of genomic DNA
using
7-red cDNA as probe. A, 1 µg of A. thaliana genomic DNA was digested to completion with various combinations
of restriction enzymes as indicated. Fragments were separated by a 0.8%
agarose gel electrophoresis. The blot was revealed using as probe the
P-labeled BamHI -KpnI fragment of
pV8/
7red, which contains the
7-red open reading frame (see
``Experimental Procedures''). B, human (10 µg, lane 1), quail (10 µg, lane 2), X. laevis (5 µg, lane 3), D. melanogaster (10 µg, lane 4), maize (10 µg, lane 5), or 3 µg of
genomic DNA from yeast ELR01 (lane 6) or strain FY1679 (lane 7) were digested to completion by EcoRI, and
the fragments were separated by electrophoresis. The blot was revealed
by low stringency hybridization with the cDNA probe as in panel
A.
Overexpression of
7-Red and Time Course of in Vivo
Sterol Conversion
To optimize expression, the cDNA encoding the
7-red was reformatted and cloned into pYeDP1/8-2, placing the
flanking sequence-free open reading frame under the transcriptional
control of a galactose-inducible GAL10-CYC1 promoter.
FY1679-28C cells were transformed by the resulting pV8/
7red. Cells
were first grown on glucose-repressed conditions in which the
plasmid-borne
7-red composite gene is silent. Following the
transfer in inducing culture conditions, the time course of the changes
in the yeast cell sterol composition was followed (Fig. 4). As
expected, during growth in glucose or in ethanol, the main sterol is
ergosterol and ergosta-5,22-dieneol and 5-eneol are hardly (ethanol) or
not at all (glucose) detectable. Following the addition of galactose,
ergosterol content rapidly decreases with a concomitant increase in
ergosta-5,22-dieneol content. A very limited formation of
ergosta-5-eneol occurs during the first 2 h following induction. This
compound nevertheless slowly accumulates with increasing induction
times (up to 9 h), while ergosta-5,22-dieneol content remains constant
and then slowly decreases. At the end of the culture, ergosterol
accounts for 5% (w/w) of the total sterols, ergosta-5-eneol for 45%
(w/w), and ergosta-5,22-dieneol for 50% (w/w). The proportion of the
other sterol intermediates could be estimated to less than 10% of the
total sterol based on GC analysis. This indicates that in vivo accumulation of ergosta-5,22-dieneol results from the direct
reduction of previously accumulated ergosterol. In contrast,
accumulation of ergosta-5-eneol requires de novo sterol
biosynthesis, and likely results from reduction of the biosynthetic
intermediate ergosta-5,7-dieneol (Fig. 5). The decrease in
ergosta-5,22-dieneol content during the late induction phase suggests
that ergosta-5-eneol is no longer a good substrate for the yeast
-22 desaturase enzyme.
Figure 4:
In vivo sterol conversion upon
expression of
7-red in yeast. FY1679-28C cells were transformed by
pV8/
7red as indicated under ``Experimental Procedures.''
Transformants were grown to stationary phase in a selective medium
similar to SGRI (culture phase labeled G). The culture was
diluted with an equal volume of YP medium complemented with ethanol
(final concentration 1.5% by volume) as carbon source and grown for
another 12 h (culture phase labeled E).
7-Red expression
was started by addition of D-galactose at a final
concentration of 20 g/liter (phase labeled L). Culture
aliquots were withdrawn at regular intervals and total sterols
extracted (alkaline hydrolysis) as described under ``Experimental
Procedures.'' Sterols ergosterol (
), ergosta-5,22-dieneol
(
), and ergosta-5-eneol (
) were analyzed by GC, and values
were calculated as percent by weight of total
sterols.
Figure 5:
Biosynthetic scheme for sterols in
7-red transformed yeast.
