(Received for publication, May 18, 1995; and in revised form, December 1, 1995)
From the
Ubiquinone (coenzyme Q) is a lipid that transports electrons in the respiratory chains of both prokaryotes and eukaryotes. Mutants of Saccharomyces cerevisiae deficient in ubiquinone biosynthesis fail to grow on nonfermentable carbon sources and have been classified into eight complementation groups (coq1-coq8; Tzagoloff, A., and Dieckmann, C. L.(1990) Microbiol. Rev. 54, 211-225). In this study we show that although yeast coq7 mutants lack detectable ubiquinone, the coq7-1 mutant does synthesize demethoxyubiquinone (2-hexaprenyl-3-methyl-6-methoxy-1,4-benzoquinone), a ubiquinone biosynthetic intermediate. The corresponding wild-type COQ7 gene was isolated, sequenced, and found to restore growth on nonfermentable carbon sources and the synthesis of ubiquinone. The sequence predicts a polypeptide of 272 amino acids which is 40% identical to a previously reported Caenorhabditis elegans open reading frame. Deletion of the chromosomal COQ7 gene generates respiration defective yeast mutants deficient in ubiquinone. Analysis of several coq7 deletion strains indicates that, unlike the coq7-1 mutant, demethoxyubiquinone is not produced. Both coq7-1 and coq7 deletion mutants, like other coq mutants, accumulate an early intermediate in the ubiquinone biosynthetic pathway, 3-hexaprenyl-4-hydroxybenzoate. The data suggest that the yeast COQ7 gene may encode a protein involved in one or more monoxygenase or hydroxylase steps of ubiquinone biosynthesis.
Ubiquinone (coenzyme Q, or Q) ()is a lipid component
of the electron transfer chain and functions in the transport of
electrons from Complex I or II to the cytochrome bc
complex found in the inner mitochondrial membrane of eukaryotes,
and in the plasma membrane of prokaryotes(1, 2) . Q
carries out this function via cycles of reduction (to form the
hydroquinone, ubiquinol, or QH
) and oxidation (to form Q).
This same redox chemistry also allows QH
to function as a
lipid soluble antioxidant, directly scavenging lipid peroxyl radicals
in a capacity similar to vitamin E(3) , and/or by its ability
to reduce tocopherol radicals and hence regenerate vitamin
E(4, 5) . QH
is found in a variety of
eukaryotic intracellular membranes and is present in lipoproteins,
where it may serve a primary function as an
antioxidant(6, 7) . Supplementation of diets with Q
results in increased levels of QH
in low density
lipoprotein particles with an increased resistance to lipid
peroxidation(8, 9) . Based on these observations,
QH
may play an important role in the protection of lipids
in cellular membranes and in lipoprotein particles and, hence, function
to prevent or slow atherosclerosis and possibly other disease processes
related to oxidative stress.
Q is synthesized from the precursors p-hydroxybenzoic acid and isoprene diphosphate in both
eukaryotes and prokaryotes(10) . The proposed pathway for the
biosynthesis of Q (Fig. 1) derives from the characterization of
accumulating Q biosynthetic intermediates in Q-deficient mutant strains
of Escherichia coli and Saccharomyces
cerevisiae(10, 11) . Q mutant strains of S.
cerevisiae are non-respiring or petite mutants(12, 13) and have been classified into eight complementation
groups, coq1-coq8(14) . Addition of Q or Q
to mitochondrial extracts prepared from each coq mutant restored NADH-cytochrome c reductase
activity to levels near that of the wild-type parental
strain(12) . Three of the complementation groups (coq1-coq3) have been characterized. In S.
cerevisiae synthesis of compound 1 is carried out by
enzymes encoded by the COQ1 and COQ2 genes(15, 16) . The COQ3 gene encodes an
O-methyltransferase thought to catalyze the synthesis of compound 5(17) . Evidence for the branched pathways between
prokaryotes and eukaryotes derives from the isolation of compound 2 (Fig. 1) in UbiB E. coli mutants(18) ,
compound 4 in coq3 mutants of S. cerevisiae(19) and compound 5 in another S. cerevisiae mutant(20) . Gibson and Young (21) analyzed other E. coli mutants and characterized UbiH, UbiE, UbiF, and UbiG mutants as accumulating
compounds 6, 7, 8, and 9, respectively.