In Vivo Analysis of
7-Red Substrate
Specificity
To investigate in more detail substrate specificity
of the
7-red, mutant strains PLC 1051, 1451, and 1061, which
accumulate sterol biosynthesis intermediates were transformed with
pV8/
7red. Main sterols accumulated by yeast mutants expressing or
not the
7-red activity are listed in Table 1. In the
22-desaturase-deficient strain PLC1051,
7-red expression
results mainly in accumulation of ergosta-5-eneol confirming that
ergosta-5,7-dieneol is a good substrate for the enzyme in vivo as predicted from the late accumulation of ergosta-5-eneol in a
similar experiment with the wild-type parental strain. Similar
experiment with mutant PLC1451, which also lacks the sterol
24(28)-reductase, led to accumulation of ergosta-5,24(28)-dieneol
(ostreasterol), the expected reduction product. In PLC1051 and 1451,
the expression of the
7-red thus changes the end product
synthesized but does not cause accumulation of biosynthesis
intermediates. In the erg6 mutant PLC1061, the sterol C-24 S-adenosyl methyl transferase, which converts
cholesta-8,24-dieneol (zymosterol) into ergosta-8,24(28)-dieneol
(fecosterol), is deficient, thus leading to zymosterol accumulation and
to a lesser extent to cholesta-5,7,24-trieneol and
cholesta-5,7,22,24-tetraeneol accumulations. Expression of the
7-red in this strain caused the reduction of the
7 double
bond of cholesta-derivatives, yielding cholesta-5,22,24-trieneol and
cholesta-5,24-dieneol but zymosterol, which bears a double bond in C-8,
remained unaffected, suggesting a high specificity of
7-red for
the C-7 position.
7-Red can thus accept in vivo a very
large range of ergosta- and cholesta- compounds carrying a
5,7-dieneol structure.
In Vitro Analysis of
7-Red Enzymatic
Properties
The subcellular location of the
7-red was
analyzed using the cholesta-5,7-dieneol as substrate since cholesterol,
the expected reduction product, is absent from yeast and well resolved
from endogenous sterols. GC profiles of sterols were examined after
incubation of cholesta-5,7-dieneol and NAPDH with microsomal fractions
from the FY1679-28C strain expressing
7-red. Two peaks
corresponding to the residual substrate (cholesta-5,7-dieneol) and to
the cholesterol formed are present and well separated from endogenous
sterols. The cholesterol peak was found to be absent when either
cholesta-5,7-dieneol or NADPH were omitted or when microsomes from a
yeast transformed with a void plasmid were used. In addition, a
negative result was also obtained when NADH was substituted to NADPH.
In our hands, the microsomal fractions exhibits the highest specific
activity (versus protein content) but some activity was also
found in lipid droplets and cytosol, suggesting a rather diffuse
subcellular location of the enzyme. The activity of
7-red toward
sterol esters was tested using sterol acetate as a model.
7-Dehydrocholesterol acetate and ergosterol acetate (200
µM) were incubated with cytosolic or microsomal fractions
from PLC1051 strain transformed with V8/
7-red. Both esters were
found to be efficiently reduced at C-7 upon incubation with the
cytosolic fractions. 7-Dehydrocholesterol ester was clearly a better
substrate than the ergosterol ester (45% versus 15% of
conversion). Similar experiments with microsomal fractions led
surprisingly to the fast hydrolysis of steryl acetate by some
endogenous esterase, a reaction that was absent when cytosolic
fractions were used. This indicates that a free hydroxyl group at the
C-3 position is not required for activity of
7-red and that fatty
acid steryl esters might be physiological substrates of this enzyme.
Genome Integration of the
7-Red Expression Cassette
and Effects on Cell Viability
Genomic integration of the
7-red expression cassette opened the way to add additional
heterologous activities in engineered cells and allowed us a better
analysis of the physiological effects of
7 reduction of sterols.
The cassette containing the GAL10-CYC1 promoter, the
7-red open reading frame, and the PGK terminator was
extracted from pV8/
7-red by PCR, and the resulting fragment was
inserted within a plasmid containing the yeast ADE2 gene (see
``Experimental Procedures''). FY1679-28C cells were
transformed by the interrupted ADE2 sequence, and homologous
recombination events were selected on the basis of the generation of ade2 clones. PCR and Southern blotting analysis (Fig. 3B) of clone ELR01 confirmed that the full
7-red expression cassette had been integrated within the ADE2 genomic locus. Galactose induction of the
7-red expression
induced a dramatic change in sterol composition as was observed with
the pV8/
7-red transformed strain, except for an even lower level
of residual ergosterol. A single integrated copy of the expression
cassette is thus sufficient to completely reduce in vivo
7-sterols. The ELR01 doubling time and the final cell density
at saturation were found to be similar to those of the parental strain
under similar culture conditions. Cell viability was analyzed and
indicated in both cases that more that 90% of cells in exponential
growth phase were viable. Together, these results indicate that
7-reduced sterols support yeast growth and cell viability as well
as unsaturated sterols under tested conditions.