Corresponding yeast mutants to these steps have not been described,
although intermediate 8 has been detected in wild-type
yeast(22) . Given the divergence of the early steps in the
pathway, it is important to fully characterize Q biosynthesis in a
eukaryote. Recent evidence suggests that the Q biosynthetic pathway in
higher eukaryotes mirrors that of S. cerevisiae, since a rat
cDNA homologue to the yeast COQ3 gene was isolated based on
its ability to restore synthesis of Q in a coq3 mutant(23, 24) .
Figure 1: The pathway of Q biosynthesis. The proposed biosynthetic pathway for Q in eukaryotes (including yeast) and in prokaryotes is thought to diverge after assembly of compound 1 (3-polyprenyl-4-hydroxybenzoate). The length of the isoprenoid chain (n) varies depending on the species and ranges from n = 6 (S. cerevisiae) to n = 10 (Homo sapiens). The other intermediates in the pathway are 2 (2-polyprenylphenol), 3 (2-polyprenyl-6-hydroxyphenol), 4 (3,4-dihydroxy-5-polyprenylbenzoate), 5 (3-methoxy-4-hydroxy-5-polyprenylbenzoate), 6 (2-polyprenyl-6-methoxyphenol), 7 (2-polyprenyl-6-methoxy-1,4-benzoquinone), 8 (2-polyprenyl-3-methyl-6-methoxy-1,4-benzoquinone or DMQ), 9 (2-polyprenyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinone), and 10 (ubiquinone-n). Intermediates 6, 7 and 9 are hypothetical in S. cerevisiae, as is intermediate 3 in E. coli. The asterisk designates the reaction catalyzed by DMQ monoxygenase.
In this work a yeast mutant from the coq7 complementation group has been shown to lack detectable Q, but produces 2-hexaprenyl-3-methyl-6-methoxy-1,4-benzoquinone (5-demethoxyubiquinone or DMQ, compound 8, Fig. 1). We demonstrate here that the COQ7 gene encodes a protein of 272 amino acids, which is necessary for growth on nonfermentable carbon sources and which restores Q biosynthesis in the coq7-1 mutant. Curiously, deletion of the COQ7 gene generates mutant strains that do not accumulate DMQ, but accumulate large amounts of 3-hexaprenyl-4-hydroxybenzoic acid (compound 1). This anomaly is discussed.
Quantitation of Q and
DMQ was by external standard injection of known quantities of Q (Sigma) using the integrated area units of identified peaks.
Concentrations of Q
standards in ethanol were determined
using E
= 15,300 M
cm
(28) . This
method provides a reasonably accurate estimate of DMQ since the two
compounds have similar spectral qualities; DMQ
in ethanol
at 271 nm, E = 14,500 M
cm
(29) .
Analytical HPLC of C-labeled yeast total lipid extracts employed a
cyanopropyl column equilibrated for at least 10 min in 98% solvent A
(hexane) and 2% solvent B (isopropanol:hexane:water:methylene chloride,
52:41:5:2) at a flow rate of 1 ml/min. Ten minutes after sample
injection (10-50 µl), the percentage of solvent B increased
linearly at 1.75 percent/min, a linear gradient was used for 20 min to
a ratio of 63:37 (solvent A:B). At 35 min buffer B reached 45%, and by
45 min it was 100% B. Base-line conditions were restored within 55 min.
Figure 5: Manipulation of cloned sequences. Panel A, restriction mapping and deletion analysis of the cloned genomic sequence rescuing the NM101 strain. A 4.8-kb BamHI fragment of the p7.8 insert was subcloned into the single-copy yeast shuttle vector pRS316 to create pNM782. The 4.8-kb BamHI fragment is represented by the top bar and shows relevant restriction sites. The remaining plasmids were created by the restriction digestion, deletion, and religation of pNM782. The plasmids were tested for functional complementation of the NM101 defect by transformation and growth on YPG plates. Those plasmids capable of restoring growth on YPG plates are shown at the left with a + sign, non-rescuing plasmids are shown with a - symbol. Restriction sites are symbolized with single letters in pNM782 and correspond to the following DNA restriction enzymes: B, BamHI; C, ClaI; E, EspI; H, HinDIII; X, XbaI; Xh, XhoI. The EspI site was mapped only within the pNMQ7 plasmid. Panel B, characterization of the 1.9-kb insert of pNMQ7 and the structure of coq7 deletion constructs. A detailed map of restriction enzyme sites within the COQ7 gene and the upstream open reading frame is shown. The upstream open reading frame was truncated by EspI and XhoI digestion of pNMQ7 creating the rescuing plasmid pNMQ71 (Panel A). A deletion within the COQ7 open reading frame employed EcoRV and StuI digestion to remove a 368-bp blunt-ended fragment, as shown by the cross-hatched area. This DNA segment was replaced with two different DNA fragments containing the LEU2 gene. LEU2 gene fragments are not drawn to scale and are contained on either BglII (2.9 kb) or PstI (4.1 kb) restriction enzyme fragments of YEp13. No difference was seen between the integration of the larger disruption cassette, pYDQ72, versus the smaller disruption cassette, pYDQ71.
Figure 4:
Identification of the accumulating
quinones in NM101 and in JM43,coq71/pNMQ71. The electron
impact mass spectra for the purified radioactive compounds, purified as
in Fig. 3, are shown. Lower panel, the fragments are
arrayed by m/z along the x axis for fraction 14
purified from NM101. The chemical structure of the intermediate and the
likely origin of the fragment ions are shown. The peak at 446.1
corresponds to a known contaminant. Upper panel, the
fragmentation pattern for fraction 10 purified from
JM43coq7
1/pNMQ71. Molecular structure of Q and the
structure of the base peak ions are shown. The y axis for both
panels is the percentage of relative intensity collected for the
represented ions in each spectrum.
Figure 7:
HPLC analysis of Q intermediates from
NM101 yeast strain and coq7 deletion strains shows that deletion of
the gene abolishes the accumulation of DMQ. A three-dimensional
profile of radioactivity present in normal phase HPLC fractions
collected from the fractionation of 400 µl of total yeast lipids
from two coq7 deletion strains (FY250,coq7-1 and
NM101,coq7
-1) and NM101 is shown; 400 µl represents
one twentieth of the labeled lipid extract from 1 liter of yeast).
Strains were labeled, extracted, and analyzed as detailed under
``Materials and Methods.'' The x axis denotes the
milliliters collected per minute, and the y axis represents
C radioactivity in 1-ml fractions (counts/min minus
background). The identity of the lipid extract analyzed is shown in the z axis, with representative symbols in the legend. The largest
peak present in the extract from the NM101 strain is labeled as DMQ.
, nm101D; &cjs2106;, fy250D;
,
NM101.
Figure 6:
COQ7 sequence analysis. Panel
A, the nucleotide sequence and deduced protein sequence is shown
for the COQ7 allele. Nucleotides are numbered from 5` to 3`.
Amino acid residues (single-letter code) are placed under the
center of each codon, and the A of the first ATG codon is designated as
+1. The asterisk at nucleotide 311 designates the single
base pair change (A instead of G) present in the mutant coq7-1 allele and predicts an Asp instead
of Gly. Panel B, the truncated sequences of the yeast COQ7
protein and a probable C. elegans homologue (accession no.
U13642, GenBank(TM) data base) are shown in an alignment generated
from DNAstar(TM) MegAlign using the PAM 250 table and the Jotun Hein
method for alignment. Identical residues are shaded. The
sequences as shown span the region of highest homology, from amino acid
92 of Coq7p and amino acid 20 of the putative C. elegans homologue. These sequences were calculated to be 44% similar and
are 42% identical. The asterisk designates the Gly
Asp change present in the
Coq7-1p.
Figure 3:
Purification and analysis of
[U-C]p-hydroxybenzoic acid-labeled
quinones. Total lipids were extracted from 2 liters of the coq7-1 strain (NM101) or from a 1-liter culture of
JM43coq7
-1/pNMQ71. The total lipid extracts were first
separated by reverse phase HPLC (not shown) as described under
``Materials and Methods.'' This step removed the predominant
C-radiolabeled intermediate 3-hexaprenyl-4-hydroxybenzoate
(compound 1, Fig. 1) from the quinone-like material. Reverse
phase fractions 25 and 26 contained the
C-labeled quinone
material and were individually further purified by normal phase HPLC as
shown in panel A (the isocratic solvent was 0.1% 2-propanol in
heptane). The relative Absorbance units (266 nm) is shown as either a solid line (NM101, fraction 25), or a dotted line (JM43coq7
-1/pNMQ71, fraction 26). Panel B,
radioactivity was monitored by scintillation counting of 10% of each
1-ml fraction of NM101 (black bars) or
JM43coq7
-1/pNMQ71 (open
bars).