DISCUSSION
The quantitative and qualitative sterol compositions and the
presence of double bonds or branched groups are known to modulate
membrane properties. This aspect has been well documented in fungi and
especially in the yeast S. cerevisiae since several mutant
strains are available(26, 42) . In a Candida
albicans mutant with an inactive lanosterol C-14 demethylase, the
accumulation of 14
-methyl sterols is responsible for a more rigid
membrane(43) . Similarly, alteration of membrane properties in erg mutants is suggested by a constantly lowered efficiency of
transformation by plasmids. In the case of the erg6, a defect
in tryptophan transport and increased drug sensitivity was also
documented(44) . As a compensation mechanism, the fatty acid
composition of membranes could be adapted to balance the changes in
membrane properties induced by accumulation of abnormal
sterols(45) . In addition to the ``bulk'' effect,
ergosterol also plays a role in the control of the yeast cell cycle.
Sterols that can satisfy this ``sparking'' effect must
possess a double bond at C-5. Unsaturation at C-22 facilitates the
sparking better than the C-7 unsaturation, and the 24
-methyl group
seems to be of low importance(19) .
The double bond at C-7,
while not important for the sparking effect, is critical for polyene
antibiotic sensitivity, which also depends on others factors, including
phospholipid composition and presence of a suitable membrane
potential(46) . The particularly high level of resistance of
the erg2 mutant (deficient in sterol
8-
7 isomerase)
confirms that the presence of
5,7-conjugated double bond is
determining. Consistently, FY1679-28C expressing
7-red is highly
nystatin-resistant (up to 80 µg/ml compared with 2 µg/ml for
parental FY1679). This resistance correlates with accumulation of
almost 95% of C-7-saturated sterols. Upon induction,
7-red rapidly
converts the whole ergosterol pool, including steryl esters to the
corresponding reduction products. This observation supports the finding
that esterified sterols could be in vivo substrates for the
enzyme. The low content of free sterols within the microsomal membranes
in comparison to the value into the plasma membrane is generally
explained by a vectorial transport process involving the synthesis of
steryl esters in microsomes and their hydrolysis by a specific lipase
at the plasma membrane(47) . Steryl esters stored into the
lipid droplets and the free sterols within the plasma membrane might
rapidly interconvert thus explaining reduction of the whole ergosterol
pool by
7-red.
Ergosta-5,22-dieneol and ergosta-5-eneol, which
are, respectively, the reduction products of ergosterol and
ergosta-5,7-dieneol, accumulate in the presence of the
7-red in
transformed FY1679-28C. Ergosta-5-, ergosta-5,24(28)-, or
ergosta-5,22,24(28)-eneols and cholesta-5,24- or
cholesta-5,22,24-eneols can be formed in vivo by reduction of
the corresponding
7-sterols in the different mutants tested. This
indicates that
7-red can tolerate a large variety of sterol side
chains while being highly regio-specific for the
7-bond reduction
in a
5,7-structure. In particular, this enzyme lacks any
8-,
14-,
24(25)-, or
24(28)-reductase activity. This raises
the question of the basis of the regio-specificity within the sterol
reductase family and suggests that the N-terminal part, which is the
more divergent sequence region within the family, might be involved in
this role. The transformed wild type strain behaves like a leaky erg5 (
22-desaturase mutant) strain since ergosta-5-eneol
but not ergosta-5,22-dieneol preferentially accumulates as an end
product of the de novo sterol synthesis. This suggests that
7-reduced sterols are bad substrates for the
22-desaturase
and that reduction of ergosta-5,7-dieneol by the
7-red is highly
competitive with the C-22 desaturation.
The deduced amino acid
sequence of the A. thaliana
7-red demonstrates that the
enzyme belongs to the same sequence and consequently structural family
as the
14- and
24(28)-reductases. Of particular interest is
the confirmation than lamin B receptor, a nuclear membrane protein
featuring a N-terminal DNA binding domain, is a member of the sterol
reductase family. A close examination of the sequence alignment
demonstrates that all highly conserved regions among
7-,
14-,
and
24(28)-reductases are also conserved in lamin B receptors.
This puzzling observation strongly suggests that lamin B receptor may
actually have a sterol reductase activity in addition to its DNA
binding role. Chicken lamin B receptor was expressed in a sterol
C-14-reductase-deficient yeast and did not complement the mutation. In
addition, no equivalent of lamin B receptor has been found in yeast to
date on the basis of the systematic sequence analysis. Yeast has no
need for
24-reductase activity, a step specifically required for
cholesterol biosynthesis. We thus propose that lamin B receptor might
be the cholesta-5,24-dieneol
24-reductase, the last missing member
of the sterol reductase family.