Figure 2:
C97 (coq7-1) accumulates a
quinone intermediate which is not Q. Panel A, a lipid extract
from the C97 yeast strain labeled with
[carboxyl-C]p-hydroxybenzoic
acid was separated by normal phase HPLC and 1-ml fractions were
collected and
C radioactivity was determined by
scintillation counting (⧫). Values are plotted as the
C radioactivity in counts/min (minus background). The superimposed line shows the gradient profile of percent polar
components (the B solvent). Panel B, a lipid extract of C97
was labeled with [U-
C]p-hydroxybenzoic
acid as described in panel A. The radioactivity detected in
fraction 8 does not coincide with a ubiquinone standard, which would be
present in fraction 6 (data not shown).
The lower panel of Fig. 4shows the EI mass spectra observed for fraction 14 (Fig. 3) from the coq7-1 strain NM101. The
spectra obtained show a fragmentation pattern consistent with that for
demethoxyubiquinone (2-hexaprenyl-3-methyl-6-methoxy-1,4-benzoquinone,
DMQ, compound 8, Fig. 1; (41) and (42) ). As expected for a quinone-containing intermediate, both
M+2 (CH
O
: 562.439117;
observed mass 562.438596; PPM -0.9) and M
(C
H
O
: 560.421753; observed mass
560.422946; PPM -2.1) ions were present. Further confirmation of
the M and M+2 ions for this compound is found in the presence of
C
ions for M and M+2, which are also within ±
10 PPM error (data not shown). Characteristic base peaks at 167 and 205
correspond to the tropylium and pyrylium ions, respectively, as
detailed above. The peak at 446.1 corresponds to a known contaminant.
Similar spectra were observed for fraction 15 from NM101 (Fig. 3, panel B) and for fractions 14 and 15 from the
respiratory competent yeast strain (Fig. 3, panel B).
Thus DMQ is detected in both the coq7-1 mutant strain
and in a respiratory competent yeast strain. Other fractions were also
analyzed by EI mass spectrometry, but produced no evidence for the
presence of either Q or Q intermediates.
The amount of DMQ found
under the UV peaks at 14-15 min in the chromatograms shown in Fig. 3was estimated by comparing the integrated areas to the
area corresponding to a known amount of Q chromatographed
under the same conditions. This method provides a reasonably accurate
estimate of the amount of DMQ since it has similar UV spectral
qualities to Q
(see ``Materials and Methods'').
The amount of DMQ accumulating in the coq7-1 mutant (109
ng of DMQ/g wet weight NM101 yeast) was found to be similar to the
amount of DMQ present in the respiratory competent strain (159 ng of
DMQ/g wet weight JM43,coq7
-1/pNMQ71). Thus the
defect responsible for the absence of Q
in the coq7-1
strain does not cause DMQ to accumulate above levels of that found in
respiratory competent yeast.
Initial determination of the DNA sequence of pNMQ7 made use of oligonucleotide primers derived from the vector sequence of pRS316. Submission of this partial sequence to GenBank revealed complete identity with the nucleic acid sequence of a truncated open reading frame (ORF C), 357 bp upstream of the UBP2 gene ((33) ; accession no. M94916). The sequence corresponding to this entire open reading frame was then determined and is shown in Fig. 6. The DNA sequence predicts a polypeptide of 272 amino acids with a predicted molecular mass of 30,924.5 daltons.
Recently other investigators made use of an
independent screen and isolated and sequenced a yeast gene CAT5 (accession no. X82930) which has complete sequence identity with COQ7 and may be involved in glucose derepression. ()These investigators also reported (accession no. X82930)
the presence of an upstream open reading frame fully encoded within the
1.9-kb sequence of the pNMQ7 plasmid (Fig. 5, panel B).
This upstream open reading frame present was truncated to create
pNMQ71. As detailed in Fig. 3and Fig. 4, the presence of
the COQ7 open reading frame in pNMQ71 restores both
respiration and synthesis of Q
in the coq7
strain, JM43,coq7
-1, indicating that the 272 amino acid
polypeptide encoded by the COQ7 gene restores Q production.
The amino acid sequence encoded by the COQ7 (CAT5) gene has no remarkable similarity to any known protein (PAM 120 or PAM 250 matrices and the available protein data bases) other than a putative Caenorhabditis elegans homologue present in the cosmid sequence cz395(44) . The yeast Coq7p and the C. elegans predicted protein sequence are 42% identical (Fig. 6, panel B).
Figure 8:
Northern analysis of the COQ7 gene shows it is induced in nonfermentative growth conditions. Two
identical panels from the same Northern blot were each probed with P-labeled DNA fragments corresponding to the COQ7 gene (A) and the COQ3 gene (C). These
same panels were subsequently reprobed with
P-labeled
plasmid DNA containing the clathrin heavy chain gene (CHC1, panels B and D)(37) . Lanes 1 and 2 indicate growth conditions of yeast from which mRNA was
collected: lane 1, growth in YPD; lane 2, in YPG.
Each RNA preparation (5 µg) was separated by electrophoresis on
1.2% agarose-formaldehyde gel. Subsequent Northern analysis was
performed as described under ``Materials and Methods'' with
the COQ7 1.9-kilobase cDNA insert of pNMQ7 (A) or
with a 2.0-kilobase cDNA of COQ3 (B). Probe specific
activities were 3.0
10
and 2.8
10
cpm/µg, respectively. The blot in panels A and C was set aside >12
P half-lives (6 months) and then
hybridized with pCHC1001 (3.5
10
cpm/µg), as
shown in panels B and D. Blots were hybridized at 65
°C and washed three times with 0.2
SSC, 0.1% SDS at 55
°C. RNA size standards (Life Technologies, Inc.) are indicated.
Autoradiographic exposure times were 12 days (A and C) and 5 days (B and D).
This study describes the characterization of coq7 mutants and the isolation of the corresponding COQ7 gene affecting the production of Q in S. cerevisiae. The coq7-1 mutant lacks detectable Q, but does synthesize 3-hexaprenyl-4-hydroxybenzoate and DMQ (compounds 1 and 8, respectively, Fig. 1). The accumulation of 3-hexaprenyl-4-hydroxybenzoate is observed in wild-type yeast and in coq3-coq8 mutants ( (26) and data not shown). The yeast COQ7 gene restores both respiration and the synthesis of Q in the coq7-1 mutant. As expected, coq7 deletion mutants fail to respire and are Q-deficient, but curiously, such mutants fail to produce any detectable DMQ intermediate. Unlike other Q biosynthetic intermediates, which are extremely air- and light-sensitive and difficult to purify(19, 26) , DMQ is fairly stable. In fact DMQ can be readily recovered from wild-type yeast (22) and has been found as an impurity in some commercial sources of Q(42) . Thus it is unlikely that our failure to detect DMQ in the coq7 deletion mutant results from the instability of DMQ. It is also unlikely that DMQ is the product of an unproductive or ``side reaction'' of Q synthesis that might predominate in the coq7-1 mutant, since Law et al.(22) have shown a precursor-product relationship between DMQ and Q in S. cerevisiae. Finally, it is notable that the levels of DMQ present in the coq7-1 mutant do not accumulate to the extent that Q accumulates in wild-type yeast, and in fact the amount of DMQ in the coq7-1 strain is about two-thirds the amount of DMQ of the rescued strain.
Based on the
presence of DMQ and the absence of Q in the coq7-1 yeast
mutant, it is tempting to speculate that the COQ7 gene encodes
a polypeptide involved in a monoxygenase or hydroxylase step with DMQ
as a substrate. However, such speculation must take into account the
presence of DMQ in coq7-1 mutants and its absence in coq7 mutants. Two models are consistent with the above
observations; (i) Coq7p serves a dual function in both the first and
last monoxygenase/hydroxylase steps, and (ii) Coq7p provides a
component essential for the formation of an enzyme complex that
converts intermediate (1) to Q (Fig. 1). In model i, the
nature of the mutation in the coq7-1 allele might
generate a partially functional Coq7-1p, which although blocked
in the conversion of 8 to 9, nonetheless allows the
conversion of 1 to 4 to some extent, resulting in the
production of DMQ. Precedent for model i is provided by examples of
P450 oxidoreductases, some of which catalyze the oxidation of both
related and unrelated substrates(45) . Alternatively, in model
ii Coq7-1p would provide a defective polypeptide creating a
defective multi-enzyme complex that produces a small amount of DMQ, but
is unable to produce Q. Deletion mutants in either model would
accumulate only compound 1 because they would be devoid of any
monoxygenase/hydroxylase activity (model i) or would fail to provide
the polypeptide component required for the Q-biosynthetic enzyme
complex (model ii). Precedent for model ii is provided by the
eukaryotic multi-subunit respiratory
complexes(46, 47) . A further example is found in the
lysosomal storage disease galactosialidosis, where the loss of a
protective protein results in a loss of the multimeric form of
-galactosidase(48) . In these examples, a single
``missing'' or mutant component results in a characteristic
drastic phenotype in which many related components are either missing,
unstable, or inactive. It is important to note that the one base pair
mutation identified in the coq7-1 allele is consistent
with either of the above models and predicts the formation of an intact
polypeptide (Coq7-1p) in which glycine 104 is replaced by
aspartate (Fig. 6). Testing of these models will require the
availability of chemical amounts of the Q-intermediates to use as
substrates for in vitro assays and antibodies to enzymes of
the Q biosynthetic pathway.
Studies of ubiquinone synthesis in E. coli have shown that the three hydroxylation reactions
involved in the aerobic synthesis of the quinone ring from p-hydroxybenzoate utilize molecular oxygen and hence are
catalyzed by monoxygenases(49) . The DMQ intermediate has been
observed in Q-deficient UbiF mutants of E. coli(41) and the UbiF gene in E. coli may
correspond to a DMQ monoxygenase. The E. coli UbiF gene has
not yet been sequenced, and homology searches with the amino acid
sequence of Coq7p revealed no highly significant similarity to any
other class of protein, including any known monoxygenase or hydroxylase
proteins. A probable C. elegans homologue was detected and was
42% identical within the sequences compared (Fig. 6). Recently
other investigators have isolated the COQ7 allele (CAT5) in a separate mutant screen and have
indicated that this protein is involved in glucose derepression. The coq7 complementation group was originally isolated as a
nuclear encoded pet yeast strain and was identified as
Q-deficient because in vitro assays of cytochrome c reductase showed that levels of activity could be returned to
almost wild type by addition of Q. From these results, it is possible
that the Coq7p functions as a regulator of glucose derepression and of
Q biosynthesis.
COQ7 mRNA is induced by heat
shock(33) . Our results demonstrate an induction of the COQ7 mRNA when the cells are grown in conditions demanding
respiratory competence. This is intriguing because of recent evidence
which suggests that heat shock, diauxic shift, and oxidative stress may
be related phenomena through the coordinate control of genes induced by
these stresses(50, 51) . Mitochondria and
mitochondrial structures of the cell do not fully form until the cell
reaches stationary phase(52) , when the cell has exhausted
fermentable carbon sources and is forced to fully develope the electron
transport chain. As cells growing in glucose-based medium pass through
the diauxic shift to respiratory metabolism, they become
thermoresistant (53) and a subset of heat shock genes are known
to be induced(50) . Two consensus heat shock elements are
present in the 5` region of the COQ7 genomic sequence at
-261 to -243 and at -34 to -15. Each of these
sequences lies in the middle of a stretch of nucleotides forming an
imperfect inverted repeat. The sequence found at -261 to
-243 is CACTTTTCCGGAAAAGGG, the 5` sequence at -43 to
-15, is TTTTCAGGAAAA. The heat shock elements are underlined. In
addition a novel heat shock response element,
CT(54) , is present in the upstream region of the COQ7 gene(-541). The observed induction of the COQ7 mRNA by heat shock and by shift to a nonfermentable carbon source
are intriguing and deserve further investigation.